A new species of the genus Truncocolumella (Basidiomycota, Boletales) found in the Hengduan Mountains of China

Abstract

Background

During surveys of hypogeous fungi in the Hengduan Mountains, south-western China, three specimens of the genus Truncocolumella were discovered in Sichuan Province.

New information

Morphological and molecular analyses revealed that the collections represent a new species, Truncocolumellapseudocolumella. This article describes the new species and discusses its relationship with the other two members of the genus.

Introduction

Truncocolumella Zeller (Agaricomycetes, Basidiomycota) is a small genus of false truffles, established by Zeller in 1939 with the type species T.citrina Zeller (Zeller 1939). The main characteristics of this genus are: basidiomata depressed-spheroid to reniform, from a rhizomorphic base and with a central point of attachment; columella stump-like to dendroid, prominent; peridium persistent, not separating from the gleba; gleba pale white when immature, brown when mature; spores smooth, ellipsoid, as seen individually, almost hyaline. Another species, Truncocolumellarubra, was named in the same article and later recombined as Gastroboletusruber (Zeller) Cázares & Trappe (Cázares and Trappe 1991). The third species in this genus, T.occidentalis (Malençon) Malençon & Zeller, was reclassified from Dodgeaoccidentalis Malençon (Malençon and Zeller 1940). Subsequently, T.citrinavar.citrina Zeller and T.citrinavar.separabilis A.H. Sm. were synonymised with T.citrina (Smith and Singer 1959). To date, only T.citrina and T.occidentalis are recognised within the genus Truncocolumella.

Truncocolumellacitrina is a common ectomycorrhizal species, frequently found in coniferous forests in North America (Zeller 1939, Smith and Singer 1959, Goodman et al. 1998, Wood et al. 1998, Massicotte et al. 2000, Binder and Hibbett 2007, Twieg et al. 2007, Sato and Toju 2019). Truncocolumellaoccidentalis, however, is rare and has only been found in Canada (Malençon and Zeller 1940). Until 2012, Truncocolumella species had not been reported outside of North America. That year, a specimen was discovered at Mt. Yala in western Sichuan Province, China. Two additional collections were later found in the same region. Western Sichuan Province, part of the Hengduan Mountains, is one of the world's most biodiverse areas. Morphological and molecular analyses revealed that these three samples represent a new species, T.pseudocolumella, described in this paper.

Materials and methods

Morphological description

The specimens were collected from the Hengduan Mountainous in China. The type and other studied specimens were deposited in the herbarium of Cryptogams of the Kunming Institute of Botany, Academia Sinica (KUN-HKAS).

Descriptions of microscopic and macroscopic characters were based on specimens (HKAS131259 [YAAS L2327], HKAS95533, and HKAS95534) following the methods of Kumar et al. (2017) and Truong et al. (2017). Sections were made with a razor-blade by hand, mounted in a 5% potassium hydroxide (KOH) solution or water and then stained with a cotton blue or lactophenol solution. Mounts were observed under an Olympus BH-2 microscope. Key colours were selected from Kornerup and Wanscher (1978).

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from the specimens using the OMEGA Plant Genomic DNA Kit. The internal transcribed spacer (ITS) rDNA region was amplified with PCR primers ITS1F and ITS4 (White et al. 1990, Gardes and Bruns 1993, Truong et al. 2017). The large subunit nuclear ribosomal DNA (LSU) region was amplified with the PCR primers LROR and LR5 (Vilgalys and Hester 1990). PCR reactions were performed on a BIO-RAD C1000TM instrument. Thermal cycles with the following settings: initial denaturation for 5 min at 94°C, followed by 32 cycles of 40 s denaturation at 94°C, annealing at 56°C for 40 s for ITS and 52°C for 30 s for LSU, extension for 1 min at 72°C and final extension at 72°C for 10 min. The PCR products were verified on 1% agarose electrophoresis gels stained with ethidium bromide. The purification and sequencing of the PCR products was conducted by Sangon Biotech Limited Company (Shanghai, China).

