Hortiboletus bubalinus (Oolbekk. & Duin) L. Albert & Dima

Biketova, Alona Yu., Svetasheva, Tatyana Yu., Taylor, Andy F. S., Simonini, Giampaolo, Gelardi, Matteo, Morozova, Olga V., Polemis, Elias, Muñoz, José A., Albert, László, Saitta, Salvatore, Wasser, Solomon P., Nevo, Eviatar, Zervakis, Georgios I., Vizzini, Alfredo & Dima, Bálint, 2025, Morphological and molecular re-assessment of European and Levantine species of the genus Hortiboletus (Boletaceae), IMA Fungus 16, pp. e 144731-e 144731 : e144731-

Hortiboletus bubalinus (Oolbekk. & Duin) L. Albert & Dima , Index Fungorum 251: 1 (2015)

Figs 8 , 9 , 10

≡ Boletus bubalinus Oolbekk. & Duin , in Oolbekkink, Persoonia 14 (3): 367 (1991)

≡ Xerocomellus bubalinus (Oolbekk. & Duin) Mikšík , Index Fungorum 182: 1 (2014)

≡ Xerocomus bubalinus (Oolbekk. & Duin) Redeuilh , Docums Mycol. 23 (no. 89): 62 (1993)

? = Xerocomus erubescens Cadiñanos & J. A. Muñoz , Belarra 9: 61 (1992)

? – ‘ Boletus populinus ’ Oolbekk. & Duin (1988) , Coolia 31 (1): 11, nom. illeg., nom. prov., Art. 53.1 (Shenzhen), non B. populinus Schumach. , Enum. Pl. 2: 384 (1803), q. e. Oxyporus populinus (Schumach.) Donk

Holotype examined here.

NETHERLANDS • North Holland: Aerdenhout, A. W. dunes, near Ezelenvlak , on sandy soil, under Pinus and Populus , 14. 10. 1983, leg. G. Oolbekkink & W. van Duin, L 0053449 (G. Oolbekkink & W. van Duin 145 – collector’s number), GenBank: ITS – PV 094766 and PV 094767 .

Description.

Basidiomes pileate-stipitate, xerocomoid, epigeal, small to medium-small sized. Ontogenetic development gymnocarpic.

Pileus (2.9 –) 3.8–10.3 (– 13.5) cm broad, firstly hemispherical then persistently convex and finally broadly pulvinate-flattened, sometimes slightly depressed at centre, regularly to hardly unevenly shaped by shallow depressions, margin even to faintly wavy-lobed, initially involute then curved downwards or rarely completely plane, not or only a little extending beyond the tubes (up to 1 mm); surface matt, dry, but slightly greasy in aged basidiomes with moist weather, markedly wrinkled and finely pruinose in the early stage of development then tomentose with age, usually not cracked or occasionally areolate towards the peripheral zone; somewhat variable in colour, at first, dark garnet red or dark reddish-brown, but typically paler towards margin (pale orange to yellowish-orange), often later gradually fading to drab tinges and becoming orange ochraceous, sometimes with scattered pinkish hues, then evenly pale ochraceous brown, chamois olive to dull ochraceous buff, often retaining small and dispersed pale-reddish to ochraceous-orange zones (especially in the depressions) and with a thin, reddish-pink marginal band; aged basidiomes can completely lack reddish tones, although some basidiomes retain bright reddish tints with age; unchanging on handling or touching or when injured; subpellis layer pale pink to whitish buff.

Tubes up to 12 mm long, at first, thin in side view then increasingly wider with age and shorter than the thickness of the pileus context, easily separable from the context above, adnate, but soon depressed around the stipe apex and shortly decurrent with a tooth, bright yellow to olive yellow then olive brown, bluing on cutting.

Pores initially forming a concave or ascending surface, later flattening, at first very small then gradually wider with age (up to 2.0 mm in diameter), compound, labyrinthine becoming angular at maturity, concolourous with the tubes; strongly bluing on bruising and later becoming dirty brownish.

Stipe (3.1 –) 4.3–7.3 (– 8.3) × 1.1–2.7 (– 3.2) cm, as long as the pileus diameter at maturity, otherwise slightly to moderately shorter or longer, central to slightly off-centre, solid, firm, dry, straight or faintly curved, at first ventricose or clavate although tapering at the very base, later more or less cylindrical, sometimes remaining inflated downwards or progressively tapering from apex down to the base, not or slightly rooting; surface smooth or with fine punctuations from apex down to the middle part, longitudinally fibrillose with age, without reticulum; usually bright yellow or somewhat reddish at the very apex, flesh pink, reddish-pink to wine red elsewhere, rarely yellow to pale yellow throughout, usually progressively and sometimes completely losing reddish tones with age similarly to the pileus surface and eventually becoming dull buff; yellow ochraceous at the very base, sometimes with brownish-olive hues, occasionally with purplish patches; unchanging or faintly bluing on pressure and finally turning brownish; basal mycelium yellow.

