Costatisporus caerulescens T.W. Henkel & M.E. Sm., in Smith et al., IMA Fungus 6(2): 312 (2015)
Etymology: Cyanescens (L. adj. A) = becoming dark blue; referring to the dark blue auto-oxidation reaction of the bruised peridium.
Diagnosis: Easily differentiated from other sequestrate taxa by the off-white to greyish yellow peridium that stains dark blue, brown acolumellate gleba, strong chocolate nutty odour, and large basidiospores with costate ornamentation.
Basidiomata subglobose to ovate and slightly lobed, 12–26 mm tall, 12–33 mm broad; surface initially off-white to light to greyish yellow (4A3–4A4, 4B3) where unstained by soil, developing increasingly deep blue (23C8–23D8–23E8) stains slowly and progressively over 5–10 minutes where squeezed or bruised, firm, softer in areas, with small, possibly invertebrate mycophagist excavations, glabrous macroscopically, under a hand lens matted tomentose, with age gelatinizing in areas to dark brown and viscid; base subtended by one to several tan hyphal cords. Peridium in longitudinal section thin, 0.3–0.7 mm, appearing single-layered macroscopically, white initially, bluing slowly but intensely on exposure in younger specimens, separable. Gleba densely loculate with brown-hispid interior surfaces under hand lens, initially brown (7E8) to reddish brown (7F6–7F7–7F8, 8F8) throughout, with age gelatinizing and darker brown (9F4–9F5–9F6), with advanced age violet brown (10F5) over outer 1/6 with locule structure breaking down and blue stains evident on glebal trama under hand lens, acolumellate, with a greyish, gelatinous sterile basal pad 1 × 5 mm. Odour strong, variously described as chocolate-nutty, musty, soapy, putty-like, or of mushroom buillon; taste indistinctive, fungoid. Macrochemical reactions: KOH reddish brown on peridium and gleba; NH4OH negative on all surfaces.
Peridium 150–615 μm thick, two-layered; outer layer 50–415 μm thick, dark yellow to brownish, of tightly interwoven hyphae; individual hyphae with yellow cytoplasmic pigment in H2O and KOH, occasionally hyaline; intercalary cells 20–92.5 × 5–10 μm, thin-walled, frequently with spiraled to ring-like extracellular encrustations; inner layer 50–250 μm thick, hyaline, of loosely interwoven to parallel hyphae, these 3–8 μm wide, thin-walled. Glebal trama composed of mediostratum and lateral stratum; mediostratum hyaline in H2O and KOH, 25–45 μm wide, often splitting at locule junctions, of parallel to loosely interwoven hyphae, these hyaline in KOH and H2O or infrequently with pale yellow cytoplasmic pigments, 2–8 μm wide, thin-walled; lateral stratum moderately to strongly diverging, 10–49 μm thick; individual hyphae hyaline in H2O and KOH, 4–7 μm wide, thin-walled, grading into interwoven subhymenium hyphae, these hyaline to pale yellow, 5–9 μm wide, thin-walled. Hymenium a palisade of basidia and basidioles. Basidia subclavate to clavate, hyaline to faintly grey in H2O and KOH, 39.0–66.4 μm long, 7.5–12.2 μm broad at apex, 3–9 μm at the centre, 2.5–5.8 μm at the base, thin-walled; sterigmata three or four, 4.5–5.5 × 1–2 μm. Basidiospores statismosporic, yellowish to light brown in H2O and KOH, inamyloid, with complex costate ornamentation of 5–10 longitudinal, somewhat spiraled main ridges running pole to pole; ridges entire or discontinuous and occasionally bifurcating, with numerous narrow, shallow, nearly perpendicular cross-ridges, subglobose to oblong with ornamentation included, 17–25 × (10–)13–20 μm (mean = 19.9 ± 1.41 × 15.9 ± 1.96 μm; Qr = 1.00–1.80(–2.30), Qm = 1.27 ± 0.20; n = 118), ovate to subfusiform and basally acuminate with ornamentation excluded, 14–21 × 8–11.5 μm (mean = 16.8 ± 1.32 × 10.0 ± 0.65 μm; Qr = 1.36–2.21, Qm = 1.69 ± 0.17; n = 100); pedicel infrequent, 0.5–4 × 1–2 μm. Cystidia and clamp connections absent.
Habit, habitat and distribution: Solitary to scattered and hypogeous to partially emergent on mineral soil/humic layer interface on the forest floor, under D. corymbosa or A. insignis; known only from the type locality and a second site ∼8 km distant in the Upper Potaro River Basin of Guyana.
Index Fungorum number: IF 812364
Notes: Costatisporus cyanescens is recognized in the field by the blue-bruising peridium with occasional mycophagist excavations, and dark brown to violet-brown, finely loculate, acolumellate gleba that gelatinizes with maturity. Micromorphologically, the basidiospore ornamentation of longitudinal main and lateral secondary ridges is distinctive. The basidiospore ornamentation is remarkably similar to that in species of the epigeous bolete genus Boletellus, which has no known sequestrate members (Singer 1986, Mayor et al. 2008, Halling et al. 2015). The similar basidiospore ornamentations of Costatisporus and Boletellus are apparently coincidental, as C. cyanescens has no close phylogenetic relationship with Boletellus, which occurs in the Xerocomoideae clade (Figure 1).