Molecular phylogenetic analyses

ITS was used for the analysis of the diversity of Truncocolumella species in this study because ITS appears to be a useful locus for the delimitation of Truncocolumella species. Thirty ITS sequences from NCBI and this study representing two species of Truncocolumella and selected accessions from the closely-related genera Chroogomphus, Gomphidius, Rhizopogon, Suillus (Table 1), including Gastroboletusvividus Trappe & Castellano, Gastroboletussubalpinus Trappe & Thiers and Gastroboletusruber (Zeller) Cázares & Trappe as outgroup taxa were used. The sequences of the Truncocolumella species generated in this study were submitted to the GenBank database. We first used the Basic Local Alignment Search Tool for the GenBank database to recheck whether the newly-generated sequences were amplified from the contaminant or not and examined clusters with closely-related sequences. DNA sequences were retrieved and assembled using SeqMan. The ITS gene was analysed using BioEdit v. 7 (Hall 2007), sequence alignments were aligned using MAFFT version 7 (Katoh and Standley 2013) and Maximum Likelihood (ML) analysis was performed using RAxML-HPC2 v. 8.2.12 (Stamatakis 2014) as implemented on the CIPRES portal (Miller et al. 2011), with the GTR+G+I model and 1,000 rapid bootstrap (BS) replicates for all genes. A reciprocal 70% bootstrap support approach was used to check for conflicts between the tree topologies from the gene. As the topology of the ML tree and the Bayesian tree are similar, the ITS1, ITS2 and 5.8s sequences were combined using SequenceMatrix (Vaidya et al. 2011) partitioned phylogenetic analyses. For Bayesian Inference (BI), the best substitution model for each partition was determined by MrModelTest 2.2 (Nylander et al. 2004). The result suggested that ITS1: JC+I, 5.8S: GTR+G+I, ITS2: K80+I+G were the best models.. Bayesian analysis was performed using MrBayes ver. 3.2.7a (Ronquist et al. 2011) on the CIPRES Science Portal (Miller et al. 2011) and four parallel runs were performed for 10 million generations sampling every 100^th generation for the single gene trees. Parameter convergence > 200 was verified in Tracer v. 1.7 (Rambaut et al. 2018). The phylogenetic clade was strongly supported if the bootstrap support value (BS) was ≥ 70% and/or a posterior probability (PP) < 0.01.

Table 1.

Taxa information and GenBank accession numbers of the sequences used in this study. Newly-generated sequences are in bold.

Species name	Voucher	Origin	GenBank No.		Reference
Chroogomphusroseolus	HKAS 50552	China	EF423620	Li et al. (2009)
Chroogomphusochraceus	OKM 25472	USA	AH009858	Miller et al. (2002)
Gastroboletusruber	OSC 79741	USA	EU697272	GenBank
Gastroboletusruber	OSC 69644	USA	EU837224	GenBank
Gastroboletusruber	OSC 74672	USA	EU837223	GenBank
Gastroboletussubalpinus	Trappe607-holotype	USA	EU231989	Dentinger et al. (2010)
Gastroboletusvividus	JLF4456	USA	MH213058	GenBank
Gomphidiusborealis	IB:NR19990532-holotype	USA	NR_132800	Miller et al. (2002)
Gomphidiusnigricans	OKM 27830	USA	AY077474	Miller et al. (2002)
Rhizopogoncolossus	MICH AHS49480-holotype	USA	AH011348	Grubisha et al. (2002)
Rhizopogondiabolicus	MICH AHS68424-paratype	USA	AH011349	Grubisha et al. (2002)
Rhizopogonellenae	MICH AHS66137-holotype	USA	AH011350	Grubisha et al. (2002)
Rhizopogonevadens	MICH AHS65484-holotype	USA	AF062927	Grubisha et al. (2002)
Rhizopogonflavidus	YAAS L2957	China	NR_158904	Li et al. (2016)
Rhizopogonjiyaozi	YAAS L2929	China	NR_158906	Li et al. (2016)
Rhizopogonochraceorubens	MICH AHS59643-holotype	USA	AF062928	Grubisha et al. (2002)
Rhizopogonsinoalbidus	YAAS L2949	China	NR_158905	Li et al. (2016)
Rhizopogonsongmaodan	HKAS 106767	China	NR_174900	Wang et al. (2021)
Suillusamericanus	C0075059F	USA	KU663184	Zhang et al. (2022)
Suillusflavidus	KM171907	Scotland	KU721177	Zhang et al. (2022)
Suillusgranulatus	KM172141	Italy	KU721242	Zhang et al. (2022)
Truncocolumellacitrina	JLF 2149	USA	MH217566	GenBank
Truncocolumellacitrina	TDB-2001	USA	L54097	Kretzer et al. (1996)
Truncocolumellapseudocolumella	HKAS131259	China	KP090063	This study
Truncocolumellapseudocolumella	HKAS131259	China	KP090064	This study
Truncocolumellapseudocolumella	HKAS95533	China	OR631922	This study
Truncocolumellapseudocolumella	HKAS95534	China	OR631923	This study
Truncocolumella sp1.	OSC 67369	USA	KT968570	GenBank
Truncocolumella sp2.	MR3D88-RHITRU1	USA	OM459721	GenBank
Truncocolumella sp3.	OSC 111948	USA	KF206337	GenBank