Context firm and tough when young, later soft textured and eventually flabby in the pileus (up to 2.3 cm thick in the central zone, up to 1.5 mm thick halfway to margin and gradually becoming thinner towards the edge), fibrous in the stipe, pinkish-red in the pileus in very young to young basidiomes, but with a thin straw yellow to whitish layer above the tubes, becoming pale flesh pink at maturity and eventually fading to dull white in aged basidiomes or on drying; pale yellowish in the stipe, but progressively duller downwards, dirty yellow ochraceous at the very base and, as a rule, devoid of carrot-orange to orange-red dots, which can rarely be present; quickly bruising from light green-blue to blue on exposure, particularly in the pileus-stipe connection zone, above the tubes and in the peripheral layers of the stipe; after a few minutes, the bluing extends quite intensely to the lower part of the stipe (particularly in young basidiomes) and later becomes drab yellowish-olive, unchanging in the pinkish-red zone underneath the pileus surface; the oxidation phenomenon is less intense in old fruiting bodies and can be absent in very old basidiomes; context pinkish in the areas eaten by slugs and brownish where eroded by maggots; subhymenophoral layer yellowish-white; exsiccate brownish and reddish tones completely disappear during drying process.

Spore print olive brown.

Odour strong, pleasant, fruit-like but with an “ iron ” component. Taste mild.

Macrochemical spot-test reactions: Melzer’s reagent: a weak fleeting-amyloid reaction on the hymenophoral trama and on the stipe and pileus context of fresh and dry basidiomes; 20 % KOH: pale to bright orange on hymenophore and context, reddish-brown on pileus and stipe surface; 25 % NH 4 OH: none; 10 % FeSO 4: none; 10 % H 2 SO 4: none on context, orange on pileus and stipe surface and hymenophore.

Basidiospores [1020 / 35 / 35] (10.7 –) 11.7 ± 0.6 (– 13.3) × (4.3 –) 4.8 ± 0.2 (– 5.3) μm, Q = (2.26 –) 2.46 ± 0.10 (– 2.70), Vm = 140 ± 20 μm 3, sometimes abnormally large – up to 19.0 × 7.5 μm, inequilateral, fusiform to ellipsoid-fusiform in side view, narrowly or moderately ellipsoid to amygdaliform in face view, smooth, apex rounded, with a short apiculus, usually with a distinct suprahilar depression and with a slightly pronounced adaxial swelling, moderately to decidedly thick-walled (up to 1 μm), straw yellow to bright yellow-coloured in water and 5 % KOH, having one, two or three large oil droplets when mature, rarely pluri-guttulate, inamyloid and with an orthochromatic reaction.

Basidia [101 / 7 / 7] (27.4 –) 36.1 ± 4.7 (– 44.2) × (8.8 –) 11.4 ± 1.4 (– 13.0) μm, subclavate to clavate, moderately thick-walled (0.5–0.7 μm), predominantly 4 - spored, sterigmata (1.5 –) 3.2 ± 0.8 (– 4.5) μm, hyaline to pale yellowish and containing straw-yellow oil guttules in water and 5 % KOH; basidioles subcylindrical to faintly clavate, similar in size to basidia.

Cheilocystidia [20 / 3 / 3] (45.4 –) 56.7 ± 10.9 (– 72.3) × (6.9 –) 8.6 ± 1.8 (– 11.5) μm, relatively common, moderately slender, projecting straight to sometimes flexuous, subcylindrical-fusiform, fusiform to ventricose-fusiform, with rounded tip, smooth, moderately thick-walled (0.5–1.0 μm), hyaline to pale yellowish in water and 5 % KOH, without epiparietal encrustations. Pleurocystidia [38 / 3 / 3] (40.9 –) 56.6 ± 8.0 (– 77.9) × (7.9 –) 11.8 ± 2.3 (– 16.5) μm, infrequent, shape, size, colour and chemical reactions similar to cheilocystidia. Pseudocystidia not observed.

Hymenophoral trama bilaterally divergent intermediate between the “ Phylloporus - type ” and the “ Boletus - type ”, but quite closer to the former, with moderately divergent and tightly arranged, non-gelatinous hyphae (lateral strata hyphae in transversal section touching or almost touching each other, 0–4 (– 5) μm apart), hyaline to very pale yellowish in water and 5 % KOH, inamyloid in Melzer’s; mediostratum axially arranged, consisting of a tightly adpressed, non-gelatinous bundle of hyphae; in Congo red, the mediostratum is concolourous with or slightly darker than the lateral strata.

Pileipellis a physalo-palisadoderm consisting of erect subparallel to parallel chains of short, stout, septate hyphae; terminal elements [1023 / 29 / 28] (17.5 –) 28.7 ± 8.1 (– 58.5) × (7.1 –) 13.2 ± 3.1 (– 20.3) μm, Q = (1.63 –) 2.31 ± 0.66 (– 4.84), versiform, usually more or less swollen, mainly bullet-shaped or acorn-shaped, less frequently broadly cylindrical, ventricose to ovoid or cystidioid, but also globose or subglobose, sometimes mucronate, more rarely peanut-shaped, conical or utriform, occasionally lanceolate or filamentous and sinuous and then particularly long (up to 86 μm long), often bifurcate, apex rounded-obtuse or pointed, moderately thick-walled (up to 1 μm), hyaline to pale yellow in water and 5 % KOH; subterminal elements usually inflated, swollen to vesiculose and broader than terminal ones (up to 38 μm broad), often branched; pileipellis elements moderately to strongly ornamented by granular or zebra-like epiparietal encrustations, more evident and pronounced on subpellis hyphae, but also present on terminal cells surface, although less outlined, brownish in 5 % KOH.

