Cryptic species diversity in polypores: the Skeletocutis nivea species complex

Abstract We propose a taxonomic revision of the two closely related white-rot polypore species, Skeletocutis nivea (Jungh.) Jean Keller and S. ochroalba Niemelä (Incrustoporiaceae, Basidiomycota), based on phylogenetic analyses of nuclear ribosomal internal transcribed spacer (ITS) and translation elongation factor EF-1α sequences. We show that prevailing morphological species concepts of S. nivea and S. ochroalba are non-monophyletic and we delineate new species boundaries based on phylogenetic inference. We recognise eleven species within the prevailing species concept of S. nivea (S. calida sp. nov., S. coprosmae comb. nov., S. futilis sp. nov., S. impervia sp. nov., S. ipuletii sp. nov., S. lepida sp. nov., S. nemoralis sp. nov., S. nivea sensu typi, S. semipileata comb. nov., S. unguina sp. nov. and S. yuchengii sp. nov.) and assign new sequenced epitypes for S. nivea and S. semipileata. The traditional concept of S. ochroalba comprises two independent lineages embedded within the S. nivea species complex. The Eurasian conifer-dwelling species S. cummata sp. nov. is recognised as separate from the North American S. ochroalba sensu stricto. Despite comprehensive microscopic examination, the majority of the recognised species are left without stable diagnostic character combinations that would enable species identification based solely on morphology and ecology.


Introduction
Species delimitation in macrofungi has traditionally been based on morphology of fruiting bodies. Yet, their structure is often relatively simple and taxonomically useful characters are scarce. Even with rigorous microscopic inspection of hyphal structures and spores, true species diversity in macrofungi appears to be concealed by limited morphological resolution.
In polypores (a form group of primarily wood-decaying Basidiomycota with poroid hymenophores), numerous DNA-based studies have reported unaccounted species diversity within previously recognised morphospecies: e.g. Antrodia crassa (Spirin et al. 2015), Heterobasidion insulare (Chen et al. 2014, Dai andKorhonen 2009), Laetiporus sulphureus (Banik et al. 2010, Vasaitis et al. 2009), Phellinus igniarius (Tomovský et al. 2010, Zhou et al. 2016 and Porodaedalea pini (Brazee and Lindner 2013). In the above-mentioned cases, the newly discovered species can be, and mostly were, characterised morphologically. It should be noted however that much of morphological taxonomy, including our work, is conducted today as follows: first sequence your material, postulate phylogenetically supported species and then see if you can spot morphological and ecological differences between these phylogenetic species.
A handful of studies have documented morphologically indistinguishable, cryptic species diversity, for instance, in the fleshy fungus Sparassis crispa (Hughes et al. 2014), the polypore genus Heterobasidion (Otrosina and Garbelotto 2010) and the corticioid genera Coniophora (Kauserud et al. 2007), Peniophorella ) and Sebacina (Riess et al. 2013). However, there is no clear distinction when a species is morphologically cryptic. In our own experience, while Antrodiella spp. and Skeletocutis spp. may be identifiable to a few experts with the aid of a phase-contrast microscope, they are cryptic to many mycologists without access to material for comparison Niemelä 2018, Miettinen et al. 2006). To us, it would appear that truly cryptic species are a small minority in polypores and corticioid fungi-morphological differences can be detected even between closely related species.
Morphological resolution is of significance when we need to link old names and unsequenced historical specimens to modern species concepts. In these cases, the question about morphologically indistinguishable, cryptic species becomes relevant.
Here, we demonstrate previously unrecognised cryptic diversity in a polypore species complex comprising the morphospecies Skeletocutis nivea (Jungh.) Jean Keller and S. ochroalba Niemelä in the family Incrustoporiaceae Jülich (Polyporales, Basidiomycota). Despite in-depth morphometrics, extensive material and the best of our expertise, we have not been able to find reliable morphological differences between most species in this complex. Species identification is thus reliant on DNA markers only and we have taken the necessary step to provide sequenced types for all known taxa whenever possible.
According to the prevailing, morphology-based circumscription, S. nivea is a cosmopolitan white-rot polypore species, distributed in tropical and temperate zones on both hemispheres, growing on dead angiosperm wood. S. nivea was originally described from the Island of Java (Indonesia) (Junghuhn 1838) and subsequently basionyms Polyporus semipileatus Peck from North America and Poria coprosmae G. Cunn. from New Zealand have been widely accepted as synonyms of S. nivea. A species currently known as S. diluta (Rajchenb.) A. David & Rajchenb. was originally described as a subspecies of S. nivea but ITS sequences have shown it to be distinct from S. nivea and more closely related to other Skeletocutis species (Vlasák et al. 2011).
S. ochroalba, described from boreal North America (Niemelä 1985), is very similar to S. nivea but grows on coniferous wood. Subsequent findings, identified as this species, have been reported mainly from Eurasia. The morphospecies S. nivea and S. ochroalba are distinguished from other species of Skeletocutis Kotl. & Pouzar by their hyphal structure, which is usually characterised as trimitic in context and monomitic in trama and the very small size of pores and basidiospores. We use the collective term S. nivea complex to refer to both morphospecies.
