A new species of Inosperma, and first record of I. afromelliolens (Inocybaceae, Fungi) from West Africa

Here, we present the newly identified Inosperma macrocarpa and the first record of I. afromelliolens from West Africa. Inosperma macrocarpa is nested in an Old World Tropical clade, based on a molecular phylogeny inferred from the sequences of ITS, LSU, RPB2, and TEF1. Complete descriptions and illustrations, including photographs and line drawings, of the new species are presented. Morphological and molecular analyses based on collections from Benin confirmed the presence of I. afromelliolens in West Africa. Toxicity analysis showed that neither species contained muscarine, which further supports the hypothesis that the ability to produce muscarine is a derived trait of Inosperma.


Morphological analysis
Specimens were photographed in the field using a digital camera (Sony ILCE 7RM3), and the colors were described based on Kornerup and Wanscher [13].Fine sections from the dried basidiomata were rehydrated and examined in 3% KOH and Congo Red for microscopic investigation.The microscopic characteristics were drawn using a drawing tube attached to a Leica DM2700 light microscope.
Microscopic characteristics were drawn at 1000× magnification and 120 spores in three collection samples for each species were measured.We measured the length (L) and width (W) of basidiospores and calculated the ratio Q = L/W.The spore dimensions are given as (a-)b-c-d (-e), where (a) represents the extreme values at the < 5th percentile, the range b-d refers to the minimum of 90% of the measured values, (c) represents the average value, and (e) represents the extreme values at the < 95th percentile.Measurements of basidiospores and basidia excluded the apiculi and sterigmata.

Sequence alignment and phylogenetic analysis
All new sequences in this study were prepared and compared with closely related Inosperma sequences retrieved from GenBank [21].All Inosperma species described from Africa were included in the phylogenetic analysis.Sequences from other genera of Inocybaceae were added, based on Matheny et al. [1] and Aïgnon et al. [4] (Table 1).Sequences of different regions (ITS, LSU, RPB2, and TEF1) were aligned separately using MAFFT v7.511 [22], and a final concatenated dataset of ITS, LSU, RPB2, and TEF1 was generated using Geneious 7.0.2[23].
The dataset was partitioned into ITS and LSU, and the different codon positions of RPB2 and TEF1 were partitioned separately, and their introns and separate models of DNA substitution were applied to the first, second, and third codon positions of the protein-coding genes.For phylogenetic analysis, substitution models and the best partitioning schemes were determined for Maximum Likelihood (ML).Substitution models for each locus were determined based on the AICc model selection criterion implemented in PartitionFinder [44].

Nomenclature
The electronic version of this article in the portable document format (PDF) in a publication with an ISSN or ISBN will represent published work according to the International Code of Nomenclature for algae, fungi, and plants.Hence, the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone; therefore, no printed copies need to be provided.
The new names contained in this work have been submitted to MycoBank, where they will be made available to the Global Names Index.The unique MycoBank number can be resolved, and the associated information can be viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www.mycobank.org/MB/.The online version of this work was archived and made available in the following digital repositories: PubMed Central and LOCKSS.

Muscarine detection
Dried samples were ground into a fine powder, 2.5-26 mg of each specimen was weighed and placed into a 5 mL centrifugation tube with 2 mL of methanol-water (5:95, v/v).The mixture was vortexed for 30 min and ultrasonically extracted for another 30 min.After 5 min of centrifugation at 10000 rpm, the total supernatant was collected, filtrated using a 0.22 μm organic filter membrane, and mixed with acetonitrile-water (7:3, v/v) to a final volume of 1 mL for UPLC-MS/MS analysis.Lentinula edodes (Berk.)Pegler was used as a blank sample.
The UPLC-MS/MS analysis was performed using an ABSCIEX Exion UPLC system coupled to an ABSCIEX Triple Quad 6500+ system (ABSCIEX).Chromatographic separation was achieved on an ACQUITY UPLC Amide column (2.1 × 100 mm, 1.7 μm, Waters, USA).Aqueous solutions of 0.05% formic acid (A) and acetonitrile (B) were used as the mobile phase solvent flowing at 0.3 mL/min.The column was eluted with 70-10% B for 3 min, followed by 10% B for 0.5 min and then by 10-70% B for 0.5 min, and 70% B for 2 min.The analytical column was set at 40˚C and the injection volume was 2.0 μL [48].The muscarine content was estimated in the UPLC-MS/MS by using standard muscarine (Sigma-Aldrich, Chemical purity � 98%), and was calculated with an external standard method based on respective calibration curves.The protonated molecular ion ([M+H] + ) of 174.2 was chosen as the parent ion, as well as two daughter ions at 57.0 and 97.0, which were used for qualitative and quantitative detection, respectively.The MS/MS conditions were as follows: ion source, electrospray ionization; curtain gas, 20 psi; collision gas, 8 psi; ionspray voltage, 5500 V; ion source temperature, 500˚C; ion source gas, 1, 50 psi; ion source gas, 2, 50 psi.Product ion confirmation (PIC) was set as follows: scan function, negative ion scanning; scan mode: multiple reaction monitoring; PIC duration for 0.21 s; collision energy at 27 V.The Analyst software (version 1.6) was used for data acquisition and processing.

