https://orcid.org/0000-0002-3014-9194
Figures
Abstract
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.
Citation: Aïgnon HL, Fan Y-G, De Kesel A, Bahram M, Ryberg M, Yorou NS (2023) A new species of Inosperma, and first record of I. afromelliolens (Inocybaceae, Fungi) from West Africa. PLoS ONE 18(10): e0290894. https://doi.org/10.1371/journal.pone.0290894
Editor: Shwet Kamal, ICAR-Directorate of Mushroom Research, INDIA
Received: April 17, 2023; Accepted: August 14, 2023; Published: October 18, 2023
Copyright: © 2023 Aïgnon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This research was funded by the International Association for Plant Taxonomy, the Percy Sladen Memorial Fund, the National Natural Science Foundation of China (Grant Nos. NSFC31860009 & 32260005), and the Swedish Research Council (VR grant 2019-04406). Some field equipment were received from IDEA WILD, the additional samplings were carried out with the financing of Rufford Small Grants Foundation (grant No. 35556-B) and the microscope type Leica DM5700 used to make the drawings was offered to the University of Parakou by Deutscher Akademischer Austauschdienst (DAAD, grant n° PKZ 300499). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Inocybaceae Jülich (Basidiomycota, Agaricales) is one of the most taxonomically diverse families of Agaricales with seven genera, including Inosperma (Kühner) Matheny & Esteve-Rav. [1]. Members of Inosperma are morphologically characterized by basidiomes of small to medium size; occasional distinctive odors such as fruity, honey, fishy, or pleasant; rimose or squamulose pileus, smooth stipe, with a bulbous base and/or bruising reaction; smooth, ellipsoid, or phaseoliform to subglobose basidiospores; thin-walled cheilocystidia; and a lack of pleurocystidia [1, 2].
This genus is monophyletic, and four distinct clades have been recognized within it: two distinct tropical old-world clades (1 and 2) [1, 3–5], the Maculata clade [6], and Inosperma sect. Inosperma [4]. Most species of Old World Tropical clade 2 are primarily from tropical Asia [5], whereas taxa of Old World Tropical clade 1 are mainly found in tropical Africa [1, 4]. This genus includes a few toxic species [7] with high levels of muscarine, including Inosperma erubescens (A. Blytt) Matheny & Esteve-Rav, the species in Inocybaceae responsible for most mushroom poisoning [5, 8, 9]. Species of Inosperma have been consistently reported in mushroom poisoning incidents in tropical Asia [10–12]; however, the toxicity of members of this genus in Africa remains poorly studied. Thus, documenting the diversity of Inosperma in tropical Africa and their toxicity can help avoid food poisoning due to mushroom consumption.
Here, based on the morphological characteristics and multigene molecular analysis using ITS, LSU, RPB2, and TEF1 sequence data, we describe Inosperma macrocarpa sp. nov. and report the first West African record of I. afromelliolens. The toxicity of both taxa was studied by analyzing their muscarine content.
Material and methods
Study area and specimen sampling
The specimens were collected between 1997 and 2022 in Benin, woodlands dominated by Isoberlinia doka Craib & Stapf and/or I. tomentosa (Harms) Craib & Stapf, or gallery forests dominated by Uapaca guineensis Müll. Arg and/or Berlinia grandiflora (Vahl) Hutch. & Dalziel, in Okpara Forest (9.270131°N, 2.715440°E), N’dali Forest Reserve (9.758857°N, 2.696819°E), Koussoucoingou gallery forest (10.173066°N, 1.196233°E), Forest reserve of Gbadji (7.952167°N, 1.967867°E) and Forest reserve of Wari Maro (9.164733°N, 2.159917°E). No specific permits were required for mushroom sample collection for the Beninese researcher in the forest reserve.
The fresh basidiomata were dried using an electric Stöckli Dörrex dryer for 24 hours at 45°C. Most specimens studied, including the holotype of the newly described species, were deposited in the Mycological Herbarium of Parakou University, Benin Republic (UNIPAR), and additional specimens of I. macrocarpa (vouchers ADK2166 and ADK2618, leg. A. De Kesel) were deposited in the Meise Botanic Garden Herbarium (BR).