In this study, we resolve the diversity within the S. nivea complex by phylogenetic analyses of two genetic markers, viz. the nuclear ribosomal internal transcribed spacer (ITS) region and the translation elongation factor EF-1α (tef1), complemented by microscopic study of specimens. We have sampled S. nivea and S. ochroalba widely over their known distributions and combine our newly generated data with previously published sequences. Based on our results, we propose a taxonomic revision of the S. nivea complex.

DNA extraction, amplification and sequencing
In total, 92 ITS and 33 tef1 DNA sequences were generated for this study. In addition, we included sequences publicly available in the International Nucleotide Sequence Database Collaboration database (INSDC). To elucidate the relationship of the S. nivea complex to other taxa in the family Incrustoporiaceae, we also amplified the nuclear large subunit (LSU) region of the rRNA operon from representatives of the S. nivea complex and retrieved LSU sequences of relevant outgroup taxa from the INSDC. LSU is highly conserved within the S. nivea complex but enables comparisons to more distantly related taxa. All of the newly generated sequences were deposited in the IN-SDC. Voucher data for all included sequences are provided in Suppl. material 1.
DNA was extracted from dried herbarium samples of basidiocarps and mycelia from agar cultures using the E.Z.N.A. Forensic DNA kit (Omega Bio-tek). Pieces of the sample were cut out with a scalpel and then homogenised with a mortar and pestle in a 1.5 ml centrifuge tube. Further steps were performed according to the kit manufacturer's protocol.
Polymerase chain reactions (PCRs) were carried out with either the Illustra Pure-Taq Ready-To-Go PCR Beads (GE Healthcare), DreamTaq Green PCR mix (Thermo Scientific) or Phire Tissue Direct PCR Master Mix (Thermo Scientific). A touchdown style PCR programme (designed by Zheng Wang http://wordpress.clarku.edu/polypeet/datasets/primer-information/) was applied for tef1 amplification. The resulting products were sequenced with BigDye v.3.1 and ABI3730XL analyser (Applied Biosystems) by Macrogen and FIMM. The electropherograms of forward and reverse sequences were aligned against each other using Sequencher v. 5.0 (Gene Codes Corporation). The aligned electropherograms were then visually inspected to ensure good sequence quality and ambiguous sequence reads were discarded. Double peaks were interpreted as true base ambiguities when they were detected in both forward and reverse sequencing electropherograms.

Alignments and phylogenetic analysis
For an outgroup analysis, a combined ITS-LSU dataset was assembled with representatives of the S. nivea complex (18 sequences) and outgroups (14 sequences from 11 species). The resulting trees were rooted with Tyromyces merulinus (a possible sister to Incrustoporiaceae) following Justo et al. (2017). The Tef1 (34 sequences) and ITS (139 sequences) datasets were analysed to investigate the phylogeny of the S. nivea complex in more detail and to delineate species within the complex. Tyromyces globosporus (JN710734) was used as an outgroup in the tef1 analysis and Skeletocutis chrysella (JQ673126) in the ITS analysis.
Sequences were aligned using PRANK v.140603 (Löytynoja and Goldman 2010) with default settings. These alignments were checked by eye and alignment errors were corrected manually. In the ITS-LSU analysis, parts of the ITS region were so divergent between distantly related taxa that they could not be credibly aligned and those sections (131/706, 18.6% of aligned positions) were excluded from the ITS-LSU analysis. The alignments and trees were deposited in TreeBASE (22552).
Phylogenetic trees were constructed using the maximum likelihood (ML) and Bayesian inference (BI) methods. ML trees were reconstructed with RAxML 8.2.10 (Stamatakis 2014) with GTR+G set as the evolutionary model and alignments partitioned according to Table 1. The rapid bootstrap with MRE-based bootstopping criterion option was used for branch support estimation.
The substitution models for BI were selected for each partition (Table 1) with JModelTest2 v.2.1.6 (Guindon andGascuel 2003, Darriba et al. 2012) prior to the run. When evaluating models, the number of substitution schemes was set to 3 and models with equal/unequal base frequencies (+F), with/without a proportion of invari-  (Ronquist et al. 2012) in two independent runs with 4 chains for 10 million generations for the analysis of ITS sequences and 1 million generations for tef1 and ITS-LSU analyses. Upon run completion, the first 25% of the trees were discarded as burn-in. By then, the average standard deviation of split frequencies had reached a value lower than 0.01.
All analyses were performed through the Cipres Science Gateway v.3.3 interface (Miller et al. 2010).
The numbers of base substitutions per site within and between the inferred species were calculated in MEGA v.7.0.25 (Kumar et al. 2016) by averaging over all sequence pairs in an analysis with Maximum Composite Likelihood model (Tamura et al. 2004). The rate variation amongst sites was modelled with a gamma distribution (shape parameter = 4). The differences in the composition bias amongst sequences were considered in evolutionary comparisons (Tamura and Kumar 2002). Standard error estimates were obtained by a bootstrap procedure (500 replicates).

