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. 2019 Aug 23;57:113–127. doi: 10.3897/mycokeys.57.35448

Diaporthe species in south-western China

Hui Long 1, Qian Zhang 1, Yuan-Yuan Hao 4, Xian-Qiang Shao 5, Xiao-Xing Wei 6, Kevin D Hyde 7, Yong Wang 1,2,, De-Gang Zhao 2,3
PMCID: PMC6717119  PMID: 31523165

Abstract Abstract

Three strains of the genus Diaporthe were isolated from different plant hosts in south-western China. Phylogenetic analyses of the combined ITS, β-tubulin, tef1 and calmoudulin dataset indicated that these strains represented three independent lineages in Diaporthe. Diaporthe millettiaesp. nov. clustered with D. hongkongensis and D. arecae, Diaporthe osmanthisp. nov. grouped with D. arengae, D. pseudomangiferae and D. perseae and Diaporthe strain GUCC9146, isolated from Camellia sinensis, was grouped in the D. eres species complex with a close relationship to D. longicicola. These species are reported with taxonomic descriptions and illustrations.

Keywords: Diaporthe , phylogeny, taxonomy, 2 new taxa

Introduction

Genus Diaporthe has been well-studied in recent years by Udayanga et al. (2011, 2012), incorporating morphological and molecular data and recommending appropriate genes to resolve species limitations in the genus. Since these revolutionary papers, 43 novel Diaporthe species have been described from China with morphological and phylogenetic evidence (Huang et al. 2013, 2015; Fan et al. 2016; Gao et al. 2014, 2015, 2016, 2017; Yang et al. 2017a,b, 2018; Yang et al. 2016; Du et al. 2016; Dissanayake et al. 2017a). Dissanayake et al. (2017b) provided an update of the genus with additional 15 species (7 new and 8 known species) from Italy based on molecular evidence. New records and species have also been reported by Hyde et al. (2016), Rossman et al. (2016), Chen and Kirschner (2017), Guarnaccia et al. (2018), Perera et al. (2018), Tibpromma et al. (2018) and Wanasinghe et al. (2018).

Three strains of Diaporthe were isolated from different medicinal plants collected in Guizhou and Guangxi during a survey of fungal diversity in south-western China. All the strains produced conidiomata containing alpha- and beta-conidia, typical of Diaporthe. This paper describes these three collections using molecular evidence, based on the analysis of combined ITS, β-tubulin, tef1 and calmoudulin datasets, as Diaporthe millettiae sp. nov. and D. osmanthi sp. nov. and D. longicicola with a new host record from Camellia sinensis.

Materials and methods

Isolation and morphological studies

The samples were collected from Guizhou and Guangxi provinces. The Diaporthe strains were isolated using the single-spore method (Chomnunti et al. 2014). Colonies, growing from single spores, were transferred to potato-dextrose agar (PDA) and incubated at room temperature (28 °C). Following 2–3 weeks of incubation, morphological characters were recorded as in Udayanga et al. (2011, 2015). Conidia and conidiophores were observed using a compound microscope (Olympus BX53). The holotype specimens are deposited in the Herbarium of Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). Ex-type cultures are deposited in the Culture Collection at the Department of Plant Pathology, Agriculture College, Guizhou University, China (GUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org) and Facesoffungi (http://www.facesoffungi.org).

DNA extraction and sequencing

Fungal cultures were grown on PDA medium until they nearly covered the whole Petri-dish (90 mm diam.) at 28 °C. Fresh fungal mycelia were scraped from the surface with sterilised scalpels. A BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416) was used to extract fungal genome DNA. DNA amplification was performed in a 25 μl reaction volume system which contained 2.5 μl 10 × PCR buffer, 1 μl of each primer (10 μM), 1 μl template DNA and 0.25 μl Taq DNA polymerase (Promega, Madison, WI, USA). Primers ITS4 and ITS5 (White et al. 1990) were used to amplify the ITS region. Three protein-coding gene fragments (β-tubulin, tef1 and calmoudulin) were amplified by the primers Bt2a/Bt2b (Glass and Donaldson 1995), CAL228F/CAL737R and EF1-728F/EF1-986R (Carbone and Kohn 1999). Gene sequencing was performed with an ABI PRISM 3730 DNA autosequencer using either a dRhodamine terminator or Big Dye Terminator (Applied Biosystems Inc., Foster 19 City, California). The sequences of both strands of each fragment were determined for sequence confirmation. The DNA sequences were submitted to GenBank and their accession numbers were provided in Table 1.

Table 1.

GenBank accession numbers of isolates include in this study.

