Микология и фитопатология, 2022, T. 56, № 5, стр. 332-349

INTRODUCTION

Suillus paluster (Peck) Kuntze and S. ochraceoroseus (Snell) Singer are two phylogenetically close and morphologically similar species. Both species have a nonviscid pileus covered with fibrillose squamules, a decurrent radiating hymenophore, and a vanishing membranous ring. S. ochraceoroseus was described by Walter Henry Snell in 1941 from Idaho in the American Northwest as Boletinus ochraceoroseus Snell. The characteristic features of this species are large fleshy fruiting bodies of pink-ocher color and bitter taste, which is enhanced by heat treatment. The North American population of this species is possibly localized in the northwest of the continent (Washington, Montana, Idaho, Oregon in USA and Alberta, British Columbia, and Canadian Northwest Territories in Canada (Snell, 1941; Pomerleau, Smith, 1962; GBIF, 2022a; Mycoportal, 2022). Specimens collected in the northwest of North America are associated with tamarack (Larix lyallii Parl. and Larix occidentalis Nutt.). Collections outside this area are rare and were made in the artificial plantation of the western American larches (Nguyen et al., 2016; GenBank ID KX213794).

Suillus paluster was described as Boletus paluster Peck by Charles Horton Peck in 1870 (Peck, 1872) from State of New York in the northeastern United States. This species is characterized by slender and small fruitbodies with red scales, a strongly radiating hymenophore with pronounced radial ribs and very large angular pores, a rather thin stem and slightly sour taste. Suillus paluster occurs in northeastern North America and associated with Larix laricina (Du Roi) K. Koch. (Peck, 1872, Pomerleau, 1964; Mycoportal, 2022).

Suillus paluster occurrences were repeatedly published from the territory of Russia from the north of the European part to Siberia and the Far East (Bolshakov et al., 2021).

We analyzed herbarium specimens from North Asia stored at LE, YSU, and MAG herbaria as well as our own collections and images of observations and specimens presented at GBIF and Mycoportal. (GBIF, 2022b; Mycoportal, 2022). A preliminary visual revision of the collections and observations of S. paluster from northeastern Eurasia (European Russia, Siberia, Far East, northern China) showed the presence of two morphological types among them: similar to the protologue of S. paluster and similar to the protologue of S. ochraceoroseus. We assumed that instead of one S. paluster species, two species, S. paluster and S. ochraceoroseus, may be present. In this case, we can observe two types of range disjunction in the species complex: the temperate Asian – East American disjunction in the range of S. paluster and the temperate Asian – West American disjunction in the range of S. ochraceoroseus.

The purpose of this work was a phylogenetic verification of the presence of two species – S. paluster and S. ochraceoroseus in North Asia, and the study of their phylogeographic relationships.

MATERIALS AND METHODS

The specimens collected by the authors in Subpolar Urals, Western Siberia and the Far East of Russia, as well as herbarium collections stored in the herbariums of Canadian National Mycological Herbarium – AAFC (DAOM), Institute of the Biological Problems of the North, Far-Eastern Branch of the Russian Academy of Sciences (MAG), Komarov Botanical Institute (LE), Royal Ontario Museum (TRTC), University of Michigan (MICH), Yugra State University (YSU) were analyzed.

Macroscopic descriptions were based on the study of both fresh and dried material as well as on photographs. Microstructures were observed at ×400 and at ×1000 in squash preparations in 5% KOH, Congo Red, and Melzer’s reagent. Up to 30 basidiospores, 10 cystidia, and 10 terminal elements of pileipellis per specimen were measured to obtain descriptive statistics. Measurements were made in ToupView V.3.7 (ToupTek Photonics) calibrated by an OMP object-micrometer (LOMO). Dimensions are given as (abs min) average min – average max (abs max), Q = average min – average max quotient (length/width ratio).

The color description is given in the RGB color model according to the cell fill mixer in MS Excel.