Microscopic study
The majority of studied materials are dried specimen collections stored in herbarium H (Helsinki). Type material and reference specimens from herbaria BPI, H, L, O and PDD were also studied. Herbarium acronyms are given according to Thiers (2017).
Pore measurements (12 per specimen) were done under a stereomicroscope (Wild M54) by counting the number of pores per 1 mm; only pores aligned in straight rows were selected for this purpose. Microscopic structures were studied and measured with Leitz Diaplan and Leica DMBL microscopes (×1250 magnification). Microscopic routines used in this study follow Miettinen et al. (2006Miettinen et al. ( , 2012. Measurements were made and illustrations were drawn in Cotton Blue using phase contrast illumination and oil immersion (with a subjective accuracy of 0.1 μm; Miettinen et al. 2006).
In microscopic descriptions, the following abbreviations are used: L -mean spore length; W -mean spore width; Q -mean L/W ratio; n -pore counts, spores or hyphae measured / number of specimens. For presenting a variation of basidiospores and hyphae, 5% of measurements were excluded from each end of the range and are given in parentheses. The respective cut-off for reported pore measures is 20%.

Sequences and phylogeny
Intragenomic variation of ITS sequences, as discussed by Lindner and Banik (2011) and Lindner et al. 2013, was found to be common and widespread in the S. nivea complex. Variation consisted mainly of single nucleotide polymorphisms (SNP) and short length variations of one or a few base pairs. In our analyses and INSDC submission, we always used the shortest alleles.
Phylogenetic analyses of the ITS-LSU dataset ( Fig. 1) show that the S. nivea complex forms a monophyletic assemblage of closely related species in the family Incrustoporiaceae. The genus Skeletocutis in its current circumscription is non-monophyletic and the S. nivea complex belongs to a clade that is separate from the type species of the genus, S. amorpha (Fr.) Kotl. & Pouzar. When the generic limits within the family are clarified in the future, the S. nivea complex should be assigned to a different genus.
Some segments of ITS sequences proved difficult to align unequivocally even within the S. nivea complex. While the composition of the clades which we interpret as species was not affected, the topologies of deeper nodes were found to be somewhat sensitive to small adjustments of the alignment in those variable segments.
Despite partially contrasting topology of inter-group relationships, the phylogenetic analyses of both tef1 (Fig. 2) and ITS (Fig. 3) datasets were concordant in grouping terminals into 13 clades that we interpret and describe here as species. Analysis of the ITS dataset revealed an additional 14 th clade (S. aff. futilis) not included in our tef1 sampling, which may represent a separate species.
All recognised species were strongly supported in the analyses of the tef1 dataset. Corresponding support from the ITS data was strong for all but S. nivea, which lacks true synapomorphic characters in relation to S. lepida and S. yuchengii in that genetic marker.
Average estimated intraspecific sequence divergence in the ITS dataset was up to 0.32% (SE=0.12) (in S. nivea) and in the tef1 dataset up to 4.2% (SE=0.8) (in S. yuchengii). Average estimated genetic distances between species varied from 1.3% (SE=0.4) between S. lepida and S. nivea, to 10.3% (SE=1.1) between S. calida and S. aff. futilis in the ITS dataset and from 3.7% (SE=0.7) between S. impervia and S. lepida, to 16.9% (SE=2.1) between S. futilis and S. ochroalba in the tef1 dataset. The genetic divergence within and between species was generally higher in tef1 than ITS sequences. The full set of estimates of genetic divergence between and within species is provided in Suppl. materials 2-4.

Morphology, ecology and distribution
Morphometrics for each species are reported in Table 2. Characteristics and ecology of the species are summarised in Table 3. Distribution maps depicting the approximate collection localities of specimens are provided in Supplement S5A-C.

Discussion
Phylogeny and species diversity in the Skeletocutis nivea complex Interspecies relationships were not clearly resolved by our data and analyses of the ITS and tef1 datasets resulted in partially contrasting topologies. Skeletocutis futilis (together with S. aff. futilis) and S. calida represent long, divergent branches which are consistently positioned as early diverging lineages in our analyses. However, the position of S. calida was found to be sensitive to slight alterations of alignment in some variable segments of ITS, alternative positions being within the crown group.
The crown group is characterised by relatively short internodes and poor interspecies resolution. However, S. lepida, S. nivea and S. yuchengii were consistently grouped together. Weak support (bs=9, pp=0.30) for S. nivea in the ITS data could be explained by incomplete lineage sorting in this genetic marker after relatively recent speciation. The loss of ancestral alleles can be expected to be slower in the widely distributed S. nivea, which probably comprises a larger population than the regionally endemic S. lepida or S. yuchengii.
Despite their ecological and morphological similarities, the conifer-dwelling species S. cummata and S. ochroalba were recovered as sister species only in the Bayesian analysis of the ITS-LSU dataset. Even then, the support was minimal (pp=0.151).