Stipitipellis a layer of slender, parallel to subparallel and longitudinally running, smooth-walled, adpressed hyphae, 5–14 μm broad, hyaline to pale yellowish in water and 5 % KOH; the stipe apex is covered by a well-developed caulohymenial layer consisting of sterile caulobasidioles, very sparse, predominantly 2 - and 4 - spored, fertile caulobasidia and scattered projecting caulocystidia, similar in shape and size to the hymenial cystidia; elsewhere, the stipe surface exhibits isolated clusters of cystidioid to clavate, sterile elements, 4–9 μm broad. Lateral stipe stratum under the caulohymenium only rarely present and obscurely differentiated from the stipe trama, of the “ boletoid ” type; at the stipe apex, a 20–30 μm thick layer consisting of divergent, inclined and running towards the external surface, densely arranged hyphae.

Stipe trama composed of subparallel to confusedly and densely arranged, loosely interwoven, filamentous, smooth, inamyloid hyphae, 2–8 μm broad.

Clamp connections absent in all tissues.

Ecology and phenology.

solitary to gregarious or even subcaespitose, growing on litter, among grass or on bare soil, mainly in moist anthropogenic environments, such as public and private gardens, urban parks, or rural areas under a large array of ectomycorrhizal trees of the families Salicaceae , Malvaceae , Fagaceae , Betulaceae , Pinaceae , including preferably Populus spp. ( P. alba , P. nigra , P. tremula , Populus hybrids), Tilia spp. ( T. cordata , T. × europaea , T. platyphyllos ), Quercus spp. ( Q. cerris , Q. ilex , Q. petraea , Q. robur ), and Fagus sylvatica , but sometimes also Picea spp. ( P. abies , P. × fennica , P. pungens ), Betula spp. ( B. pendula ), Carpinus betulus , Corylus avellana , Castanea sativa , Carya ovata , and Pinus radiata ; some collections of H. bubalinus have been found in mixed forest with the presence of Pinus spp. ( P. nigra , P. pinea , P. sylvestris ), Cedrus ( C. atlantica , C. deodara ), Larix decidua , Salix alba , and Alnus glutinosa , but there are no finds in monodominant forest communities formed by these tree species alone ( Oolbekkink (1991); Ladurner and Simonini (2003); Albert (2004); Muñoz et al. (2008); Musumeci (2008); Assyov and Stoykov (2011); Wilgan and Leski (2022), GP; Kasparavičius and Iršėnaitė (2023); Assyov in litt.; Cooper and Park in litt., GP; Klofac in litt.; Saar et al. unpubl., GP; pers. obs., GP). The main fruiting period is from spring (late April) to late autumn and early winter in Mediterranean area (November to early December).

Known distribution (see Fig. 4 b).

This is a species with a natural circumboreal distribution, widespread in Europe and Western Asia, also found in the USA and introduced in New Zealand (based on sequences data from INSDC, see phylogenetic analysis), and much more frequent than previously assumed (Fig. 4 b ). Reported to date from Asia: Turkey ( Allı et al. 2019); Australia and Oceania: New Zealand ( GBIF 2024; Cooper and Park in litt., GP); Europe: Austria ( Montag 2009; Šutara et al. 2009; Klofac in litt.; this study, GP), Belgium ( Vadthanarat et al. (2019), as Xerocomellus porosporus, GP ), Bulgaria ( Assyov and Stoykov 2011), Czechia ( Šuhaj and Mikšík 2016; Mikšík 2017), Estonia (Saar unpubl., GP – UDB 024777), Finland (this study, GP), France ( Musumeci 2008; Péan and Mornand 2009; this study, GP), Germany ( Kaldorf et al. (2004), as Uncultured Boletaceae, GP ; Klofac (2007); this study, GP), Greece (this study, GP); Hungary ( Albert 2004; Kaposvári 2013; this study, GP), Italy ( Gelardi 2009, 2010; this study, GP), Kosovo * ( Ramshaj et al. 2021), Lithuania ( Kasparavičius and Iršėnaitė 2023), Netherlands ( Oolbekkink and van Duin 1988; Oolbekkink 1991; Ladurner and Simonini 2003; Noordeloos 2007; Noordeloos et al. 2018; this study, GP), Norway ( Knudsen and Taylor 2008, 2012; this study, GP), Poland ( Wilgan and Leski (2022), as Xerocomellus chrysenteron , GP – ON 129125), Portugal (Azores Islands, probably introduced) ( Iglesias et al. 2021; Souto et al. 2024), Russia ( Kapitonov (2013); Svetasheva et al. (2018); Bolshakov et al. (2021); Pham et al. (2024), GP; this study, GP), Serbia ( Uzelac 2009; this study, GP), Slovakia (Mikšík in litt.), Spain ( Muñoz et al. 2008; this study, GP), Sweden ( Taylor and Eberhardt 2006; Knudsen and Taylor 2008, 2012; Jeppson and Knuttson 2017; this study, GP), Switzerland (this study), United Kingdom ( Hills 2008; Burnham and Kibby 2011; Ainsworth et al. 2013; Kibby 2017; this study, GP); North America: USA ( Nuhn et al. (2013), as Xerocomellus cf. rubellus, GP ; Frank et al. (2020), as Hortiboletus sp. , GP).

Additional examined material.