PCR ITS1–5.8S–ITS2 products were obtained without DNA extraction using the standard protocol of Thermo Scientific Phire Tissue Direct PCR Master Mix kit and amplification with ITS1-F and ITS4-B primers (Gardes, Bruns, 1993). For the PCR TEF1α products primers EF1-983F and EF1-1567R (Rehner, Buckley, 2005) were used. Amplified products were sequenced using BigDyeH Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California). The sequences were assembled in CodonCode Aligner V.9.0.1 (CodonCode Corporation) and manually interpreted to correct the ambiguous bases.

For phylogenetic inferences, 33 ITS sequences were used, of which 9 were obtained by the author in the course of this work, the rest were downloaded from the international NCBI GenBank database, as well as 22 TEF1α sequences (19 downloaded from NCBI GenBank and 3 obtained by the authors). GenBank ID, Herbarium Numbers and Country of origin are listed in Table 1. The datasets were aligned in MAFFT online v. 7 (http://mafft.cbrc.jp/alignment/server) (Katoh et al., 2019). Phylogenetic differences were measured using Hamming dissimilarity in UGENE v.37 (Okonechnikov et al., 2012) in the ITS (33 sequences, 509 bp including alignment gaps) and TEF1α (22 sequences, 506 bp including alignment gaps) datasets. ITS and TEF1α Bayesian phylogenetic trees (Fig. 1a, 2) were generated in BEAST v1.10.4 (Suchard et al., 2018) using the GTR + I + G model, strict model of molecular clock without calibration, random starting tree and 10 million generations. Bayesian phylogeographic tree ITS (20 sequences, 501 bp including alignment gaps), was generated in BEAST v2.6.4 (Drummond, Bouckaert, 2014), using bModelTest (Bouckaert, Drummond, 2017), 10 M of generations.

Fig. 1.

Bayesian phylogenetic tree ITS, 10 M of generation, generated in BEAST v. 1.10.4, GTR + I + G model: I – Boletinus clade; II – Suillus clade. Specimens ecology and geography: A – North Asian larch range (Larix sibirica, L. gmelinii, L. cajanderi, + L. kaempferi), B – West American larch range (L. occidentalis, L. lyallii), C – East American larch range (L. laricina). Posterior probability above the branches (below 0.6 not shown). Specimens voucher ID/GenBank ID. Country and region of origin and/or taxonomic name in leaves. Newly generated sequences are given in bold. *In the plantings outside of natural range of North Asian larch Larix sibirica. **In the plantings outside of natural range of West American larch L. lyallii.

Fig. 2.

Bayesian phylogenetic tree TEF1α, 10 M of generation, generated in BEAST v. 1.10.4, GTR+I+G model: I – Boletinus clade; II – Suillus clade. Specimens ecology and geography: A – North Asian larch range (Larix sibirica, L. gmelinii, L. cajanderi, + L. kaempferi); B – West American larch range (L. occidentalis, L. lyallii). Posterior probability above the branches (below 0.6 not shown). Specimens voucher ID/GenBank ID, Country and region of origin or taxonomic name in leaves. Newly generated sequences are given in bold.

RESULTS

Phylogenetic analyses of ITS and TEF1α regions show a well-supported S. ochraceoroseus/S. paluster clade in both trees. Both species are also represented by well-supported subclades. The latter, in turn, diverge into groups corresponding to American and Asian populations. ITS and TEF1α trees have the same topology (Figs 1, 2). The Hamming distance between species clades of S. ochraceoroseus and S. paluster are 1–2% according to ITS (4–9 bp from 506), according to TEF1α 1% (4–6 bp from 509). However, the interspecific and intraspecific distance may overlap and depend on geography. S. ochraceoroseus/S. paluster clade is part of the boletinoid group, which also includes other species with similar morphology, S. cavipes (Klotzsch) A.H. sm. et Thiers and Suillus asiaticus (Singer) Kretzer et T.D. Bruns, formerly belonging to the section Boletinus (Smith and Thiers, 1964). S. cavipes is the closest sister species, Hamming distance from S. ochraceoroseus/S. paluster clade (ITS) is 4% (18–20 bp). The Hamming distance (ITS) to the most externally similar species, S. asiaticus, is 5% (23–28 bp).