Our results with the S. nivea complex are in line with other molecular systematic studies in Polyporales (e.g. Carlson et al. 2014) who conclude that tef1 is more variable between species than ITS and provides greater resolution in separating species. Still, unique character state combinations can be identified from ITS Table 2. Pore and spore measurements of species in the Skeletocutis nivea complex. Spore measures in bold-face are accumulated statistics from specimens below.  (oro)boreal Eurasia annual; basidiocarps small, pileus surface and margin slightly pubescent; on downed conifer logs, usually Picea S. ochroalba boreal N America potentially perennial; basidiocarps small, pileus surface and margin slightly pubescent; on downed logs of Picea sequences even for the most closely related species in the S. nivea complex. Intraspecific sequence variation in tef1 was generally larger but roughly corresponding to the variation observed in the ITS region. However, in S. yuchengii, the tef1 sequences show striking divergence in contrast to ITS. To clarify interspecies relationships within the S. nivea complex, additional genetic markers such as the rpb1 will need to be sequenced.

Geographic patterns of diversity
Our sampling is concentrated in the northern temperate zone, where most species of the S. nivea complex appear to be restricted to a single continent or region. This is in accordance with other comparable studies on polypores (e.g. Otrosina and Garbelotto 2010, Spirin et al. 2015, Han et al. 2016, Song and Cui 2017, Spirin et al. 2017. Notable exceptions here include S. semipileata, which has a circumpolar distribution and S. nivea, which ranges longitudinally from northern China to New Zealand.
The greatest species diversity is found in Eurasia and particularly in East Asia with three species unique to the region: S. lepida in Northeast Asia and S. unguina and S. yuchengii in southern China. S. futilis is thus far known only from northern Europe but the closely related S. aff. futilis occurs in North America. S. cummata and S. nemoralis have continent-wide distributions in Eurasia.
North American species include S. calida in southern U.S.A. and S. aff. futilis in northern U.S.A. The conifer-dwelling S. ochroalba represents the North American parallel of the Eurasian S. cummata with boreal, continent-wide distribution.
Wide and disjunct distributions of species like S. semipileata, S. nivea and S. coprosmae indicate that species distributions are not necessarily limited by dispersal ability. Spores in the S. nivea complex are exceedingly small and their theoretical dispersal ability along air currents is practically unlimited (Wilkinson et al. 2012). Yet, the actual dispersal ability may be severely limited by the survivability of the delicate spores during long distance transport and their ability to establish after deposition on a new substrate (Norros et al. 2015).
The generalist ecology of S. semipileata may have facilitated its dispersal over the northern hemisphere, while some of the more restricted species may be limited by low establishment probability imposed by stricter specialisation. Geographic structuring within S. semipileata, particularly evident in the tef1 data -where differentiation between North America and Eurasia and furthermore East Asian and European populations emerges -suggests that gene flow across long distances in this species is somewhat restricted. Similarly, geographic isolation is likely driving the differentiation between S. futilis and S. aff. futilis, be that inter or intraspecific.
Our sampling from the low latitudes and southern hemisphere was sporadic but yielded a proportionately large number of species, most of which were represented by only one or a few specimens. Specimens collected from a relatively small area in Yunnan, southern China fall into three species, two of which (S. unguina and S. yuchengii) are known only from that area. The only two African specimens, both from Uganda, proved to represent separate species, S. impervia and S. ipuletii. Another new species, S. afronivea Ryvarden, morphologically close to the S. nivea complex, was recently described from Uganda (Ryvarden 2018). Sequence data for S. afronivea is not available, so its affinity to the S. nivea complex cannot be verified. However, the larger spore size distinguishes S. afronivea as separate from S. impervia, S. ipuletii and most other species of the S. nivea complex. We anticipate that further studies are likely to reveal even more diversity within the S. nivea complex. Potential hotspot areas include the montane forests of the tropics as well as the temperate forests in mid-latitudes. For instance, Western North America and large parts of the southern hemisphere were not sampled in this study.

Ecology and host tree associations
Ecologically, S. nivea complex can be divided into conifer-dwelling species (S. cummata and S. ochroalba) and angiosperm-dwelling species. The lack of support for a sister species relationship between S. cummata and S. ochroalba suggests that host switching may have happened more than once during the diversification of the S. nivea complex. Both S. cummata and S. ochroalba have remarkably similar fruiting body morphology characterised by small size, pileate form with pubescent pileus surface and the occasional salmon colour on the pore surface as well as spore dimensions that are distinct from most angiosperm-dwelling species. Better phylogenetic resolution of the S. nivea complex would be required to discern whether these shared traits represent homologies or independently derived ecological adaptations.
The host range of the conifer-dwelling species remains unresolved. They are most commonly found on logs of Picea, but S. cummata in the Russian Far East has been collected from Abies and Larix as well. We also studied a specimen from China (L. Ryvarden 21394 (H)), labelled as S. nivea from Pinus, but close inspection revealed the spores to be too small for S. cummata. The specimen was not sequenced so its true species identity remains unverified. In North America, Overholts (1953) and Lowe (1966) report S. semipileata (P. semipileatus) as sometimes growing on conifers such as Picea, Pinus and Thuja. It is possible that the referenced specimens belong to S. ochroalba, but we have not confirmed their identity. However, we have confirmed one Finnish collection of S. semipileata (O. Miettinen 21003 (H)) from Juniperus communis, proving that occasional crossovers of angiosperm-dwelling species to conifer hosts sometimes occur.