AUSTRIA • Tyrol: Innsbruck, Unikreuzung , under Betula and Corylus , 31. 07. 1997, leg. H. Ladurner, det. A. Biketova, IB 1997886 ( GP) ; FINLAND • Nylandia: Helsinki, Elaeintarha , 60°11'17"N, 24°56'13"E, on lawn near P. tremula , 26. 08. 2013, leg. P. Hoeijer, det. A. Biketova & P. Hoeijer, H 6045679 ( GP) ; • Loviisa, Pernaja churchyard , 60°26'20"N, 26°02'46"E, under Q. robur , 09. 09. 2011, leg. P. Hoeijer, det. A. Biketova & P. Hoeijer, H 6032689 ( GP) ; • Porvoo, Kirkkotori, near the church , 60°23'49"N, 25°39'36"E, in lawn under T. × europaea , 16. 08. 2009, leg. P. Hoeijer, det. A. Biketova & T. Niskanen, H 6036888 ( GP) ; FRANCE • Île-de-France: Essonne, Massy , 48°39'22"N, 2°29'34"E, 80 m, fourteen young to mature basidiomes in the grass close to Quercus sp. and other broadleaved tress, 03. 06. 1994, leg. G. Redeuilh, det. G. Simonini, GS 1332 ; • Grand Est: Haut Rhin, Rixheim , 47°44'40"N, 7°23'49"E, 253 m, twelve young to mature basidiomes in a grassy ground close to Betula tree, 07. 10. 2005, leg. E. Musumeci, det. G. Simonini, GS 10414 ( GP) ; GERMANY • Hesse: Giessen, Giessen Botanical Garden, next to the pond , mixed woodland, 10. 06. 1998, leg. & det. A. Taylor, K-M 000168095 (AT 1998006; GP) and AT 1998007 ; GREECE • Central Macedonia: Kilkis, Mt. Belles , 41°18'36"N, 23°3'1"E, under F. sylvatica , 10. 09. 2009, leg. G. Konstantinidis, det. G. Konstantinidis, E. Polemis, B. Dima & A. Biketova, GK 3993 ( GP) ; • ibid. (5 km away from GK 3993 location), under F. sylvatica , 10. 09. 2009, leg. G. Konstantinidis, det. G. Simonini, GK 3982 (GS 10502) ; HUNGARY • Bács-Kiskun: Lakitelek-Tőserdő , in park under T. cordata , 02. 10. 2022, leg. & det. L. Albert, AL 22-82 ( GP) ; • Tompa, in the garden next to St. Anne’s Church , 46°10'58"N, 19°32'05"E, under Quercus sp. and Acer sp. , two young basidiomes, 22. 06. 2018, leg. & det. A. Biketova K-M 001435679 (AB B 18-287) ; • Budapest: Soroksár, Botancial Garden , under P. alba , 18. 10. 2022, leg. L. Albert & B. Nagy, det. L. Albert, B. Nagy L. & B. Dima, AL 22-91 ( GP) ; • Csongrád-Csanád: Szeged, in front of the Ferenc Móra College , in road border, under Tilia sp. , 08. 06. 2013, DB 5078 ; • Szeged, near Fesu str. 11 , 46°14'35.3"N, 20°09'34.2"E, three mature basidiomes, under Q. robur and T. cordata , 06. 06. 2018, leg. & det. A. Biketova, K-M 001435684 (AB B 18-286) ; • Szeged, next to the Institute of Biology of the University of Szeged , 46°14'45.7"N, 20°10'03.6"E, under T. cordata , a single mature basidiome, 06. 06. 2018, leg. & det. B. Dima, K-M 001435688 (AB B 18-285) ; • Nógrád: Börzsöny Mts, near Diósjenő , 47°57'05.5"N, 18°59'21.0"E, under C. betulus , one young and one mature basidiome, leg. & det. L. Albert & A. Biketova, K-M 001435711 (AB B 18-399, AL 18-73) ; • ibid., 47°57'37.7"N, 19°00'31.6"E, in Q. petrea , Q. cerris , and C. betulus forest, two mature basidiomes, 24. 06. 2018, leg. & det. A. Biketova & B. Dima, K-M 001435709 (AB B 18-398) ; • ibid., under C. betulus and S. alba , 11. 09. 2022, leg. L. Albert, det. L. Albert & B. Dima, AL 22-75 ( GP) ; • Pest: Üllő , under Populus hybrids, 03. 08. 2012, leg. L. Albert, det. L. Albert & B. Dima, AL 12-28 ( GP) ; • ibid., under Populus hybrids, 25. 07. 2012, leg. L. Albert, det. L. Albert & B. Dima, AL 12-288 ( GP) ; • ibid., 29. 05. 2014, leg. L. Albert, det. L. Albert & B. Dima, AL 14-19 ; • ibid., 04. 09. 2015, leg. L. Albert, det. L. Albert & B. Dima, AL 15-54 ( GP) ; ITALY • Emilia-Romagna: Reggio nell’Emilia, parco del Popolo , 44°42'14"N, 10°37'49"E, 53 m, two mature basidiomes in the grass, close to T. platyphyllos and P. alba trees, 17. 05. 1999, leg. & det. G. Simonini, MCVE 18373 (GS 2077) ; • ibid., 44°42'12"N, 10°37'49"E, 53 m, a single young basidiome in the grass, close to P. alba trees, 05. 06. 2016, leg. & det. G. Simonini, GS 10694 ; • Friuli-Venezia Giulia: Agordo, Belluno, Col di Foglia , 46°16'28"N, 12°02'39"E, 739 m, edge of a mixed forest, 30. 07. 2016, leg. E. Bizio, det. G. Simonini, GS 10842 ( GP) ; • Lazio: Rome, Via Appia Antica , 41°50'14"N, 12°32'20"E, 56 m, four very young to young basidiomes collected on loamy, very moist grassy soil under T. platyphyllos , Populus nigra var. italica , and Q. ilex , with the presence of C. deodara , Eriobotrya japonica , Cupressus sempervirens , and Fraxinus ornus in the garden of a private property, 23. 10. 2008, leg. & det. M. Gelardi, MG 147 ; • ibid., a single mature basidiome (this basidiome belonged to the previous collection, but was left on the ground and collected five days after), 28. 10. 2008, leg. & det. M. Gelardi, MG 147 - bis ; • ibid., three mature and one aged basidiomes, 03. 11. 2008, leg. M. Gelardi, det. M. Gelardi & A. Biketova, MCVE 25582 (MG 163, GP) ; • ibid., five basidiomes in various developmental stages, 08. 10. 2009, leg. and det. M. Gelardi, MG 257 ; • ibid., five middle-age basidiomes (three out of which growing connate at the very stipe base), 10. 