﻿New species of Tropicoporus (Basidiomycota, Hymenochaetales, Hymenochaetaceae) from India, with a key to Afro-Asian Tropicoporus species

﻿Abstract The Inonotuslinteus complex, predominantly reported from East Asia, Mesoamerica and Caribbean countries, was circumscribed into Tropicoporus as one of the new genera, based on morphological and phylogenetic data. The present paper describes four new species of Tropicoporus from India. Morphological characteristics and phylogenetic analyses, based on ITS and nLSU data, delimited the new species, which are named T.cleistanthicola, T.indicus, T.pseudoindicus and T.tamilnaduensis. The pairwise homoplasy index (PHI) test was done to confirm the distinctive nature of the new species. The traits of Indian species remain distinct from one another, except for the pileate basidiome with the mono-dimitic hyphal system, cystidioles and broadly ellipsoid basidiospores. Descriptions, illustrations, PHI test results and a phylogenetic tree to show the position of the new species are provided. In addition, an identification key to Tropicoporus in Asia and an African species is given.


Introduction
The morpho-taxonomy and phylogenetic analyses, based on the nLSU and ITS genetic markers, revealed that the Inonotus linteus complex comprises two clades and are respectively treated as two new genera, Sanghuangporus and Tropicoporus (Zhou et al. 2015).Tropicoporus is characterised by their annual to perennial, resupinate, effused-reflexed to pileate basidiome with glabrous, uncracked to radially cracked pilear surface, homogeneous to duplex context, with or without a black line.A mono-dimitic or dimitic hyphal system with simple septate generative hyphae, presence or absence of cystidioles, presence MycoKeys 102: 29-54 (2024), DOI: 10.3897/mycokeys.102.117067Sugantha Gunaseelan et al.: New species of Tropicoporus from India of hymenial setae with smooth, fairly thick-walled to thick-walled, yellowish, subglobose to ellipsoid basidiospores are microscopic characteristic features of Tropicoporus (Zhou et al. 2015;Wu et al. 2022).
A total of forty-eight Tropicoporus species have been recorded in MycoBank with fifteen new species and thirty-three new combinations (as of 12 January 2024).Two new species, namely Tropicoporus excentrodendri L.W. Zhou  Baumgartner were reported across the world, based on the morphological and molecular data (Wu et al. 2015;Coelho et al. 2016;Salvador-Montoya et al. 2018;Brown et al. 2019;Lima et al. 2022) Wu with twenty-four new combinations were reported, based on the combined dataset of ITS and nLSU sequences (Wu et al. 2022).Of the forty-eight legitimate Tropicoporus species, only T. nullisetus was reported without setae (Lima et al. 2022).
Tropicoporus linteus (also known as Phellinus linteus) is used as a renowned Chinese medicine.Due to the presence of P. linteus polysaccharides (PLPs), it may play a vital role in anti-aging, anti-bacterial, anti-inflammation, anti-tumour, anti-oxidant, hepatoprotective and hypoglycaemic processes (Chen et al. 2019).On the other hand, Tropicoporus tropicalis has been reported to cause diseases in humans (Sutton et al. 2005;Haidar et al. 2017;Gupta et al. 2022).
In India, hymenochaetoid fungi from Himachal Pradesh were studied (Kaur et al. 2022).Fourteen hymenochaetoid members were documented from Tamil Nadu (Natarajan and Kolandavelu 1998).Nevertheless, studies on Indian hymenochaetoid fungi, based on molecular data have not been attempted, which makes it difficult to understand their evolutionary history, phylogenetic relationships and accuracy of species delimitation.This study is the first attempt to describe new Tropicoporus species from India, based on morphology and molecular evidence.In addition, an identification key to Afro-Asian Tropicoporus is given.