Several of the angiosperm-dwelling species are so far known only from one or a few specimens and detailed substrate data were often not available. Our records of S. lepida, S. nemoralis and S. semipileata suggest that individual species are able grow on a wide diversity of woody angiosperms. A preference for Fraxinus wood is evident in S. nemoralis, whereas S. semipileata appears to be rather indifferent in this respect. All species can be found on thin branches, but some (S. nivea and S. semipileata) have also been recorded from coarse woody debris (>20 cm Ø). The examples presented above indicate that ecological specialisation amongst the angiosperm-dwelling species is relatively weak. However, generalisations from common and widespread species, such as S. semipileata, are likely to be biased. Some degree of niche partitioning could be expected at least locally, where two or more species co-occur.
The observed pattern of overlapping species distributions in the S. nivea complex indicates that effective hybridisation barriers are in place between species. The mechanisms of reproductive isolation and the evolutionary processes that have led to their formation are thus far unverified. The existence of possible innate reproductive barriers that prevent hybridisation on shared substrates could be investigated by mating experiments. However, in order to conduct such studies, fresh material will need to be collected to establish living cultures of the species in the S. nivea complex. Further ecological study is also required to elucidate possible higher resolution patterns of distribution and substrate use. Special care should be taken to record detailed collection data including the species, size class, quality, position and decay stage (following e.g. Söderström (1988)) of the substrate.

Crypticity in the Skeletocutis nivea complex
After macroscopic and microscopic study of 60 specimens representing 13 species (more than 700 pores, 1700 spores and 3000 hyphae measured), the majority of the species in the S. nivea complex are left without reliable morphological diagnosis. Intraspecific variability in basidiocarp phenology appears to be too wide to infer interspecific differences. On the other hand, the microscopic structures of the hyphal system are remarkably uniform across species. Easily measurable quantitative characters such as the spore dimension provide only minimal differentiation, if any. Identification is particularly problematic amongst the angiosperm-dwelling species, many of which clearly have overlapping distributions; only S. (aff.) futilis is distinguished by distinctly larger spores.

Taxonomy
A collective description of the S. nivea complex is provided below. Individual species descriptions focus on relevant specifications for each species.

Skeletocutis nivea complex
Description. Basidiocarps (Fig. 4) annual to sometimes perennial; half-resupinate (resupinate with a pileate edge) to resupinate; hard when dry; surface of pileus white to ochraceous, sometimes turning black when old (Fig. 4C); pore surface cream coloured with ochraceous tints, bluish or greenish colour sometimes develops in the tubes (Fig.  4E); context and subiculum with coriaceous consistency and whitish colour; pores 6-10 per mm; tube layer darker than context.
Hyphal structure: context and subiculum seemingly trimitic (Fig. 5C); hyphae are parallel near cap surface, forming a homogenous, coriaceous texture; skeletal hyphae prevailing, unbranched, thick-walled and often solid, refractive; generative hyphae relatively scarce, clamped, sometimes with (unevenly and irregularly) thickened walls and rarely with sandy encrustation, rarely producing generocystidia (encrusted tips of generative hyphae) with thorny encrustation; 'binding hyphae' (Fig. 5C-D) 1-2-4 μm wide, arbuscule-like, simple-septate side-branches of generative hyphae, thin-walled to solid and refractive, developing later than skeletal hyphae and sometimes missing in young parts of context/subiculum but becoming dominant in older parts, sometimes filling up the old tube layer.
Trama (Fig. 5G) monomitic to dimitic; hyphae interwoven, tightly subparallel; generative hyphae 1 -3 μm wide, usually prevailing, clamped, thin-walled or sometimes with slightly thickened walls; skeletal hyphae ( Fig. 6A-C) looking different from those in context and subiculum, sparse, sometimes apparently missing, originating from tramal generative hyphae, winding and irregularly wide (up to 5+ μm) with spacious lumen, walls usually only slightly thickened, slightly refractive; generative hyphae in dissepiment  Niemelä (1985)). A spores B encrusted tomentum hyphae arising from dense cortical tissue C section through context, showing generative and skeletal hyphae and ramified side-branches resembling binding hyphae D ramified arbuscule-like binding hypha, arising from a generative hypha E dissepiment edge hyphae F cystidioles and basidioles G vertical section through a dissepiment edge, showing trama, hymenium with a hyphal peg and sparsely encrusted dissepiment edge hyphae. edges ( Fig. 5E and G) ca. 2 μm wide, thin-walled, slightly undulating, often somewhat irregularly shaped towards the tips, bare to richly encrusted with sandy crystals.
Basidiospores (Fig. 7) narrowly allantoid, 2.5-4.0×0.4-0.9 μm, Q'=3.4-7.0, IKI-, CB-(contents CB+). Discussion. The tramal hyphal structure in S. nivea and S. ochroalba has traditionally been described as monomitic. However, our microscopic study revealed two distinct hyphal types existing in the trama of all species in the S. nivea complex. Amongst the normal clamped and thin-walled generative hyphae, there are usually at least some notably wider and slightly thick-walled hyphae which seem to lack clamps. We call these special hyphae tramal skeletal hyphae. They appear to originate from the generative hyphae in the trama and reach down almost to the pore mouths. Usually the lack of clamps, greater width and thicker walls help to tell them apart from generative hyphae in the trama. Although the tramal skeletal hyphae are usually wide and only slightly thick-walled, some specimens of S. nivea had narrower and solid skeletal hyphae in the trama.