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.
DNA extraction and PCR and sequencing
Genomic DNA was extracted from dried specimens using a NuClean Plant Genomic DNA kit (ComWin Biotech, Beijing, China). The full ITS region and parts of LSU, RPB2, and TEF1 were amplified. We produced amplicons using the primers ITS1F/ITS4 for ITS [14, 15], LR0R/LR7 for LSU [16–18], bRPB2-6F/bRPB2-7.1R for RPB2 [18], and EF1-983F/EF1-1576R for TEF1 [20]. All PCR products were sent to the Beijing Genomics Institute (Beijing, China) for purification and sequencing using the same primers as those used for PCR.
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].
ML analysis was performed using IQ-TREE v2.2.0 [45]. Ultrafast bootstrapping (UFBoot) was performed using 1000 replicates [46]. Sequences of Inocybe (Fr.) Fr., Nothocybe Matheny & K.P.D. Latha, and Pseudosperma Matheny & Esteve-Rav. were used for rooting [47].
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.
Results
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 annotated with a formal name and was determined to be a previously undescribed species (Inosperma macrocarpa; Fig 1).
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.
Phylogenetically, I. macrocarpa is nested in Old World Tropical clade 1, closely related to G1842 from Zambia, with strong support (99% SH-aLRT values, 100% ML ultrafast bootstrap).
Taxonomy
Inosperma afromelliolens Eyssart. & Buyck, Cryptog. Mycol. 42(5): 69 (2021), Figs 2, 3 and 6F.
Description. Pileus was 7–25 mm in diameter, conical, umbo, plane to convex, surface slightly rimose, dry, radially fibrillose, generally smooth with some fissured margins, honey-yellowish (3A2), with no color change on bruising or cutting. Lamellae 1 mm deep, entire, sub-horizontal, white, with slightly flocculose edges, pale smooth, and obtuse at the margin. Stipe:19–35 × 1–4 mm in diameter; white, cylindrical, central, uniform, slightly enlarged at the base, fibrillose, base slightly bulbous, flesh white. Odor and taste were not distinctive. Basidiospores were smooth, ellipsoid to cylindrical, pale yellow, (8) 8.3–10.3–12 (12.2) × (4) 4.3–5.1–6 (6.1) μm, Q = (1.5) 1.6–2.0–2.4 (2.5). Basidia 28–40 × 7–12 μm, clavate, 2–4 sterigmates. Cheilocystidia 35–42 × 8–15 μm, cylindrical to clavate, thin-walled, and hyaline. Pleurocystidia absent. Pileipellis a cutis with cylindrical, smooth, thin-walled hyphae, 3–7 μm diam. Stipitipellis a cutis, regularly arranged, hyphae 5–10 μm diam., parallel, filamentous. Caulocystidia 20–40 × 7–9 μm diam, piriform, sometimes utriform, observed at the top of the stipe. Clamp connections were common in all tissues.
A. Basidiospores, B. Basidia, C. Cheilocystidia, D. Caulocystidia, E. Pileipellis F. Stipitipellis. Scale bars: 5 μm (A); 10 μm (B–F).
A–F. Inosperma afromelliolens (HLA0754). A. Basidiospores B. Basidia, C. Cheilocystidia, D. Caulocystidia, E. Pileipellis F. Stipitipellis. Scale bars: 5 μm (A-B); 10 μm (C–F).
Habitat. Woodlands dominated by Isoberlinia doka and I. tomentosa. Occurrences from June to September.