Morphological features of the species S. ochraceoroseus and S. paluster are presented in Table 2. Images of fruiting bodies in situ are shown in Fig. 3. Microstructures demonstrating interspecies diagnostically important differences between S. ochraceoroseus and S. paluster are shown in Fig. 4.

Fig. 3.

Basidiocarps of Suillus ochraceoroseus (a–c) and S. paluster (d–f) in situ: a – YSU-F-11775; b – observation (no specimens collected), Subpolar Urals, 28.07.2020 (photo by E. Zvyagina); c – observation, Magadan, 22.07.2011 (photo by N. Sazanova); d – observation, Magadan, 28.07.2011 (photo by N. Sazanova); e – MAG 5845; f – MAG 4957. Bars – 1 cm.

Fig. 4.

Interspecies diagnostically important differences between Suillus paluster (a) and S. ochraceoroseus (b). Bars: Sp (spores) – 10 μm, Pp (pileipellis) – 20 μm, Fb (fruiting body) – 1 cm.

Species from sister clades of S. cavipes and S. asiaticus differ from S. ochraceoroseus and S. paluster in having a hollow stipe. S. phylopictus Rui Zhang, X.F. Shi, P.G. Liu et G.M. Muell. and S. spraguei (Berk. et M.A. Curtis) Kuntze, also having a dry scaly brown-red surface of the cap and stem, are distinguished by a wooly fibrous partial veil, pubescent stipe, brown in KOH cystidia, collected in bundles and immersed in gelatinous exudate.

Analysis of Asian and American specimens showed that S. paluster differs from S. ochraceoroseus in slender fruiting bodies, prominent radial ribs of the hymenophore protruding over very large pores up to 5 mm, wider ellipsoid spores, and the structure of the pileipellis (Fig. 5). S. paluster has trichoderm from free septate hairs with elongated pointed ends, S. ochraceoroseus has plagiotrichoderm from glued septate hyphae, sometimes with pointed ends. The scales on the surface of the cap of S. ochraceoroseus are formed by raised patches of glued hyphae of the pileipellis. In addition, there are a number of less obvious differences. The color of the surface of the fruit bodies of  S. paluster is more evenly red without changing from pink to ocher, the flesh is more yellow, the taste is mild without bitterness, the preferred habitats are wet, waterlogged with sphagnum.

Fig. 5.

Bayesian phylogeographic tree ITS + Location, 10 M of generation, generated in BEAST v. 2.6.4. Posterior probability above the branches (below 0.6 not shown). Specimens voucher ID/GenBank ID, Country and region of origin in leaves. Newly generated sequences are given in bold. Discrete geography of the specimen’s origin locations in color: 1 – North Asia, 2 – Northwest North America, 3 – Northeast North America. *In the plantings outside of natural range of North Asien larch Larix sibirica. **In the plantings outside of natural range of Western American larch (L. lyallii).

S. ochraceoroseus is varying degrees of pink-buff with light yellow, sometimes bluish flesh, slightly bitter to acrid taste, and grows in a variety of environmental conditions.

We did not find significant intraspecific differences in morphological characters between populations from North Asia and North America.

Pronounced and stable morphological differences between species S. paluster and S. ochraceoroseus may indicate the genetic isolation. However, in this case, morphological differences are combined with a relatively small genetic distance between species with a comparable distance between populations of the same species. According to Genealogical concordance phylogenetic species recognition (GCPSR) (Taylor et al., 2000), the concordance of trees of different genes at the junction of species level branches arises as a result of the fixation of previously polymorphic loci due to genetic isolation and is a reliable criterion for species recognition. Therefore, despite the fact that the nucleotide differences between the sequences of S. ochraceoroseus and S. paluster do not reach the psychological threshold of 3%, the concordance of the ITS and TEF1α phylogenetic trees is at the level of the divergence of S. ochraceoroseus and S. paluster, good support for the branches of S. ochraceoroseus and S. paluster in both trees, significant differences in the morphology of the hymenophore, spores, and pileipellis make it possible to distinguish between these species.

DISCUSSION

According to the ITS phylogeographic tree (Fig. 5), the separation of S. ochraceoroseus and S. paluster may have originated in North America. The sequences of the Northwestern American clade of S. ochraceoroseus share substitutions with the Asian clade of the species and the Northeast American clade of S. paluster and a similar Hamming distance (3–5 and 4–5 differences per 501 bp).