Taxon treatments

Truncocolumella pseudocolumella

L. Li, S.H. Li & Y. Wang sp. nov.

0CB67103-CD0B-598F-B525-F13FCBC73E8A

851721

Materials

1. Type status: Holotype. Occurrence: catalogNumber: HKAS 131259; recordNumber: L2327; recordedBy: Shu-Hong Li; associatedSequences: KP090063, KP090064; occurrenceID: C1F523CC-76F3-573A-8065-139715C2CC02; Taxon: taxonID: MB 851721; scientificNameID: Truncocolumellapseudocolumella; Location: continent: Asia; country: China; stateProvince: Sichuan; municipality: Ganzi; locality: Yala Snow Mountain; verbatimElevation: 3772.4 m; verbatimLatitude: 30°31′N; verbatimLongitude: 101°37′E; Identification: identificationID: HKAS 131259; identifiedBy: Lin Li; Event: year: 2012; month: August; day: 19; habitat: in the forest of Quercusguyavifolia H. Lév.

2. Type status: Other material. Occurrence: catalogNumber: HKAS 95533; recordedBy: Shan-Ping Wan; associatedSequences: GenBank: OR631922; occurrenceID: E10A3DB1-A334-5221-8BED-5D60FE32F4DA; Taxon: scientificName: Truncocolumellapseudocolumella; Location: higherGeography: Hengduan Mountains area; country: China; stateProvince: Sichuan; county: Ganzi; Identification: identificationID: HKAS 95533; identifiedBy: Lin Li; Event: year: 2014; month: August; day: 22; habitat: in the forest of Pinus

3. Type status: Other material. Occurrence: catalogNumber: HKAS 95534; recordedBy: Shan-Ping Wan; associatedSequences: GenBank: OR631923; occurrenceID: 9ACD3595-0888-58BC-9057-6F0AECF08031; Taxon: scientificName: Truncocolumellapseudocolumella; Location: higherGeography: Hengduan Mountains area; country: China; stateProvince: Sichuan; county: Ganzi; Identification: identificationID: HKAS 95534; identifiedBy: Lin Li; Event: year: 2014; month: August; day: 22; habitat: in the forest of Pinus

Description

Basidiomata irregularly depressed-globose to pyriform, with a yellowish-tawny (4C8) rhizomorphic base, 1.5–3.0 cm in diameter, the surface typically smooth and dry in appearance, light yellowish-tawny (4C5) with yellowish-brown (4C8) rhizomorphs, colour unchanged on bruising or exposure, elastic, very mature basidiomata deliquesce like Rhizopogon (Fig. 1A). Odour light and pleasant.

Figure 1.

Truncocolumellapseudocolumella. A Basidiomata; B, C Dry peridium turning black when stained with 5% KOH; D A section of basidiomata in 5% KOH; E A section of the hymenium in lactophenol cotton blue; G-I Basidia in 5% KOH (H. 4-spored basidia, I. 2-spored basidia); F, J Basidiospores in 5% KOH. Scale bars: A = 1 cm; B, C = 0.5 cm; D = 50 μm; E, G, H, I = 20 μm; F, J = 10 μm.