11. 2009, leg. & det. M. Gelardi, MG 284 ; • ibid., a single mature basidiome, 29. 04. 2010, leg. & det. M. Gelardi, MG 300 ; • ibid., several very young to mature basidiomes, 21. 09. 2010, leg. & det. M. Gelardi, MG 341 ; • ibid., several very young to mature basidiomes, 17. 11. 2011, leg. & det. M. Gelardi, MG 451 ; • ibid., several very young to mature basidiomes, 15. 10. 2013, leg. & det. M. Gelardi, MG 567 ; • Rome, Consorzio Olgiata, Isola 32 B 3 , 42°01'29"N, 12°21'40"E, 133 m, several basidiomes in all developmental stages collected in loamy soil, on needle litter and among the grass under C. atlantica with the presence of T. platyphyllos and P. pinea in the garden of a private property, 11. 10. 2022, leg. & det. M. Gelardi, MG 950 ; • Liguria: La Spezia, Deiva Marina , 44°13'16"N, 09°31'22"E, 12 m, artificially irrigated urban flowerbed, five young to mature basidiomes in the grass, soil pH = 7.5, close to T. cordata , 17. 06. 1994, leg. & det. G. Simonini, GS 1138 ; • ibid., artificially irrigated urban flowerbed, 10 young to mature basidiomes in the grass, soil pH = 6.9. close to T. cordata , 18. 06. 1994, leg. & det. G. Simonini, MCVE 17711 (GS 1142) ; • ibid., artificially irrigated urban flowerbed, 12 young to mature basidiomes in the grass, soil pH = 6.9, close to T. cordata , leg. & det. G. Simonini, 20. 06. 1994, MCVE 17712 (GS 1143) ; • Lombardia: Brescia, Valsaviore, Cevo , 46°04'49"N, 10°22'47"E, 1190 m, on very dry, acid soil on a slight slope facing south, eleven very young to mature basidiomes collected in the grass and on decomposed litter of needles and dry leaves, in a sub-alpine mixed broadleaved / conifer thicket dominated by C. avellana and P. abies with the presence of Juniperus sp. , ferns and nettles near a grassy pasture, with trees of S. alba , Fraxinus sp. , and Populus nigra standing in the middle of the pasture, all within 30 m from the place of collection, 16. 08. 2009, leg. M. Gelardi & S. Gelardi, det. M. Gelardi, MCVE 25592 (MG 226) ; • ibid., a single mature basidiome, 20. 08. 2009, leg. & det. M. Gelardi, MG 330 ; • ibid., 1230 m, a few hundred metres away from the previous collections, five middle-aged to mature basidiomes growing under a single young P. abies tree in the centre of a grassy clearing surrounded by a P. abies forest with the presence of L. decidua (without presence of broadleaved trees), 18. 08. 2009, leg. & det. M. Gelardi, MG 231 ; • Sicily: Messina, Monti Peloritani, Mt. Dinnammare, Sentiero Ziriò , 38°10'36.7"N, 15°28'12.1"E, 800 m, five young to mature basidiomes growing with Cedrus sp. , Q. ilex , Pinus spp. , Pseudotsuga menziesii , and Juglans regia , 25. 09. 2019, leg. & det. S. Saitta, TUR-A 209256 ( GP) ; • ibid., several basidiomes, 24. 09. 2020, leg. & det. S. Saitta, TUR-A 209574 ( GP) and TUR-A 209575 ( GP) ; • Trentino-South Tyrol: Trentino, Lake Pudro, Pergine Valsugana , 46°04'42"N, 11°13'16"E, 510 m, several young to mature basidiomes growing along a trackside in a riparian environment under A. glutinosa , Populus nigra and P. tremula , 26. 08. 2010, leg. M. Floriani & M. Gelardi, det. M. Gelardi, MG 338 ; • Veneto: Sacca Sessola lagoon island, Venezia , 45°24'17"N, 12°19'23"E, 1 m, 14 basidiomes in the grass, close to Cedrus , other conifers and Populus , 13. 10. 1990, leg. E. Bizio, det. G. Simonini, GS 1125 ; NETHERLANDS • South Holland: Leiden, Johan Wagenaarlaan , several partial fairy rings on lawn on sandy clay soil under Tilia , no other trees nearby, 05. 09. 1988, leg. & det. C. Bas, L 0069896 (Bas C. 8633; GP) ; NORWAY • Oslo Co.: Oslo, Bygdoy, Ingstadasen , 59°54'29.8"N, 10°41'15.1"E, under Tilia and Corylus , 31. 08. 2017, leg. E. Bendiksen, det. B. Dima, O-F- 254512 ( GP) ; • Oslo, Tøyen, Botanical Garden , 59°54'59.8"N, 10°46'17.4"E, under Tilia , 06. 07. 2004, leg. A. Molia & E. W. Hanssen, det. A. Taylor, O-F- 270491 ( GP) ; • ibid., 59°55'16"N, 10°46'55"E, 05. 09. 2001, leg. G. Gulden, det. B. Dima, O-F- 73245 ( GP) ; • ibid., under Tilia , 07. 09. 2004, leg. G. Gulden, det. A. Taylor, O-F- 67109 ( GP) ; • ibid., 59°54'58"N, 10°46'08"E, Picea abies dominated forest, 01. 08. 2014, leg. A. K. Wollan, det. A. K. Wollan, A. Taylor & B. Dima O-F- 248341 ( GP) ; • Østfold Co.: Moss, Melløsparken , 59°25'26"N, 10°40'05"E, under Fagus and Betula in park, 01. 09. 2019, leg. E. W. Hanssen & R. Braathen, det. B. Dima, O-F- 307797 ( GP) ; • Telemark Co.: Kragerø, Jomfruland , 58°52'52"N, 9°36'40"E, under Corylus , Quercus , Pinus , and Salix , 01. 10. 2022, leg. B. Rian & M. S. Olsen, det. B. Dima, O-F- 204266 ( GP) ; RUSSIA • Pskov Oblast: Pushkinogorsky District, A. S. Pushkin Museum-Reserve “ Mikhaylovskoe ” , 57°03'08"N, 28°55'04"E, on a grassy edge of mixed coniferous-deciduous forest, 11. 09. 2018, leg. O. Morozova, det. O. Morozova & T. Svetasheva, LE F- 315962 ( GP) ; • Tsevlo Village, remains of an abandoned park , on soil under old-growth T. cordata , 07. 