The distribution of the species is shown in Fig. 6. The American part of the range of S. ochraceoroseus is confined to the distribution of the two Western American larches, Larix lyallii and L. occidentalis (Little, 1971). An illustration of the modern distribution of larches can be seen in the article by Semerikov and Lascoux (1999). The modern range of these larches does not contact with the range of L. laricina, the host of Suillus paluster. However, the extant American larch populations originate from glacial refugia that were located south of the ice sheet (Whitlock, 1995). More data from both North America and Eurasia are needed to reconstruct the migration pattern and timing. However, there is evidence that both North Asian lineages of S. ochraceoroseus and S. paluster, which apparently formed independently of each other, are derivatives from the two North American species (Fig. 3).

Fig. 6.

Suillus ochraceoroseus and S. paluster distribution combined Peck (1873), Snell (1941), Slipp, Snell (1944), Pomerleau, Smith (1962), Pomerleau (1964), GBIF (2022 a, b), Mycoportal (2022), GenBank (Table 1), herbarium collections LE, MAG, YSU and Tatiana Bulyonkova personal collection. Distribution of Larix taxa adapted from Semericov and Lascoux (1999).

In the case of S. ochraceoroseus, we can see a well-supported North Asian lineage and a somewhat weaker lineage from western North America. The North Asian lineage is represented by sequences of specimens collected in the western (Western Siberia), eastern (Kamchatka), and southern parts of North Asia (Northern China and Japan). The northwestern North American lineage of S. ochraceoroseus contains sequences of specimens collected in Idaho and Washington (USA). Both groups contain sequences of the ‘alien’ specimens, originating from other geographical regions. Together with the North Asian sequences, the MK573971 sequence of LE216155 collected in the artificial plantation of Asian larches [Larix gmelinii (Rupr.) Kuzen., L. sibirica Ledeb.] Lindulovskaya Roshcha in the Leningrad Region (Russia) is grouped. The Northwest American lineage contains the KX213794 sequence of specimen S191 from the Washington (DC, USA), collected according to the annotation in the park under L. lyallii (Nguen et al., 2016). In both cases, the host plant grew outside the boundaries of its natural range.

The Suillus paluster clade in the ITS tree consists of a well-supported North Asian lineage and a weakly supported clade of northeastern North American specimen sequences.

Therefore, in one complex of closely related species, we can observe two examples of Asian – North American disjunction: temperate Asian – East American disjunct species S. paluster and temperate Asian – West American disjunct species S. ochraceoroseus.

According to a recent checklist of agaricoid and boletoid species in Russia (Bolshakov et al., 2021), S. ochraceoroseus is mentioned now for the first time.

Below we provide descriptions of the morphology of the Asian specimens of S. paluster and S. ochraceoroseus.

Suillus ochraceoroseus (Snell) Singer, Persoonia 7(2): 319, 1973. ≡ Boletinus ochraceoroseus Snell, Mycologia 33(1): 35, 1941. ≡ Fuscoboletinus ochraceoroseus (Snell) Pomerleau et Smith, Brittonia 14: 158, 1962.

Iconography: Pomerleau and Smith (1962: 159, pl. 1 as F. ochraceoroseus).

Basidiocarps boletoid. Pileus 6.5–11 (12) cm diam, convex at first, then flat or with raised edge. Surface dry and scaly, squamules fibrous, bright pink (R138G41B48) or ocher pink (R170G89B96) on a light pink and yellow background. Fragments of grayish-pink (R207G174B195) membranous partial veil remain along the edge of the cap, darkening with time. Hymenophore boletinoid, strongly deccurrent with angular pores up to 2–3 mm wide, dirty buff (R219G213B147). Flesh thick, light yellow, pinkish-yellow, sometimes turning blue when cut. Stipe cylindrical, thick, central, 1–1.5 cm in diameter, 5–8 cm tall, covered with a network of decurrent hymenophore, pinkish-buff (R198G134B96) in the upper part, naked, dry, pink (R169G98B116) with light stains (R210G190B189) under the ring. Stipe flesh solid, yellow, in some cases bluing. Partial veil membranous, covered on the outside with a grayish-pink bran-like bloom, forming a thin membranous vanishing ring. Smell indistinct, taste bitter, intensifying with heat treatment, almost disappearing upon drying.