Morphological analyses
Eight specimens were collected from parts of Eastern Ghats and the plain region of Tamil Nadu, southern India.Macro-morphological characteristics such as shape, size of basidiome, perennial or annual, colour, texture, margin (acute or obtuse), context (homogenous, duplex with or without black line), tube layer (colour, length, stratification) and pores (size and shape) were examined in the fresh sample and recorded.Colour descriptions were based on the Methuen Handbook (Kornerup and Wanscher 1978)  , free-hand sections of dry specimens were mounted in water, 5% potassium hydroxide (KOH) (v/w), cotton blue (CB) and Melzer's reagent (IK).Sections were studied and photos were taken at magnification up to 1000× using a LABOMED OPTIC-CX BINO LED microscope.The drawings were made using LABOMED CxL2 compound microscope.Microscopic measurements and illustrations were made in 5% KOH solution.Basidiospores measurements (as minimum-mean-maximum) and Q values (length/width ratios) were recorded.The following abbreviations are used: IKI ̄ (inamyloid), IKI + (amyloid), CB ̄ (acyanophilous), CB + (cyanophilous), L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = (variation in the L/W ratios; and basidium length excludes the lengths of the sterigmata) and n = number of spores measured.For measuring the spores, an average of 50 spores were considered.Specimens in this study were deposited in the Madras University Botany Laboratory (MUBL), Centre for Advanced Studies in Botany, University of Madras, India.

Genomic DNA extraction, PCR amplification and sequencing
Extraction of total genomic DNA from mycelium and dried basidiome followed the protocol of Doyle and Doyle (1987), modified by Góes-Neto et al. (2005).The ITS and nLSU regions were amplified and sequenced with the primers ITS1/ITS4 and LR0R/LR7, respectively (Vilgalys and Hester 1990;White et al. 1990).The polymerase chain reaction (PCR) procedure for ITS was as follows: initial denaturation at 95 °C for 3 min, followed by 32 cycles at 95 °C for 30 s, 52 °C for 30 s and 72 °C for 1 min and a final extension of 72 °C for 3 min.The PCR procedure followed for nLSU was as follows: initial denaturation at 94 °C for 1 min, followed by 34 cycles at 94 °C for 30 s, 45 °C for 30 s and 72 °C for 1.5 min and final extension at 72 °C for 10 min.The PCR products were sequenced at Eurofins Genomics India Pvt. Ltd., Karnataka, India.

Genealogical concordance phylogenetic species recognition analysis
Genealogical concordance phylogenetic species recognition analysis (GCPSR) by the pairwise homoplasy index (PHI) test was used to determine the recombination level within closely-related species (Bruen et al. 2006).The data were analysed by the software SplitsTree 4 (Bruen et al. 2006;Huson and Bryant 2006).
The relationships between closely related taxa were visualised by constructing split graphs from concatenated datasets, using the LogDet transformation and splits decomposition options.If the PHI test value is (Φw) ≤ 0.05, it indicates significant recombination within the dataset.This is an important method to provide further evidence to justify a species.All results are shown in Fig. 1.

Phylogenetic analyses
In total, eight new sequences of the ITS and seven new sequences of the nLSU regions were generated and submitted to GenBank (  1).The combined nLSU and ITS dataset were aligned and the multiple sequence alignment consists of 1,820 characters (914 for nLSU and 902 for ITS) of which 1,017 were constant, 962 were variable and 570 (31%) were parsimony informative.The best-fit evolutionary model (GAMMA+P-Invar Model) was estimated by jModelTest 2.1.10for the combined datasets.The Maximum Likelihood (ML) trees were constructed using raxmlGUI 2.0 with 1,000 rapid bootstrap inferences (BS).The Bayesian analysis was run for 2,000,000 generations and the average standard deviation reached 0.010166.The phylogenetic topology was selected from Bayesian analysis.The Maximum Likelihood bootstrap values ≥ 60% and the Bayesian posterior probabilities ≥ 0.90 are summarised in Fig. 2. Etymology.The specific epithet cleistanthicola (Lat.)refers to the host Cleistanthus collinus.
Hyphal structures.Hyphal system monomitic in the context and dimitic in the trama, tissue darkening with KOH without hyphal swelling.
Hyphal structures.Hyphal system monomitic in the context and dimitic in the trama, tissue darkening with KOH without swelling Context.Generative hyphae, thin to thick-walled, hyaline to golden yellow, simple septate, rarely branched, 2-5 μm diam.
Habitat and distribution.Basidiomes were found on living trees of Fabaceae members (Albizia amara and Prosopis cineraria), distributed in Kalvarayan Hills, Kallakurichi District, Tamil Nadu, India.
Habitat and distribution.Basidiomes were found on living trees of Fabaceae members (Albizia amara and Peltophorum pterocarpum), distributed in Kalvarayan Hills, Kallakurichi District, Tamil Nadu, India.
Hyphal structures.Hyphal system monomitic in the context and dimitic in the trama, tissue darkening with KOH without hyphal swelling.
Habitat and distribution.Basidiomes are found on living trees of Madhuca longifolia and Prosopis cineraria, distributed in Cuddalore District, Tamil Nadu, India.