Sometimes the tramal hyphal structure is dominated by the skeletal hyphae but sometimes they seem to be missing completely or occur only sporadically in otherwise monomitic tramal structure (at least in S. nemoralis and S. semipileata). They can also be difficult to detect when the whole tramal structure becomes sclerified and generative hyphae also develop thickened walls, which was observed in some specimens of S. nivea. In general, clear detection of tramal skeletal hyphae is easiest in a squash mount from very thin longitudinal slices of the tube layer which have been properly thinned to an almost disintegrated state.
The nature of the arbuscule-like 'binding hyphae' has been discussed by David (1982) and Niemelä (1985) and both express some reservations about using the term 'trimitic' to describe the S. nivea complex. They point out that the 'binding hyphae' in the morphospecies S. nivea and S. ochroalba originate as clampless side-branches of the generative hyphae and, hence, they are not binding hyphae proper, such as those of Trametes. David (1982) studied the staining reactions of the hyphal walls and noted that the walls of the 'binding hyphae' are congophilic and non-metachromatic whereas the walls of the skeletal hyphae are non-congophilic and metachromatic. Our observations confirm that all species in the S. nivea complex appear to be similar in this respect.
Description. Basidiocarps annual; half-resupinate; up to 1.5 cm wide and 2 mm thick; hard when dry but easy to break apart; pilei thin, protruding up to 5 mm; margin incurved; upper surface minutely rough, matted, white to cream coloured when young, turning ochraceous; context up to 1.5 mm thick, faintly zonate in longitudinal section with thin dark lines separating layers of growth; tube layer up to 0.5 mm thick; pores 8-10(-11) per mm.
Distribution and ecology. The species is known only from two specimens from southern U.S.A., collected from warm temperate deciduous forests where specimens were growing on rather thin twigs of unidentified woody angiosperm.
Etymology Description. Basidiocarps possibly perennial; resupinate to half-resupinate; up to 6 cm wide and 8 mm thick; hard when dry, breaking apart neatly; pilei fleshy, protruding up to 1.7 cm; margin blunt with narrow, sterile ridge on the underside; upper surface minutely rough, matted, white to cream coloured when young, turning ochraceous brown and finally blackish with age; pore surface sometimes with greenishgrey tints deep within the tubes in pileate part; context and subiculum whitish-cream colour to light greyish-brown near contact with substrate (in thick basidiocarps); context up to 5 mm thick, azonate; tube layer from 0.5-1.5 up to 6 mm thick and zonate in perennial basidiocarp, lighter horizontal zones appear where tubes are filled with arbuscule-like 'binding hyphae'; pores (6-)7-8(-9) per mm.
Hyphal Discussion. After examining the type, we have chosen to use a previously published name Poria coprosmae as the basionym for this Australasian species. P. coprosmae was described by Cunningham (1947) from Westland, New Zealand. He (Cunningham 1965) later concluded that his P. coprosmae was the same as Polyporus semipileatus Peck but he treated them mistakenly under the name Tyromyces chioneus (Fr.) P. Karst., as explained by Buchanan and Ryvarden (1988).
In their type studies of Polyporaceae species described by Cunningham, Buchanan and Ryvarden (1988) place P. coprosmae in the genus Ceriporiopsis Dom., rejecting placement in Incrustoporia or Skeletocutis based on the absence of encrusted hyphae. Rajchenberg (1995), on the other hand, found the hyphal structure in the holotype and other collections of P. coprosmae in PDD more in line with that of S. nivea. Our studies of the holotype confirm this view with the addition that we also observed encrusted generocystidia and thin-walled skeletal hyphae in the trama, which are characteristic for the S. nivea complex. Macroscopically, the only other studied specimen from Tasmania looks quite different from the type. However, we could not identify any clear microscopic differences and cannot rule out the possibility that the macroscopic differences represent variation between developmental stages. Nevertheless, considering the level of crypticity in the S. nivea complex, we have reservations in stating that our sequenced specimens are truly conspecific with the old type. Thus, we refrain from assigning an epitype for now.
S. nivea occurs in the North Island of New Zealand and it is possible that these two species could overlap as S. nivea has been shown to extend respectively far into the temperate zone in the northern hemisphere. The type specimen is a thin and resupinate basidiocarp on a fallen branch of a Coprosma shrub. The Tasmanian specimen, on the other hand, has evidently been growing on coarse wood and is unique in having a clearly perennial habit with a zonate tube layer. Description. Basidiocarps annual; half-resupinate to pileate; up to 3 cm wide and (pilei) up to 1 cm thick; hard when dry but easy to break apart; pilei nodulous or thick but steeply sloping, protruding up to 1 cm; margin of pileus curved downwards, blunt, with narrow, woolly ridge on the underside; upper surface matted to minutely pubescent, white to cream coloured when young, turning ochraceous with almost orange hues; pore surface with ochraceous or sometimes salmon/peach coloured tints, sometimes a greenish-grey tint is visible in the tubes; context and subiculum finally coriaceous but looser and fibrous near cap edge and surface; context faintly zonate in longitudinal section with thin dark lines separating layers of growth; tube layer up to 1 mm thick; pores (5-)7-8(-13) per mm.