Specimens examined. Benin, Borgou Province, N’dali Region, in Forest Reserve of N’dali, 8.5456667°N, 2.8403333°E, on soil in woodlands dominated by I. doka, 04 July 2017, Leg. Aïgnon HL., Voucher (HLA0407); GenBank accession: ITS (MT534296) and LSU (MT560736); ibid. 01 September 2017, Leg. Aïgnon HL., Voucher (HLA0468); GenBank accession: ITS (MN096191), LSU (MN097883), and RPB2 (MN200774); ibid., voucher (HLA0469); GenBank accession: ITS (MT534294) and LSU (MT560738/ MT536757); ibid. Tchaourou Region:92546667°N, 27230000°E, on soil in the forest of Okpara in a woodland dominated by I. doka, June 7, 2017, leg. Aïgnon HL., voucher (HLA0355) GenBank accession: ITS (MT534291); ibid., June 30, 2017, leg. Aïgnon HL., Voucher (HLA0405); GenBank accession: ITS (MT534292) and LSU (MT560737); ibid. 9.2446277°N; 2.7262333°E on soil in Okpara forest in a woodland dominated by I. doka, 07 July 2021, leg. HL. Aïgnon, a voucher specimen (HLA0754), was deposited at UNIPAR. GenBank accession numbers: ITS (OQ300372), LSU (OQ300369), RPB2 (OQ435246); TEF1 (OQ441164).
The voucher specimens (HLA0355, HLA0405, HLA0407, HLA0468, HLA0469, and HLA0754) examined in West Africa were morphologically, anatomically, and phylogenetically close to I. afromelliolens (Table 2).
Inosperma macrocarpa Aïgnon & Yorou sp. nov.
MycoBank No. MB 846514
Syn. Inocybe gbadjii De Kesel nomen provisorum, in Boa, Wild Edible Fungi, A global overview of their use and importance to people (Rome): 104 (2004) (nom. inval., art. 39.1(a) ‐ Shenzhen).
Figs 4, 5 and 6A–6E, 6G and 6H
A–H. Inosperma macrocarpa (HLA0791), microscopical characters in KOH and Congo Red. A. Basidiospores B. Basidia, C. Cheilocystidia, D. Caulocystidia, E. Pileipellis F. Stipitipellis, G. Pileipellis hyphae H. Stipitipellis hyphae. Scale bars: A–B = 5 μm, C–D = 10 μm, E–H = 20 μm.
A–F. Inosperma macrocarpa (HLA0791). A. Basidiospores B. Basidia, C. Cheilocystidia, D. Caulocystidia, E. Pileipellis F. Stipitipellis. Scale bars: A–B = 5 μm, C–F = 10 μm.
Macromorphology of: A–E, G–H. Inosperma macrocarpa (A–E = HLA0930, G = HLA0791, H = HLA0920) and F. Inosperma afromelliolens (HLA0407). Scale bar: 2.5 cm (A–F) and 1 cm (G–H).
Diagnosis. Inosperma macrocarpa differs from all Inosperma species known from tropical Africa by its larger basidiomata (37–86 mm), plano-convex to convex, and slightly conical pileus.
Holotype. Benin, Atacora Province, Boukombé Region, Koussoucoingou, 10.173066°N, 1.196233°E, in a gallery forest dominated by Uapaca guineensis and Berlinia grandiflora, September 16, 2021, Leg. Aïgnon HL., Voucher (HLA0791 deposited at UNIPAR) GenBank accession numbers: ITS (OQ300373), LSU (OQ300370), RPB2 (OQ435244), and TEF1 (OQ441169).
Etymology. macrocarpa (L.) refers to large basidiocarps.
Description. Pileus was 37–86 mm in diameter, wide, plano-convex to convex, slightly conical with age, yellowish white (3A2) at the margin, dark yellowish white in the middle (4A2), no color change on bruising or cutting, surface wavy, margin incurved, streaked, flesh uniform, surface dry, and glabrous to fibrillose. Lamellae 2 mm deep, crowded, adnexed, 30 reaching stipe, with some tiers of yellow-brown lamellula (5B5). Stipe 49–65 mm high, 8–12 mm in diameter, white, cylindrical, central, equal, straight, sometimes entirely fibrillose at the top, inseparable from the cap, sometimes curved, base slightly bulbous to marginally bulbous, flesh white to yellowish (3A2). Stipitipellis was glabrous to fibrous. Odor and taste were initially neutral, then reminiscent of almonds. Basidiospores (6.6) 8.0–9.8–10.5 (12) × (3.6) 5.0–6.2–6.6 (8) μm, Q = (1.2) 1.3–1.6–2.0 (2.2), smooth, ellipsoid. Basidia 24–35 × 7–12 μm, clavate, 2–4 sterigmates. Cheilocystidia 26–35 × 6–15 μm diam, clavate to piriform, sometimes subutriform. Pleurocystidia absent. Pileipellis a cutis of cylindrical hyphae, 5–8 μm broad, filamentous with incrusting pigment. Stipitipellis a cutis, regularly arranged with subparallel hyphae 4–15 μm diam, septate, filamentous, no reaction with KOH. Caulocystidia 15–33 × 6–11 μm diam, utriform, observed on the upper third of the stipe. Clamp connections were common in all tissues.
