Three new species of Inosperma (Agaricales, Inocybaceae) from Tropical Africa

Abstract Here, we describe three new species of Inosperma from Tropical Africa: Inosperma africanum, I. bulbomarginatum and I. flavobrunneum. Morphological and molecular data show that these species have not been described before, hence need to be described as new. The phylogenetic placements of these species were inferred, based on molecular evidence from sequences of 28S and RPB2. Additional analysis using ITS dataset shows interspecific variation between each species. Phylogenetic analyses resolve I. flavobrunneum in Old World Tropical clade 1 with weak support, I. bulbomarginatum is sister of Old World Tropical clade 1 and I. africanum is indicated as sister to the rest of Inosperma. Complete description and illustrations, including photographs and line drawings, are presented for each species. A new combination of Inocybe shawarensis into Inosperma is also proposed.


Introduction
Inocybaceae Jülich (Basidiomycota, Agaricales) is a family of ectomycorrhizal species, forming symbiotic association with more than 23 families of vascular plants (Matheny et al. 2020). The family is diverse with an estimated 1050 species distributed worldwide (Matheny and Kudzma 2019;Matheny et al. 2020). The number of species described will continue to increase as new habitats are explored (Matheny and Watling 2004;Esteve-Raventós 2014;Manimohan 2015, 2016;Matheny et al. 2017;Naseer et al. 2018;Jabeen and Khalid 2020).
Recently, Inocybaceae was revised to include seven genera, Auritella Matheny & Bougher, Inocybe (Fr.) (Matheny et al. 2020). Inosperma is represented by more than 70 known species that are distributed in Africa, Asia, Australasia, Europe, North America and northern South America (Matheny et al. 2020). Typically, the species of the genus are characterised by a radially fibrillose and rimose or squamulose pileus; smooth, ellipsoid or phaseoliform basidiospores; and absence of metuloid hymenial cystidia. In addition, many species of Inosperma have odours that are fruity, pleasant, like honey, fishy, pelargonium or otherwise distinct (Matheny et al. 2020). Phylogenetically the genus is monophyletic with four major clades: the Maculata clade (Larsson et al. 2009), I. sect. Inosperma and two clades from the Old World tropics (Pradeep et al. 2016;Matheny et al. 2020).
In this study, we describe three new species of Inosperma from West Africa, based on morphological characters, as well as analysing their phylogenetic position using multigene molecular analysis of 28S and RPB2 sequences data.

Morphological analyses
Specimens were photographed in the field with a digital camera Sony FE. Colour codes are described using Kornerup and Wanscher (1978). For anatomical analyses, samples of specimens were rehydrated and examined directly in 3% potassium hydroxide (KOH) and Congo red. Drawings of microscopic characters were made with the aid of a drawing tube attached to a Leica DM2700. Microscopic characters were drawn at magnification 1000×. Spore measurements were made from 40 spores for each species. We measured length (L) and width (W) of the basidiospores and calculated the ratio Q = L / W. Measurements of basidiospores and basidia excluded the apiculus and sterigmata, respectively and are given as (a-)b-c(-d), where (a) = extreme minimum value, range b-c contains minimum of 90% of the calculated values and (d) = extreme maximum value as used in Aïgnon et al. (2021).