09. 2019, leg. & det. L. B. Kalinina, LE F- 330285 ( GP) ; • Saint-Petersburg: Summer Garden, central part , 59°56'42"N, 30°20'06"E, on a lawn under T. cordata , 21. 07. 2017, leg. T. Svetasheva, det. A. Biketova & T. Svetasheva, LE F- 332077 ( GP) ; • ibid., in an open lawn with a single T. cordata , 21. 07. 2017, leg. T. Svetasheva, det. A. Biketova & T. Svetasheva, LE F- 332078 ( GP) ; • ibid., on a lawn under T. cordata and Acer platanoides , 14. 07. 2020, leg. O. Morozova, det. O. Morozova & T. Svetasheva, LE F- 315839 ( GP) ; • ibid., in a grassy site under Q. robur , T. cordata and Sorbus aucuparia , 20. 07. 2017, leg. and det. T. Svetasheva, LE F- 332081 ; • Summer Garden, northern part “ Cross Arcade ” bosquet , 59°56'47.6"N, 30°20'01.2"E, on a lawn under P. abies (the nearest T. cordata tree being 10 m away), 21. 07. 2017, leg. T. Svetasheva & O. Morozova, det. A. Biketova & T. Svetasheva, LE F- 332079 ( GP) ; • Summer Garden, between the central part and the “ Cross Arcade ” bosquet , 59°56'43"N, 30°20'07"E, under T. cordata and P. abies planting, among mosses on soil, 14. 07. 2020, leg. and det. T. Svetasheva & O. Morozova, LE F- 315852 ( GP) ; • ibid., on a lawn ca. 10–15 m from P. abies , 21. 07. 2017, leg. T. Svetasheva & O. Morozova, det. A. Biketova & T. Svetasheva, LE F- 332080 ( GP) ; • ibid., under P. abies and T. cordata , 06. 08. 2020, leg. O. Morozova, det. O. Morozova & T. Svetasheva, LE F- 315838 ( GP) ; • Summer Garden, site 5 , 59°56'48"N, 30°20'10"E, under T. cordata , 10. 09. 2015, leg. E. Shalakitskaya, det. T. Svetasheva, LE F- 315840 ; • Peter the Great Botanical Garden, near the building “ Kizhi ” , 59°58'9.4"N, 30°19'20.7"E, along a path, under T. cordata and C. avellana , 22. 07. 2011, leg. & det. O. Morozova & T. Svetasheva, LE F- 254151 ( GP) ; • Peter the Great Botanical Garden, near the Main Herbarium building , 59°58'9.7"N, 30°19'14.5"E, on a lawn under T. cordata , 21. 07. 2009, leg. & det. O. Morozova & T. Svetasheva, LE F- 254144 ( GP) ; • ibid., under T. cordata , 21. 09. 2010, leg. O. Morozova, det. T. Svetasheva, LE F- 332075 ( GP) ; • Tatarstan Republic: Elabuzhsky District, “ Bolshoy Bor ”, site 26 , 55°45'34"N, 52°16'57"E, mixed coniferous-deciduos forest, 29. 08. 2013, leg K. Potapov, det. T. Svetasheva, LE F- 315842 ; • Kazan, Gorky Central Park , under Tilia and Betula planting, 11. 08. 2015, leg. K. Potapov, det. T. Svetasheva, LE F- 315844 ; • Zelenodolsky District, Volzhsko-Kamsky Nature Reserve, near “ Dolgoye ” bog , 55°54'54.0"N, 48°46'37.2"E, a single basidiome on the soil in deciduous forest ( Quercus , Tilia , Betula ), 24. 08. 2016, leg. M. Djakov, det. T. Svetasheva & G. Simonini, LE F- 311914 (GS 10807; GP) ; • Tula Oblast, Tula, Platonovsky Park , 54°9'6"N, 37°34'36"E, broadleaved forest, under Q. robur and T. cordata , 27. 07. 2016, leg. & det. T. Svetasheva, LE F- 343931 ; • Udmurt Republic: Votkinskji District, 2 km SW of Novyi , 56°48'13"N, 54°00'45"E, 95 m, on sandy ground on the roadside, in P. tremula forest with T. cordata and U. glabra , 13. 07. 2014, leg. V. I. Kapitonov, det. G. Simonini, GS 10506 ( GP) ; • Izhevsk City , 56°52'32"N, 53°13'26"E, 159 m, two mature basidiomes on needle litter, in a city park close to P. × fennica forest with T. cordata , Acer negundo and Populus sp. , 21. 09. 2013, leg. V. I. Kapitonov, det. G. Simonini, GS 10514 ( GP) ; SERBIA • Central Serbia: Mačva District, Banja Koviljača , 44°30'40"N, 19°9'33"E, 167 m, two young to middle-aged basidiomes growing on the ground among grass and moss along a pathway, under T. cordata , in a mixed broadleaf forest dominated by C. betulus , F. sylvatica , and Q. cerris , 08. 08. 2020, leg. A. Biketova & B. Dobrić, det. A. Biketova & B. Dima, K-M 001435682 (AB B 20-368; GP) ; SPAIN • Cantabria: Valle de Cabuérniga, Los Tojos , 43°09'34"N, 4°14'56"W, 399 m, three specimens growing on acidic soil, in a short slope close to C. avellana and Populus nigra , 06. 09. 2005, leg. J. Muñoz, det. J. Muñoz & A. Taylor, JAM 0593 ( GP) ; SWEDEN • Södermanland Co.: Stjärnhov, Solbacka Golf Course , under a single Betula sp. , 14. 07. 2002, leg. & det. A. Taylor, AT 2002036 ( GP) ; SWITZERLAND • Zurich: Schwamendingen , 47°23'48"N, 8°34'14"E, 550 m, in the grass in an urban wood, close to P. abies and F. sylvatica , 20. 08. 1985, leg. C. Lavorato, det. G. Simonini, GS 1541 ; UNITED KINGDOM • England: Berkshire, Silwood Park near Ascot, SU 946686 , under Carpinus , Tilia , and Cornus , 27. 09. 2006, leg. A. Hills, det. A. Taylor, K-M 000172000 (AH 2006071; GP) ; • Greater London, Richmond, RBG Kew, along the way from Elizabeth Gate to Jodrell Lab , 51°29'01.3"N, 0°17'28.5"W, two mature basidiomes, among grass on the open lawn, in vicinity of Magnolia spp. , Q. ilex , and Pinus nigra var. maritima , 19. 10. 2021, leg. A. Biketova, det. A. Biketova & G. Kibby, K-M 001435675 (AB B 21-381; GP) .