The basidiospores of South-East Asian sequestrate Rhodactina (Boletaceae) species are longitudinally ridged but lack the intervening secondary ridges observed in C. cyanescens, are purple in water mounts, and dextrinoid (Pegler et al. 1989, Yang et al. 2006). Although no 28S, RPB1, or RPB2 sequences were available for the genus Rhodactina, comparison of the ITS1 sequence from Rhodactina incarnata with that of C. cyanescens did not indicate a close relationship. The tropical Asian genus Durianella has sequestrate basidiomata that undergo a deep blue colour change upon exposure, but also have a well-developed columella, fibrillose exoperidial warts, and echinulate basidiospores (Desjardin et al. 2008).
Species of the sequestrate genera Rossbeevera (East Asia, Australasia) and Chamonixia (mostly North Temperate) feature longitudinal ridging of the basidiospores and often undergo a blue, green, or blackish colour change upon bruising or exposure (Smith & Singer 1959, Lebel et al. 2012, Orihara et al. 2012b). Basidiospore ornamentation in Rossbeevera lacks intervening subridges and has 4–5 short, broad longitudinal ridges which contribute to their slight to stellate polar angularity (Lebel et al. 2012, Orihara et al. 2012b), contrasting with the 5–10 spiraled, acute ridges of C. cyanescens that impart a consistently stellate polar shape. Furthermore, Rossbeevera is phylogenetically distant from C. cyanescens (Figure 1). The ridged basidiospore ornamentation of Chamonixia species superficially resembles that of C. cyanescens, but their longitudinal ridges are straight with rounded margins and lack intervening subridges (Smith & Singer 1959).
Although the longitudinally ridged basidiospore ornamentation of C. cyanescens bears some resemblance to that seen in Gautieria (Gomphales) and Austrogautieria (Hysterangiales), no species of these genera undergo a blue colour change upon bruising or exposure (Zeller & Dodge 1918, Stewart & Trappe 1985). Additionally, Gautieria species differ from C. caerulescens in having gently rounded basidiospore ridges that terminate before the poles, a persistent dendroid columella, and globose cells in the peridium (Dodge & Zeller 1934). While Austrogautieria species are acolumellate and have apically convergent basidiospore ridges with subacute margins, only one species, the Australian A. manjimupana, overlaps with C. caerulescens in the number of basidiospore ridges (5–10); all other species of Austroguatieria have 8–14 ridges (Stewart & Trappe 1985). Austrogautieria manjipumana differs from C. cyanescens in the smaller (14–20 × 8–13 μm), more ellipsoid basidiospores that lack intervening subridges (Stewart & Trappe 1985). Additionally, Gautieria and Austrogautieria, as members of the Phallomycetidae, are phylogenetically distant from Boletales (Giachini et al. 2006, Hosaka et al. 2006).
In the phylogenetic analysis presented here, C. cyanescens is well supported as sister to Sutorius (formerly the Tylopilus eximius complex), which is a genus of dark maroon to purple, epigeous bolete species occurring in the Americas, Australasia, and sympatrically with C. cyanescens in Guyana (Fulgenzi et al. 2007, Halling et al. 2012). The molecular-based analysis may reflect evolutionary reality, but Costatisporus and Sutorius are very different morphologically. Sutorius species have robust, pileate-stipitate basidiomata with exposed hymenophores that bruise cinnamon-brown, ballistosporic basidia, and smooth, fusiform basidiospores, among many other different micromorphological features (Fulgenzi et al. 2007, Halling et al. 2012). The Costatisporus-Sutorius clade indicated here is supported as sister to the recently characterized, mostly epigeous genus Neoboletus (Fig. 1) (Wu et al. 2014, 2015). While the sole sequestrate species of Neoboletus, N. thibetanus, stains blue upon exposure, it is easily distinguished from C. cyanescens by its notable stipe and bright yellow peridium. In the future it will be necessary to sequence additional loci from species within this emerging clade to shed light on the putatively strong relationships between Sutorius, Neoboletus, and C. cyanescens.
Figure 1. Maximum likelihood (ML) phylogram (-ln = 57979.037507) based on RPB1, RPB2, and 28S ribosomal DNA sequences depicting phylogenetic relationships of the Boletaceae and new sequestrate Guyanese taxa (in red bold). ML bootstrap support values greater than 70 are shown above the nodes. Other sequestrate taxa are indicated in bold with solid black dots preceding their binomials. Previously identified clades with multiple species from the same higher taxon are collapsed into triangles for visual simplification as is a clade of outgroup taxa from several non-Boletaceae lineages of Boletales.