Basidiospores (2.8-)2.9-3.4(-3.9)×0.5-0.8(-0.9) μm, L=3.1 μm, W=0.66 μm, Q'=(3.3-)3.8-6.0(-6.6), Q=4.68, n=270/9. Distribution and ecology. Boreal, Eurasian taiga; known from Fennoscandia, Czech Republic and Far East. The species seems to be rather rare in Europe but possibly more common in the Far East where Spirin (H) has collected it abundantly. The species has been found growing on fallen spruce logs (Picea abies, P. jezoënsis) but also on Abies nephrolepis and Larix sp. Discussion. S. cummata is most notably distinguished from other Eurasian species in the S. nivea complex by its occurrence on conifer wood. Spores of S. cummata are also larger than those of angiosperm-dwelling species apart from S. futilis. The pubescence on pileus surface in pileate specimens provides an additional identification cue. Very wide but thin-walled tramal skeletal hyphae seem to be particularly pronounced in this species. All distinctive features of S. cummata are shared with the North American conifer-dwelling species S. ochroalba.  Description. Basidiocarps annual; half-resupinate; small, up to 5 mm wide and 1.5 mm thick; hard when dry but easy to break apart; pilei very small and nodulous; upper surface white when young, turning yellowish-brown; context and subiculum white; tube layer up to 0.2 mm thick; pores 6-8 per mm.
Distribution and ecology. The species is known only from the type specimen which was collected from a Betula stand on a disturbed site near the seashore in Helsinki, Finland (hemiboreal zone) where it was growing on rather thin twigs of Sorbus aucuparia.
Discussion. While macroscopic features may be quite scanty, characteristic trimitic-looking subiculum, skeletal hyphae in trama and encrustations of dissepiment edge hyphae reveal S. futilis to be a member of the S. nivea complex. S. futilis can be distinguished from other species in the complex by thicker spores.
In our analyses, S. futilis constitutes a sister taxon to the rest of the S. nivea complex. The clade also includes S. aff. futilis in North America. Owing to the limited material available, we refrain from judging whether they represent geographic variation within one species or vicariant sister species. The voucher specimens of S. aff. futilis (Lindner DLL2009-067; -068 (CFMR)) are in a rather poor condition, but it seems that the small size of basidiocarps and thick spores are as characteristic for S. aff. futilis as they are for S. futilis. Description. Basidiocarps annual; half-resupinate; up to 6 mm thick; hard when dry, breaking apart neatly; pilei fleshy, protruding up to 5 mm; margin blunt; upper surface almost smooth, matted, white to cream coloured when young, turning ochraceous brown and finally blackish with age; context and subiculum whitish-cream to light greyish-brown; context up to 5 mm thick, zonate in longitudinal section with thin dark lines separating layers of growth; tube layer from up to 1 mm thick; pores (7-)8-9(-10) per mm.
Distribution and ecology. The species is known only from the type specimen, collected from Bwindi Impenetrable National Park in Uganda where it was reportedly growing on rotting branches.
Specimen examined. UGANDA. Western Reg.: (holotype, see above). Description. Basidiocarps annual; half-resupinate; up to 2.5 cm wide and 6 mm thick; hard when dry, breaking apart neatly; pilei fleshy, protruding up to 5 mm; margin blunt; upper surface almost smooth, matted, white to cream coloured when young, turning ochraceous brown; pore surface cream coloured with greyish tint deep within the tubes; context and subiculum whitish-cream to light greyish-brown; context up to 5 mm thick, faintly zonate in longitudinal section with thin dark lines separating layers of growth; tube layer up to 1.5 mm thick; pores 9-10(-11) per mm.
Distribution and ecology. The species is known only from the type specimen, collected from Kibale National Park in Uganda, where it was reportedly growing on a leaning, dead branch.
Etymology. Named in honour of the pioneering Ugandan mycologist Perpetua Ipulet, who collected the type of this species.
Etymology Basidiocarps annual; resupinate to half-resupinate; bone hard when dry, breaking apart neatly; resupinate basidiocarps up to 10+ cm wide; pilei nodulous to shelfshaped, often laterally fused and fleshy, protruding up to 1.3 cm; sterile margin often quite pronounced especially in resupinate part; upper surface slightly rough, matted, white to cream coloured when young, turning ochraceous and finally blackish with age; pore surface sometimes with a greenish-grey or turquoise tint emerging within the tubes especially in the pileate part but sometimes in scattered blotches; context sometimes faintly zonate in longitudinal section; tube layer up to 2 mm thick; pores (6-)7-8(-10) per mm.