Notes.

This bolete does not pose any problem from both the nomenclatural and taxonomic viewpoint. Originally described as a member of Boletus , it was then recombined in Xerocomus ( Redeuilh 1993) , in Xerocomellus ( Mikšík 2014) , and, finally, in Hortiboletus ( Dima 2015) .

This species was introduced by G. T. Oolbekkink and W. W. van Duin in a publication by Oolbekkink (1991) dealing with the ornamentation of Xerocomus basidiospores observed at SEM. The colour picture of X. bubalinus in Ladurner and Simonini (2003) refers to a topotypical collection; however, the picture was published with overexposed red tones, whereas the original image is much more brownish. A collection with features consistent with the original concept by Oolbekkink and Duin was described in detail from France (Alsace), in association with Betula on sandy soil ( Musumeci 2008).

Based on the extended original description ( Oolbekkink 1991) and our data, the basidiospore size of H. bubalinus is unusual for Hortiboletus , with a very high Q value = 2.46 ± 0.10 in average (up to 2.7) when compared with those of H. rubellus – 2.31 ± 0.12. Robinson et al. (2011) indicated frequent bifurcating or even trifurcating terminal elements of pileipellis. This feature was also highlighted in the line drawings of the pileipellis in Gelardi (2010). In addition, pileipellis subterminal elements are often wider than terminal ones, with typically inflated, branched and sometimes broadly rounded cells up to 30 μm broad ( Gelardi 2009, 2010).