Basidiospores (6.8) 7.4–10.5 (11.2) × 2.8–3.6(3.7) μm, Q = 2.4–3.2, nearly cylindrical yellow in KOH. Basidia (16.8)20.0–29.2(29.9) × (3.2) 4.8–7.4(8.2) μm four-spored, club-shaped, hyaline or light yellow in KOH. Cystidia (26.9)37.8–74.5(82.1) × (7.2)7.7–12.2(13.8) μm, cylindric, subfusiform, subclavated, sometimes with outgrowths or septa, hyaline or yellowish in KOH. Pileipellis – plagiotrichoderm, consisting of septate hyphae with pointed terminal hyphae (40.5)42.1–92.8(99.0) × (8.8)9.6–17.2(19.3) µm, forming scales of tightly glued hyphae, ocher in KOH.

Habitat and distribution. In larch and mixed forests. Forms mycorrhiza with Larix. The Asian population enters Eastern Europe in the west along with plantings of Larix sibirica, occupies Siberia, the Far East and Japan in the north and east, and Northern China in the south. The American population is localized in the northwestern United States.

Specimens examined: Russia, Leningradskaya Oblast, Vyborgskiy rayon, Wildlife Sanctuary “Lindulovskaya Roshcha”, near the Roshchino station, route number 1, 60.236077°N, 29.544979°E, edge of bilberry Scots pine forest with spruce, roadside in artificial plantation of Larix sibirica, 19.08.1997, coll. O.V. Morozova (LE 216155, GenBank ITS MK573971); ibid., 60.236077°N, 29.544979°E, under Larix, 03.08.1952, coll. E. Kosinskaya (LE 4503) (Vasilkov, 1952, as Boletus paluster); Yaroslavskaya Oblast, Breytovskiy Rayon, Zakharyino, Darvinovskiy zapovednik, 57.93832°N, 40.28116°E, 01.08.1951., coll. T. Kutova (LE 4511) (Vasilkov, 1952, as Boletus paluster); Yamalo-Nenetskiy Avtonomnyy Okrug, Labytnangi, 66.65553°N, 66.38592°E, 01.08.1962, coll. B. Vasilkov (LE 4504); ibid., 66.65553°N, 66.385918°E,.07.08 1962, coll. E. Nezdoiminogo (LE 4505); Khanty-Mansiyskiy Avtonomnyy Okrug, Berezovskiy Rayon, Neroyka village vicinities, 64.54848°N, 59.6407°E, Larix-dominated forb sparse forest, 12.08.2020, coll. E. Zvyagina (YSU-F-11779); ibid., Sovetskiy Rayon, Malaya Sos’va Nature Reserve, Khangokurt, 61.958009°N, 64.241868°E, Pinus-dominated forest with larch, 16.08.1990, coll. A. Vasina (LE 312269) (Zvyagina, Vasina, 2015, as Suillus paluster);, ibid., road Khanty-Mansiysk – Sovetskiy, 61.22826°N, 64.16629°E, pine and larch forest with dwarf shrubs and feather mosses in ground cover, 22.08.2010, coll. E. Zvyagina (YSU-F-11775, GenBank ITS MK573964); ibid., Kondinskiye Ozera Nature Park, 60.92161°N, 63.68796°E, pine and larch forest with dwarf shrubs and feather mosses in ground cover, 24.08.2010, coll. E. Zvyagina (YSU-F-11776); ibid., Malaya Sosva Nature Reserve, Belaya Gora rangers station, 61.790608°N, 64.516273°E, pine and larch forest with dwarf shrubs and lichens in ground cover, 15.08.2013, coll. E. Zvyagina (YSU-F-11778) (Zvyagina, Vasina, 2015, as Suillus paluster); ibid., Surgutskiy Rayon, Aitromyegan river, 61.690167°N, 74.353101°E, mixed taiga, 