Etymology. Refers to the distribution area of the species in the nemoral zone. Discussion. S. nemoralis shares its wide distribution in Eurasia with similar-looking S. semipileata. Both species tend to form rather large, half-resupinate basidiocarps with fleshy pilei. S. nemoralis has slightly larger spores and pores than S. semipileata, but the distinction is probably too small for definitive identification.
In Europe, S. nemoralis does not reach as far to the northeast as S. semipileata and appears to be missing in continental Finland. At its north-eastern outpost in Åland Islands, S. nemoralis is rather common, especially in old coppice meadows where it prefers the wood of Fraxinus. Description. Basidiocarps annual; half-resupinate; hard when dry, breaking apart neatly; pilei nodulous to shelf-shaped, sometimes laterally fused and quite fleshy, up to 2 cm wide and 5 mm thick, protruding up to 1.3 cm, often connected to wider resupinate part; upper surface almost smooth to slightly rough, matted, white to cream coloured when young, turning ochraceous and finally blackish with age; pore surface often with a greenish-grey or turquoise tint emerging within the tubes particularly in the pileate part but often in scattered blotches; context and subiculum coriaceous, white; context sometimes faintly zonate in longitudinal section; tube layer up to 1 mm thick; pores (7-)8-10(-13) per mm.
Basidiospores ( Discussion. S. ochroalba is most notably distinguished from other North American species of the S. nivea complex by its occurrence on conifer wood. Surface of the pileus is characteristically pubescent in this species. The spores of S. ochroalba are also thicker than those of North American angiosperm-dwelling species apart from S. aff. futilis. Resemblance to the Eurasian conifer-dwelling S. cummata is strong both in phenology and microscopic structure. Description. Basidiocarps annual; resupinate to half-resupinate; hard when dry, breaking apart neatly; resupinate basidiocarps up to 10+ cm wide; pilei nodulous to shelf-shaped, often laterally fused, up to 4 mm thick and protruding up to 1.5 cm, sometimes rather fleshy but often thin and sharp-edged with slightly incurved margin or with narrow, sterile ridge on the underside; upper surface slightly rough, matted, white to cream coloured when young, turning ochraceous and finally blackish with age; pore surface cream coloured with ochraceous or rarely faint salmon coloured tints, often a greenish-grey or turquoise tint emerges within the tubes particularly in the pileate part, sometimes in scattered blotches; context and subiculum coriaceous, white; context sometimes faintly zonate in longitudinal section; tube layer up to 2 mm thick; pores (7-)8-9(-11) per mm.
Distribution and ecology. The species is known only from the type specimen, collected from Yunnan, China, where it was growing as small individual pilei on thin twigs of unidentified woody angiosperm.
Etymology. Derived from unguis (Lat.), claw, nail; refers to nail-like basidiome caps. Specimen examined. CHINA. Yunnan: (holotype, see above). Description. Basidiocarps annual; resupinate to half-resupinate; small, up to 2.5 cm wide and 3 mm thick; hard when dry but easy to break apart; pilei nodulous, protruding up to 3 mm; margin blunt; upper surface minutely rough, matted, white to cream coloured when young, turning ochraceous; pore surface cream coloured with yellowish to ochraceous tints; context up to 2.7 mm thick faintly zonate in longitudinal section with fuzzy, ochraceous lines separating layers of growth; tube layer up to 0.3 mm thick; pores 8-10(-11) per mm.
Distribution and ecology. The species is known from three specimens from Yunnan, China, collected from twigs of unidentified woody angiosperm.
Etymology. In honour of the Chinese polypore researcher, Prof. Yu-Cheng Dai. Specimen examined. U.S.A. Maine: (holotype, see above) Discussion. P. hymeniicola is a poorly known species from North America which has sometimes been associated with the S. nivea complex (P. semipileatus by Lowe (1947Lowe ( , 1966). Niemelä (1998) studied the type specimen and concluded that the dimitic trama with solid skeletal hyphae does not match with the S. nivea complex. Even though we have observed some specimens of S. nivea with such hyphal structure, they were not observed in North American material. Furthermore, the basidiocarp of the type specimen grew on a dead basidiocarp of another polypore species, unlike any of our studied material of the S. nivea complex. The species would appear to be related to S. stellae and related species (Incrustoporia).
Discussion. Cunningham (1950) mentions the name P. alboniger as the label of a herbarium specimen he determined to be P. atromaculus (see below). We consider the name to be invalid as it lacks proper description (ICBN Melbourne Art. 38.1 & 39.1).
Discussion. Cunningham (1965) considered P. atromaculus to be a synonym of S. nivea (or Tyromyces chioneus, as he called it). The name refers to Tasmanian collections of L. Rodway. Lloyd (1922) used the name P. atromaculatus in reporting his determinations to Rodway. Referencing Lloyd (1922), Stevenson and Cash (1936) published Polyporus atromaculus Lloyd in herb. accompanied by a description and specification of a type specimen in a list of fungus names proposed by Lloyd. It is doubtful whether their intention was to validate the name but the description remains invalid (ICBN Melbourne Art. 39.1) and we reject the name. Ryvarden (1990) appears to share this view as he does not include P. atromaculus in his type studies of Polyporus species described by Lloyd.