Based on the original diagnosis of X. erubescens Cadiñanos and Muñoz (1992) , it shares with H. bubalinus some morphological features: the pinkish pileal context, the pileipellis structure, lack of reddish tints on both pileus (which is sometimes present in H. bubalinus ) and stipe surfaces, and lack of orange-red dots in the stipe base. However, spores of X. erubescens are significantly larger [15.4–19.0 (– 20.3) × 5.7–7.2 (– 7.7) μm] according to the original diagnosis ( Cadiñanos and Muñoz 1992), but they were re-analysed (isotype collection IB 19920820 (BAR 1656 / 92) and IB 19980600) by Ladurner and Simonini (2003), who obtained significantly smaller sizes [(11.0 –) 13.4 ± 1.04 (– 15.5) × (4.4) 5.0 ± 0.31 (– 5.9) μm], when compared with spore sizes of some H. bubalinus collections (e. g. holotype L 0053449). Moreover, abnormally large spores (up to 19.0 × 7.5 μm) occur in some H. bubalinus collections, including its holotype (L 0053449).

In our multilocus analysis (Fig. 1 ), H. bubalinus forms a highly-supported terminal clade (BS = 94, PP = 1.00). In both ML and BI trees, it clusters in a highly-supported subgeneric clade (BS = 99, P P = 1.00), together with H. arduinus and three to four undescribed species: one from North America ( USA) and two to three from Asia ( China, Japan, and South Korea). However, there are minor differences in topology of this subgeneric clade in multilocus ML and BI trees. In multilocus ML phylogeny, H. bubalinus clusters as a sister clade to Hortiboletus sp. 7 (represented by LSU, tef 1 - α, and rpb 2 sequences of a single collection HKAS 51292) with weak statistical support (BS = 61). In multilocus BI phylogeny, H. bubalinus clusters together with a common branch of Hortiboletus sp. 4 , Hortiboletus sp. 5 , and Hortiboletus sp. 7 (all represented by only ITS sequences) with weak support ( PP = 0.89).

Based on multilocus and four single-locus sequence data, two other European Hortiboletus species are significantly distant from H. bubalinus (Figs 1 , 2 ). The closest species are H. engelii and H. hershenzoniae with a consistent, ca. 13 % dissimilarity (> 100 nucleotide and indel differences). The ITS of H. bubalinus has been difficult to sequence with the classical Sanger method due to frequent polymorphisms, particularly in short insertion of AC repeats in ITS 1 region, which vary in length within the species from 14 to 26 bp and even within the same genome with maximum observed difference of 12 bp (in JAM 0593). Therefore, in some cases, cloning has been performed to obtain full ITS sequences (e. g. the holotype). Furthermore, H. bubalinus has one of the longest tandem repeats in the ITS 2 region among other Hortiboletus species (see Table 1 ). Based on different alignment parameters of TRF analysis, the length of this long tandem repeat is estimated to be from 225–228 bp (2-7 - 7) to 231–253 bp (2-3 - 5). All species in H. arduinus / H. bubalinus subgeneric clade are characterised by such long minisatellite-like insertion in the ITS 2 region.

Figures

Figure 1. ML phylogenetic tree of Hortiboletus generated from a multilocus (ITS + LSU + tef 1 - a + rpb 2) dataset. BS values ≥ 50 % and PP values ≥ 0.8 indicated at nodes. Thickened branches indicate high statistical support (BS ≥ 70 % and PP ≥ 0.95). Species names of collections follow current identification, except original names of types of Boletus harrisonii and B. rubellus var. flammeus. Hortiboletus species present in Europe and Levant are indicated by light red-coloured fields. Collections with newly-generated sequences are indicated in bold. Two-letter country codes (ISO 3166-1 alpha- 2) denote the origin of specimens. At the end of the annotations to some collections, abbreviations are indicated in brackets: e – epitype, h – holotype, p – paratype, env – environmental sample.

Figure 2. Phylogenetic trees of Hortiboletus from separate ML analyses of each locus: ITS, LSU, tef 1 - a, and rpb 2. BS values ≥ 50 % and PP values ≥ 0.8 are indicated at nodes. Thickened branches indicate high statistical support (BS ≥ 70 % and PP ≥ 0.95). Pie charts underneath each label are the percent of total characters in the alignment that are parsimony-informative.

Figure 4. Distribution map of a H. rubellus and b H. bubalinus, based on the current study and sequence data from public repositories and associated publications.

Figure 8. Size distributions of a spores and b terminal cells of pileipellis in Hortiboletus using the “ isoprobability ellipse ” method. The distributions of average spore sizes in collections are shown with one standard deviation.

Figure 9. Hortiboletus bubalinus basidiomes: a holotype collection L 0053449; b AL 12-28; c AL 22-75; d MCVE 25582; e TUR-A 209256; f LE F- 315962; g GS 10842; h LE F- 332081; i LE F- 332077. Photos: (a) B. Dima; (b-c) L. Albert; (d) M. Gelardi; (e) S. Saitta; (f) O. V. Morozova; (g) E. Bizio; (h) T. Yu. Svetasheva; (i) E. S. Popov.

Figure 10. Microscopical features of H. bubalinus: a basidiospores of collection MCVE 18373 in L 4; b basidia in Congo red (GS 10414); c pileipellis in Congo red; d pleurocystidia in Congo red, left collection GS 10072, others – GS 10414; e tuft of cheilocystidia in Congo red (GS 10414). Scale bar: 10 μm. Photos: G. Simonini.