Species Culture no. GenBank no.
ITS tef1 β-tubulin calmoudulin
Diaporthe alleghaniensis CBS 495.72 KC343007 KC343733 KC343975 KC343249
D. ambigua CBS 114015 AF230767 GQ250299 KC343978 KC343252
D. anacardii CBS 720.97* KC343024 KC343750 KC343992 KC343266
D. arecae CBS 161.64 KC343032 KC343758 KC344000 KC343274
D. arengae CBS 114979 KC343034 KC343760 KC344002 KC343276
D. baccae CBS 136972 KJ160565 KJ160597 MF418509 MG281695
D. beilharziae BRIP 54792 JX862529 JX862535 KF170921
D. betulae CFCC 50470 KT732951 KT733017 KT733021 KT732998
D. bicincta CBS 121004 KC343134 KC343860 KC344102 KC343376
D. biguttusis CGMCC 3.17081 KF576282 KF576257 KF576306
D. celastrina CBS 139.27 KC343047 KC343773 KC344015 KC343289
D. celeris CBS 143349 MG281017 MG281538 MG281190 MG281712
D. charlesworthii BRIP 54884m* KJ197288 KJ197250 KJ197268
D. cinerascens CBS 719.96 KC343050 KC343776 KC344018 KC343292
D. cotoneastri CBS 439.82 FJ889450 GQ250341 JX275437 JX197429
D. decedens CBS 109772 KC343059 KC343785 KC344027 KC343301
D. elaeagni CBS 504.72 KC343064 KC343790 KC344032 KC343306
D. ellipicola CGMCC 3.17084 KF576270 KF576245 KF576291
D. eres CBS 138594 KJ210529 KJ210550 KJ420799 KJ434999
D. foeniculina CBS 187.27 KC343107 KC343833 KC344075 KC343349
D. goulteri BRIP 55657a KJ197289 KJ197252 KJ197270
D. helianthi CBS 592.81 KC343115 GQ250308 KC343841 JX197454
D. hongkongensis CBS 115448 KC343119 KC343845 KC344087 KC343361
D. inconspicua CBS 133813 KC343123 KC343849 KC344091 KC343365
D. longicicola GUCC9146 MK398676 MK480611 MK502091 MK502088
D. longicicola CGMCC 3.17091 KF576267 KF576242 KF576291
D. macinthoshii BRIP 55064a* KJ197290 KJ197251 KJ197269
D. millettia GUCC9167 MK398674 MK480609 MK502089 MK502086
D. oncostoma CBS 589.78 KC343162 KC343888 KC344130 KC343404
D. osmanthusis GUCC9165 MK398675 MK480610 MK502090 MK502087
D. perseae CBS 151.73 KC343173 KC343899 KC344141 KC343415
D. phragmitis CBS 138897 KP004445 KP004507
D. pseudomangiferae CBS 101339 KC343181 KC343907 KC344149 KC343423
D. pseudophoenicicola CBS 462.69 KC343184 KC343910 KC344152 KC343426
D. rosicola MFLU 17.0646 NR157515 MG829270 MG843877 MG829274
D. saccarata CBS 116311 KC343190 KC343916 KC344158 KC343432
D. stitica CBS 370.54 KC343212 KC343938 KC344180 KC343454
D. vaccinii CBS 160.32 AF317578 GQ250326 KC344196 KC343470
Valsa ambiens CFCC 89894 KR045617 KU710912 KR045658

Ex-type isolates were labeled with bold.

Phylogenetic analyses

DNA sequences from our three strains and reference sequences downloaded from GenBank (Dissanayake et al. 2017a, b), Guarnaccia et al. (2018) and Wanasinghe et al. (2018) were analysed by maximum parsimony (MP) and maximum likelihood (ML). Sequences were optimised manually to allow maximum alignment and maximum sequence similarity, as detailed in Manamgoda et al. (2012). MP analyses were performed in PAUP v. 4.0b10 (Swofford 2003), using the heuristic search option with 1,000 random taxa additions and tree bisection and re-connection (TBR) as the branch swapping algorithm. Maxtrees = 5000 was set to build the phylogenetic tree. The characters of the alignment document were ordered according to ITS+tef1+β-tubulin+CAL for GUCC9165 and GUCC9167 and tef1+β-tubulin for GUCC9146 with equal weight and gaps were treated as missing data. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated for each tree generated. The resulting Phylip file was used to make ML and Bayesian trees by the CIPRES Science Gateway (https://www.phylo.org/portal2/login.action) and RAxML-XSEDE with 1000 bootstrap inferences.