Habit. In small or large groups, scattered on soil.
Habitat. Woodland forest dominated by Isoberlinia doka and/or I. tomentosa and Uapaca togoensis and gallery forest dominated by Uapaca guineensis and/or Berlinia grandiflora. Occurrences from June to September.
Geographical distribution. Currently known: Benin
Edibility. The new species is used for consumption in Borgou Province with the local name osousou kaka in the Nagot language and Zou province with the local name kocholé in the Fon language.
Additional specimens examined. Benin, Atacora Province, Boukombé Region, in the gallery forest of Koussoucoingou, 10.176230°N–1.203339°E, on soil in woodlands dominated by gallery forests with Uapaca guineensis and Berlinia grandiflora, September 15, 2021, Leg. Aïgnon HL., voucher (HLA0787), GenBank accession numbers: ITS (OQ300390), LSU (OQ286290), RPB2 (OQ427873), and TEF1 (OQ441166); ibid., 10.175111°N, 1.202631°E, leg. Aïgnon HL., voucher (HLA0788), GenBank accession numbers: ITS (OQ300391), LSU (OQ286291), RPB2 (OQ435242), and TEF1 (OQ441167); ibid., Leg. Aïgnon HL., voucher (HLA0790), GenBank accession numbers: ITS (OQ300392), LSU (OQ286292), RPB2 (OQ435243), and TEF1 (OQ441168); ibid., 10.174022°N, 1.203330°E, Leg. Aïgnon HL., Voucher (HLA0790), GenBank accession: ITS (OQ300392), LSU (OQ286292), RPB2 (OQ435243), and TEF1 (OQ441168); ibid., 16 September 2021, Leg. Aïgnon HL., voucher (HLA0790), GenBank accession numbers: ITS (OQ300392), LSU (OQ286292), RPB2 (OQ435243), and TEF1 (OQ441168); ibid., Leg. Aïgnon HL., voucher (HLA0792), GenBank accessions: ITS (OQ300393), LSU (OQ286293), RPB2 (OQ435245), and TEF1 (OQ441170); ibid., Borgou Province, Tchaourou region, Okpara forest, 9.2446277°N, 2.7262333°E, on soil in woodlands dominated by Isoberlina doka, August 18, 2022, leg. HL. Aïgnon, specimen voucher (HLA0917); ibid., leg. HL. Aïgnon, specimen voucher (HLA0920); ibid., August 22, 2017, leg. HL. Aïgnon, specimen voucher (HLA0456); ibid., Wari Maro, Forêt classée de Wari Maro, 9.164733°N, 2.159917°E, on soil in woodlands dominated by Isoberlina doka and Uapaca togoensis, June 20, 1998, leg. A. De Kesel, voucher (ADK2166, deposited at BR, BR 5020112676592); ibid., Zou Province, Savalou, near Ouèssè, Reserve forest of Gbadji, 7.952167°N, 1.967867°E, on soil close to an inselberg, in a woodland dominated by Isoberlina doka and Uapaca togoensis, June 19, 1997, leg. A. De Kesel, voucher (ADK2618, deposited at BR 5020115701772).
Reprinted from [AIGNON] under a CCBY license with permission from [AIGNON], original copyright [2022].
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 + 4.18331e4 (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.
Acknowledgments
We are grateful to Ms. Deng Lun-Sha and Mr. Gao Jia-Long for their help with sequencing and detection.
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