Phylogenetic analysis
Approximately 45 new sequences were submitted to GenBank.The sequences used for the phylogenetic analyses are presented in Table 1.The ITS locus was present in 110 taxa and the alignment had 909 sites; the LSU locus was present in 127 taxa and the alignment had 1470 sites; the RPB2 locus was present in 105 taxa and the alignment had 778 sites; and the TEF1 locus was present in 51 taxa and the alignment had 1113 sites.Multigene molecular analysis of ITS, LSU,, and TEF1 sequence data grouped the newly sampled specimens into two separate clades, each with short branches within the clade relative to the branch leading to the clade (Fig 1).One clade also included a specimen of Inosperma afromelliolens and we concluded that our samples were conspecific to that specimen.The other clade did not include any specimens  Habitat.Woodlands dominated by Isoberlinia doka and I. tomentosa.Occurrences from June to September.

Toxin detection
The weight of the tested samples was 0.01236 ± 0.009 (Table 3).After comparing the retention time (0.89 min) and relative deviation (5.11%) with standard muscarine in the allowance of ± 25% relative range, muscarine was not detected in the four samples of I. macrocarpa and the one sample of I. afromelliolens.The calibration curve for muscarine generated during validation was y = 16702.81879x+ 4.18331e 4 (r = 0.99505) for muscarine concentrations in the range 2-100 ng/mL (y = peak area, and x is = muscarine concentration, r = correlation coefficient).

Discussion
Here, we present Inosperma macrocarpa as a novel species.Based on the morphological and molecular similarities between the collections from West Africa and Inosperma afromelliolens  collected elsewhere, to date only known in Zambia, our data indicate that the distribution range of I. afromelliolens is broader than previously reported.Based on their morphological characteristics, collections from West Africa (voucher specimens HLA0468, HLA0469, and HLA0355.HLA0405 and HLA0754) were similar in size (7-25 mm versus 10-30 mm), color, and anatomical features to the collections of I. afromelliolens (voucher PC0088778) (Table 3).This is in agreement with our phylogenetic analysis, which did not show any differences between these collections (Fig 1).Inosperma macrocarpa presents morphological characteristics of taxa from the genus Inosperma, especially a radially rimose, fibrillose, or squamulose pileus and the absence of pleurocystidia [1].Molecular analysis based on the combined data of ITS, LSU, RPB2, and TEF1, confirms its position in Inosperma.I. macrocarpa is nested in Old World Tropical clade 1 and is close to the undescribed collections of Inosperma sp.G1842, and Inosperma sp.BB3233 from Zambia, with weak (57%) SH-aLRT values and strong (98%) ML Ultrafast bootstrap support.Morphologically, I. macrocarpa is close to I. cookei a European species; however, in terms of pileus size (37-86 mm), I. macrocarpa is closest to I. erubescens.
The Inocybaceae have many muscarine species, but the genus Inosperma has a few toxic species [5,8,9] that have been systematically reported in incidents of mushroom poisoning in tropical Asia [7,10,11].In Africa, poisoning due to the consumption of wild mushrooms is often not reported and is difficult to assess [49].In particular, no cases of mushroom poisoning have been officially reported in Benin, although mushrooms from families that include toxic species, such as Inocybaceae are consumed in this region.The first published records of Inosperma macrocarpa date back to twenty years back [50,51].These records, provisionally named Inocybe sp. and Inocybe gbadjii (ADK2166 and ADK2618, respectively), indicate that this taxon is consumed and appreciated by the local people.This prompted us to perform a toxicity analysis, which revealed a negative result for muscarine content in these taxa as well as in I. afromelliolens (Table 2).
This study is the first to focus on the toxicity of Inosperma species in Africa and increase the diversity of taxa in Inosperma to eight species, five of which are distributed in West Africa: I. africanum, I. bulbomarginatum, I. flavobrunneum [4], I. afromelliolens [52], and I. macrocarpa.Inosperma afromelliolens is widely distributed and present in East Africa in Zambia, along with I. boeticum, I. submaculatum [52], and I. misakaense [53].Our data suggest that the diversity of Inosperma in Tropical Africa is greater than the currently known, and many species remain to be identified.

Fig 1 .
Fig 1. ML tree of ITS, LSU, RPB2, and TEF1 sequences showing the placement of Inosperma macrocarpa.Values above or below branches indicate bootstrap proportions SH-aLRT support � 80% / ultrafast bootstrap support � 95%.Origin of species is given after the name of each taxon.The new species and the new records are in red.https://doi.org/10.1371/journal.pone.0290894.g001