Peridium 102–160 μm thick, not separating or evanescent from the gleba at maturity, composed of two layers: outer layer 52–73 μm thick, reddish-tawny (7D7) interwoven hyphae of 1–1.5 μm diameter. The inner layer 40–86 µm thick, consisting of brownish-hyaline nearly parallel interwoven hyphae of 0.5–1.5 μm diameter (Fig. 1D). The boundary between the inner and outer layers gradually transitioning, with the changes in hyphae arrangement direction and hyphae colour. The clamp connection clearly visible, dry peridium becoming black (4F4) when encountering 5% KOH (Fig. 1B and C). Gleba pale white when immature, light brown to tawny at maturity, unchanging on bruising or exposure, cavities relatively small, spongy, lacking obvious columnar or dendroid radiating columella (Fig. 1A). Trama (56.0–) 62.5–85.0 μm, composed of hyaline almost parallel hyphae. Hymenium present on cavities surface. Basidia narrowly clavate, (15.0–) 17.2–19.5 (–22.4) × 3.5–6.5 (–7.0) μm, 2-4-spored. Sterigmata 2–3 μm (Fig. 1E, G, H and I). Basidiospore ellipsoid, smooth, 7.5–10.0 (–11.0) × 4.0–5.0 (–5.6) μm, grey to brownish (7D5) in mass, as seen individually, almost hyaline, typically 1–2 guttulate, infrequently 3-guttulate (Fig. 1F and J), changing to blue in lactophenol cotton blue, not obvious discolouration in Melzer's reagent.

Diagnosis

Differs from other species in the genus Truncocolumella in the basidiomata devoid of any columnar.

Etymology

Pseudocolumella, referring to the absence of the columella.

Distribution

China, Sichuan Province.

Ecology

Gregarious in the soil associated with Quercusguyavaefolia and Pinus sp.

Notes

Notes: The genus Truncocolumella currently includes three species: T.citrina, T.occidentalis and the newly described T.pseudocolumella in this study. Based on the original literature descriptions of the species T.citrina (Zeller 1939, Smith and Singer 1959) and the re-examination of the type specimens of T.citrina and T.occidentalis by Malençon and Zeller (Malençon and Zeller 1940), we compared the key distinguishing features of these three species. In terms of macroscopic characteristics, T.pseudocolumella differs from other species in the genus Truncocolumella in the basidiomata devoid of any columnar. Furthermore, the basidiomata of T.citrina have a diameter of 2–4 cm, which is comparable in size to those of T.pseudocolumella (1.5–3 cm), but they are two to three times larger than T.occidentalis. The basidiomata surfaces of T.citrina are distinctly citrine yellow, those of T.pseudocolumella are yellowish-tawny, while T.occidentalis has white basidiomata, although the colour of T.occidentalis remains uncertain due to preservation in alcohol. Microscopically, T.citrina has ellipsoid spores measuring 6–10 × 3.5–5.0 μm, with individual spores appearing nearly hyaline. Similarly, T.pseudocolumella has ellipsoid spores measuring 7.5–10.0 × 4.0–5.0 μm, also nearly hyaline, showing close resemblance between the two. According to Malençon's description (Malençon and Zeller 1940), the spores of T.citrina are clearly shorter, more oval and of more irregular shape than those of T.occidentalis, in which these bodies are long-elliptic or sometimes subcylindrical. Molecular analysis further demonstrates that T.pseudocolumella is distinct from T.citrin, with high support for their separation into different species.

Analysis

Phylogenetic analysis

The ML and Bayesian analyses of the 30 ITS sequences are shown in Fig. 2 with associated bootstrap supports for branches.

Figure 2.

Phylogeny derived from a Maximum Likelihood (ML) analysis of the nrDNA-ITS sequences from Suillineae, including Truncocolumella species, using Gastroboletus as outgroup. Values next to nodes present Maximum Likelihood bootstrap support values (BS), left and Bayesian posterior probabilities (PP), right. The names of novel species and samples with newly-generated sequences are in bold.