Discussion
Recently, the Inonotus linteus complex has gained attention because of its medicinal values and as an emerging potential pathogen in plants (Dai et al. 2009;Dai 2010), humans (Sutton et al. 2005;Haidar et al. 2017;Gupta et al. 2022) and dogs (Hevia et al. 2019).Zhou et al. (2015) Brown et al. 2019;Wu et al. 2022).Tropicoporus is characterised by its annual to perennial, resupinate, effused-reflexed to pileate basidiomes with mono-dimitic, dimitic hyphal system, ellipsoid to subglobose basidiospores.To date, twenty-three legitimate species are accepted under Tropicoporus, of which eleven were from tropical American countries, seven were from East Asian countries and one each from Africa, Costa Rica, Cuba and French Guiana.
The Bayesian phylogram illustrated in the present study is consistent with the previous studies (Coelho et al. 2016;Salvador-Montoya et al. 2018;Brown et al. 2019;Lima et al. 2022).The four new Tropicoporus species from Tamil Nadu, India, fit well within the Tropicoporus clade but formed a unique, distinct lineage that was the sister clade to T. rudis (earlier treated as Xanthochrous rudis).The T. rudis clade consists of strictly African collections (92% BS, 1.00 BPP) in the phylogeny (Fig. 1).This clade, in turn, forms the sister clade to a clade composed of T. stratificans, T. substratificans and T. linteus with 62% BS, 0.96 BPP.
The Eastern Ghats has discontinuous mountain ranges with hills ranging from 1,100 to 1,600 m with luxuriant vegetation of tropical evergreen to deciduous, thorn forest or scrub jungle that harbours diverse groups of wood rot fungi.This is the first report of the genus Tropicoporus from the Eastern Ghats of Tamil Nadu with three novel species, viz.T. cleistanthicola, T. indicus and T. pseudoindicus.

Figure 1 .
Figure 1.Split graphs show the results of the PHI test of the new species, Tropicoporus indicus, T. tamilnaduensis, T. pseudoindicus, T. cleistanthicola and their most closely-related species T. rudis, using LogDet transformation and split decomposition options.The PHI test result Φw ≤ 0.05 indicates that there is a significant recombination within the dataset.

Figure 2 .
Figure 2. Molecular phylogeny of four new Indian Tropicoporus species and other hymenochaetoid species inferred from combined ITS and nLSU sequences.The topology is from the Bayesian analysis.Maximum Likelihood bootstrap values and Bayesian posterior probabilities, above 60% and 0.9, respectively, are labelled at the nodes.The newly-generated sequences are coloured and bold; the type specimens are in bold.
The species pseudoindicus signifies the close morphological and phylogenetic relationships with the species Tropicoporus indicus.

Table 1 .
Names, strain numbers, countries of collection and the corresponding GenBank accession numbers of the sequences used in this study.

Table 2 .
Synoptic comparison of characteristics amongst species of the newly-reported Tropicoporus from India.