DNA extraction, PCR and sequencing
Genomic DNA was extracted from dried specimens by QIAGEN® plant mini kit following the manufacturer's instructions and PCR products were cleaned using ExoSAP-IT (Bell 2018). The internal transcribed spacer regions (ITS), portions of the nuclear large subunit ribosomal RNA gene (28S) and DNA-directed RNA polymerase II subunit (RPB2) were amplified. For sequencing of the ITS region, we used the primers ITS1F and ITS4 (White et al. 1990;Gardes and Bruns 1993), for LSU we used LR0R, LR7 and internal primers LR5 and LR3R (Vilgalys and Hester 1990;Cubeta et al. 1991;Rehner and Samuels 1995) and for RPB2, we used primer pairs b6F and b7.1R (Matheny 2005). PCR products were cleaned and sequenced at Macrogen Inc. (Macrogen Europe B.V., Amsterdam, Netherlands) using the same primers as those used for PCR.
For phylogenetic analysis, the dataset of 28S and RPB2 was generated using Geneious 7.0.2 (Drummond et al. 2010) and partitioned in 28S, RPB2 codon position 1, RPB2 codon position 2, RPB2 codon position 3 and the intron in RPB2 separately (Suppl. material 1). We tested for the best partitioning scheme and best model for each partition using Modelfinder (Kalyaanamoorthy et al. 2017). It was indicated that keeping all the  partitions was the best way to proceed. Maximum Likelihood (ML) analysis was performed with IQTREE 1.6.12 (Nguyen et al. 2015). Branch support was assessed with 1000 replicates of ultrafast bootstrap replicates and approximate likelihood ratio test [aLRT] and Shimodaira-Hasegawa [SH]-aLRT (SH-Alrt) test with 1000 replicates (Hoang et al. 2017). For Bayesian Inference (BI) analyses, GTR models with gamma-distributed rate heterogeneity and a proportion of invariant sites parameter were assigned to each partition as indicated above, using MrBayes 3.2.7 (Ronquist et al. 2012), set as follows: lset applyto = (all), nst = 6, rates = invgamma, ngammacat = 4, sampling frequency = 1000 and the command "unlink" was used to unlink parameters across characters on partitioned datasets. Two independent Markov Chain Monte Carlo (MCMC) processes were executed, each in four chains for 20 million generations. Posterior probabilities (BPP) were calculated after burning the first 25% of the posterior sample and ensuring that this threshold met the convergence factors described above. The sequences from Pseudosperma lepidotellum (Matheny & Aime) Matheny & Esteve-Rav., P. pluviorum (Matheny & Bougher) Matheny & Esteve-Rav., Pseudosperma sp. PBM3751 and Pseudosperma sp. TR194-02 were used as outgroup taxa. We also produced trees using ITS database only to show interspecific variation between each species.
Taxonomic key to species of Inosperma from West Africa Notes. This species is placed in the old Inosperma clade which became the genus Inosperma, but the combination is not made in the study of Matheny et al. (2020). The new combination is based on molecular phylogenetic data and sequencing the type of Inocybe shawarensis (Naseer et al. 2018).

Discussion
The new species exhibit the overall characteristics often observed in Inosperma. These characters include; pileus radially rimose, fibrillose or squamulose and absence of pleurocystidia (Matheny et al. 2020). They can be distinguished from other Inosperma species by their remarkable characteristics. In addition, I. africanum is common in West Africa and I. bulbomarginatum presents a large distribution and was recognised in Zambia in the collections of Bart Buyck (Matheny et al. 2009). However, the low sequence divergences between the sequences (2.2%-2.5%) of ITS and 0.3% of 28S allows us to confirm the wide distribution of I. bulbomarginatum. Phylogenetically, I. africanum is nested in Inosperma with full support (99% SH-aLRT values, 100% ML Ultrafast bootstrap, 1 BPP) and I. bulbomarginatum is indicated as the sister of Old World Tropical clade 1 with full support (100% SH-aLRT values, 100% ML bootstrap, 1 BPP). Sequences of Inosperma bulbomarginatum from West Africa and Zambia formed a subclade. Inosperma flavobrunneum is nested in Old World Tropical clade 1 and has sister species undescribed in a collection from Zambia, BB3233, G1842 and PC96013. ML and BI analysis, using 28S and RPB2 sequences data, shows most nodes well resolved; for example, the node uniting Old World Tropical clade 2 with the crown group of Inosperma is supported with 0.97 BPP, but with weak ML bootstrap as shown in Pradeep et al. (2016); based also on combined data of 28S and RPB2, this node is with weaker support < 50% ML bootstrap.
The position of each of these new species is confirmed by single data from ITS (Fig. 6). There are several collections from undescribed species in Inosperma (e.g. Inosperma sp. G1842, Inosperma sp. BB3233, Inosperma sp. PC 96073, Inosperma sp. PC96013, Inosperma sp. PC96082, Inosperma sp. PC96080 and Inosperma sp. Zam07) that are of African origin, thereby attesting the need for further studies of this genus on this continent. Previously, in Inosperma, only one species, Inosperma misakaense, has been described from Africa before this study (Matheny and Watling 2004). So, this study reinforces the diversity of Inosperma in Tropical Africa which now amounts to four described species.
n° 226-2014-1109) for funding molecular analysis and the Deutscher Akademischer Austauschdienst (DAAD, grant n° PKZ 300499) for granting the University of Parakou with a microscope, type Leica DM5700, that enabled us to perform microscopic investigations. Anneli SVANHOLM, Bobby SULISTYO and Brandan FURNEAUX (Systematic Biology programme, Department of Organismal Biology, Uppsala University) are thanked for their assistance during molecular analyses. We also thank Kassim TCH-AN ISSIFFOU and Evans CODJIA (MyTIPS Research Unit, University of Parakou) for their assistance during field data collection. P. Brandon MATHENY (Department of Ecology and Evolutionary Biology, University of Tennessee, USA) and an anonymous reviewer are thanked for their corrections and suggestions to improve our paper.