27.07.2010, coll. S. Babyuk (YSU-F-11781, GenBank ITS MK573966); ibid., Ugut village, left bank of Ugutka river, 60.50823°N, 74.05491°E, pine and larch forest with dwarf shrubs and lichens in ground cover, 30.08.2011, coll. E. Zvyagina (YSU-F-11780); ibid. 60.48260°N, 74.06948°E, pine after fire forest, 05.09.2013, coll. E. Zvyagina (YSU-F-11777); Respublika Buryatiya, Barguzinskiy Rayon, Bukhta Sosnovka, North-East Baykal, 54.83253°N, 109.67543°E, Larix sibirica, Pinus sibirica Du Tour mixed forest, 15.08.1966, coll. E. Nezdoiminogo (LE 4510) (Nezdoyminogo, 1969 as Boletinus paluster); ibid., Severo-Baykalskiy Rayon, Davsha, 54.35456°N, 109.50222°E, Larix sibirica crowberry-bearberry forest, 08.08.1969, coll. E. Nezdoiminogo (LE 4511); Magadanskaya Oblast, Khasynskiy Rayon, Myakit vicinities., 61.37474°N, 152.01646°E, in artificial plantation of Pinus sylvestris L., mixed with Larix cajanderi, 13.08.2015, coll. N. Sazanova (MAG4290); ibid., Olskiy Rayon, Raduzhniy, 59.70061°N, 150.153051°E, larch forest, 09.09.2002, coll. N. Sazanova (MAG2001); ibid., zakaznik “Kavinskaya dolina”, 59.68719°N, 147.49039°E, edge of larch forest, 19.08.2017, coll. N. Sazanova (MAG4965); ibid., ruchey Omylen, 59.76876°N, 148.21369°E, mixed forest, 23.08.2017, coll. N. Sazanova (MAG4975); ibid., Magadan vicinities, 59.55950°N, 150.812274°E, in Larix cajanderi Mayr forest with Pinus pumila (Pall.) Regel and Betula middendorffii Trautv., 01.09.1953, coll. B. Vasilkov (LE 4502); ibid., 59.55950°N, 150.812274°E, 1953, coll. A. Vaskovskiy (LE 4402), Kamtchatski Kray, Bystrinsiy Rayon, Esso village vicinities, Bystrinskiy National Park, left bank of the Uboyny Stream, 55.99806°N, 158.72444°E, in Larix cajanderi forest, 06.08.2005, coll. A. Kovalenko, O. Morozova, N. Psurtseva (LE262192 Ex55 (Morozova, Popov, 2008 as Boletinus paluster), GenBank ITS MK573968); Khabarovskiy Kray, Okhotskiy Rayon, zakaznik “Kava”, the valley of the river Kava near the creek Ikrimun (p. 88), 59.64011°N, 147.13524°E, in Larix cajanderi forest with Pinus pumila Regel and Betula middendorffii Trautv., 18.08.2017, coll. N. Sazanova, (MAG4805); United States of America, Idaho, E fork of Lake Fork Creek, Idaho National Forest, 44.9225°N, –115.9217°E, On humus under Pinus and Larix, 19.07.1941, coll. and det A. Smith (Smith 15665, MICH 62024).

Suillus paluster (Peck) Kuntze, Revis. gen. pl. (Leipzig) 3(3): 536, 1898. ≡ Boletus paluster Peck, Ann. Rep. Reg. N.Y. St. Mus. 23: 132. 1872. ≡ Boletinus paluster (Peck) Peck, Bull. N.Y. St. Mus. 2 (8): 78, 1889. ≡ Boletinellus paluster (Peck) Murrill, Mycologia 1 (1): 8, 1909. ≡ Fuscoboletinus paluster (Peck) Pomerl. et A.H. Sm., Mycologia 56 (5): 708, 1964. ≡ Suillus paluster (Peck) Kretzer et T.D. Bruns, Mycologia 88 (5): 784, 1996.

Iconography: Peck (1873: pl. 6, fig. 4–7 as Boletus paluster), Pomerleau (1964: 709, pl. 1 as F. paluster).