In the phylogenetic tree, the 25 ITS sequences from Suillineae revealed the phylogenetic relationship of two species of Truncocolumella and five Gastroboletus sequences are used as outgroups. Since there are few Truncocolumella sequences in GenBank and these sequences belong to only one species T.citrina, we selected sequences of Suillineae for phylogenetic analysis. The analytics include three sequences of the genus Suillus; nine sequences of the genus Rhizopogon; two sequences of the genus Chroogmophus; two sequences of the genus Gomphidus; and nine sequences of the genus Truncocolumella. Two clades were revealed in the genus Truncocolumella; Clade I includes five sequences of T.citrina from the USA; Clade II includes four sequences of a new species, T.pseudocolumella from China. The phylogenetic analysis shows that the new species is distinct from T.citrina, the type species of Truncocolumella. In addition to the ITS sequences used in this phylogenetic analysis, the LSU sequences were amplified from the newly-recorded specimens in this study and uploaded to NCBI for future study.

Discussion

Truncocolumella is an ancient, small genus with only two known species, T.citrina and T.occidentalis, which were found exclusively on the west Pacific coast of North America until the discovery of the third member, T.pseudocolumella, almost a century later in Sichuan, China. Truncocolumella is closely related to the epigeous fungi Suillus and both genera are currently classified within the family Suillaceae. The genus Truncocolumella is characterised by its prominent, stump-dendroid-like columella, a key morphological feature. However, the new species T.pseudocolumella has almost no columella, making it similar to species of Rhizopogon, although molecular analysis clearly places it within the genus Truncocolumella. In addition to the absence of columella in the basidiomata of T.pseudocolumella, which is a notable distinguishing feature, T.citrina and T.pseudocolumella also differ in several morphological characteristics: the basidiomata surface of T.citrina is distinctly citrine yellow (Zeller 1939, Smith and Singer 1959), while that of T.pseudocolumella is yellowish-tawny. The peridium of T.citrina is 70–100 μm thick, whereas that of T.pseudocolumella is thicker, reaching 102–160 μm and is differentiated into two layers based on the orientation of hyphae and changes in hyphal colouration. Additionally, the sterigmata of T.citrina are 3–5 μm in length, while those of T.pseudocolumella are shorter, measuring 1–2 μm in length. Similarly, based on the re-examination of the morphological structures of the type specimens of T.citrina and T.occidentalis by Malençon and Zeller (Malençon and Zeller 1940) and their descriptions, it is evident that T.pseudocolumella has larger basidiomata with a yellowish-tawny surface compared to T.occidentalis. Additionally, the spores of T.occidentalis are long-elliptic, sometimes almost subcylindrical and are longer than those of both T.citrina and T.pseudocolumella. Truncocolumellaoccidentalis was reclassified from Dodgeaoccidentalis and its description is based on a holotype specimen that was preserved in alcohol for 17 years. Since then, no additional reports of this species have been recorded. Unfortunately, molecular data are not available. It is hoped that future collections of this species will help clarify its identity.

Truncocolumellapseudocolumella differs significantly from the North American species, which can be attributed to bio-evolutionary geographical isolation and the unique ecological environment of the Hengduan Mountains in China. Truncocolumellacitrina is found in coniferous forests of the Pacific Northwest mountains at altitudes around 1200 m (Zeller 1939, Smith and Singer 1959), whereas T.pseudocolumella is distributed in alpine Quercusguyavifolia and Pinus forests at 3700 m in the Hengduan Mountains region of China.

Author contributions

Conceptualisation, Lin Li, Shan-Ping Wan and Yun Wang; methodology, Lin Li; formal analysis, Lin Li; investigation, Lin Li; resources, Shu-Hong Li and Yun Wang; data curation, Lin Li; writing—original draft preparation, Lin Li; writing—review and editing, Naritsada Thongklang, Zong-Long Luo and Shu-Hong Li; funding acquisition, Shu-Hong Li. All authors have read and agreed to the published version of the manuscript.