Results

Phylogenetic analyses

Three Diaporthe strains isolated from different plant hosts were sequenced. PCR products of 456–465 bp (ITS), 292–303 bp (tef1), 666–690 bp (β-tubulin) and 336–345 bp (CAL) were obtained. By alignment with the single gene region and then combination according to the order of ITS, tef1, β-tubulin and CAL with Valsa ambiens (CFCC 89894), only 1833 characters were obtained, viz. ITS: 1–492, tef1: 493–801, β-tubulin: 802–1469, CAL: 1470–1833, with 500 parsimony-informative characters. This procedure yielded eleven parsimonious trees (TL = 2169, CI = 0.58, RI = 0.71, RC = 0.41 and HI = 0.42), the first one being shown in Figure 1. All Diaporthe species clustered together, although without credible support for bootstrap and BPP values (Figure 1). Phylogenetic analysis of strains GUCC9165 and GUCC9167, using the four gene loci, confirmed them as well-resolved species (Figure 1). Strain GUCC 9165 formed an independent branch adjacent to D. arecae and D. hongkongensis (MP: 100%, ML: 94% and BPP: 1). Strain GUCC 9167 grouped with the branch which included D. arengae, D. perseae and D. pseudomangiferae (MP: 92%, ML: 98% and BPP: 1). Strain GUCC 9146 was aligned to the branch having D. longicicola and D. rosicola in the Diaporthe eres species-complex (Figure 2), with high statistical support (MP: 84%, ML: 93% and BPP: 1). This strain also showed a close relationship to D. eres and D. cotoneastri. In addition, we also compared the DNA base pair differences between our strains and related species in different gene regions (Suppl. material 1: Table S1). In Diaporthe strain GUCC9165, the four genes had 64 base pair differences with D. arecae and 119 with D. hongkongensis, the main differences being with β-tubulin and tef1. Strain GUCC9167 had 52 base pair differences with D. arengae, 61 with D. perseae and 64 with D. pseudomangiferae, wherein the base distinction was primarily in the β-tubulin and tef1 gene region. The β-tubulin sequences of D. eres and D. longicicola were apparently shorter than in strain GUCC 9146. The CAL sequences of D. rosicola were shorter than GUCC 9146. The DNA sequence of CAL for Diaporthe longicicola was not available (Gao et al. 2015). Integrating available DNA information, we discovered that 28 base pair differences were shown between GUCC 9146 and D. eres, 51 between GUCC 9146 and D. cotoneastri, 26 between GUCC 9146 and D. rosicola and 22 (only three genes) between GUCC 9146 and D. longicicola. Meanwhile, the phylogenetic analysis, based on only tef1 and β-tubulin for the D. eres species-complex (Figure 2), also indicated that GUCC 9146 clustered with D. longicicola and D. rosicola which obtained support values of MP: 99%, ML: 100% and BPP: 1 and maintained a closer relationship with D. longicicola.

Figure 1.

Figure 1.

Parsimonious tree obtained from a combined analyses of an ITS, β-tubulin, calmoudulin and tef1 sequence dataset. MP, ML above 50% and BPP values above 0.90 were placed close to topological nodes and separated by “/”. The bootstrap values below 50% and BPP values below 0.90 were labelled with “-”. The tree is rooted with Valsa ambiens (CFCC89894). The branch of our new Diaporthe species is in pink.

Figure 2.

Figure 2.

Parsimonious tree obtained from a combined analyses of a β-tubulin and tef1 sequence dataset (TL = 265, CI = 0.89, RI = 0.76, RC = 0.68 and HI = 0.11). MP, ML above 50% and BPP values above 0.90 were placed close to topological nodes and separated by “/”. The bootstrap values below 50% and BPP values below 0.90 were labelled with “-”. The tree is rooted with Diaporthe decedens (CBS 109772).

Taxonomy

Diaporthe millettiae

H. Long, K.D. Hyde & Yong Wang bis sp. nov.

18E3940B229E5A739D4216010B5CBA6D

MB829563

Figure 3

Figure 3.

Figure 3.

Diaporthe millettiae (GUCC9167). a–b upper (a) and lower (b) surface of colony on PDAc–d conidiomata e–f conidiophores, conidiogenous loci and conidia g β-conidia h α-conidia. Scale bars: 20 µm (e, f), 10 µm (g, h).

Diagnosis.

Characterised by larger J-shaped β-conidia.

Type.

China, Guangxi Province, Nanning City, from leaves of Millettia reticulata, 20 September 2016, Y. Wang, HGUP 9167, holotype, ex-type living culture GUCC 9167.

Description.

Colonies on PDA attaining 9 cm diam. after 10 days; coralloid with feathery branches at margin, adpressed, with apparent aerial mycelium, with numerous irregularly zonate dark stromata, isabelline becoming lighter towards the margin; reverse similar to surface, with zonations. Conidiomata pycnidial, multilocular, scattered, abundant on PDA after 3 wks, subglobose to irregular, 1.5–1.8 mm diam., ostiolate, with up to 1 mm necks when present. Conidiophores formed from the inner layer of the locular wall, sometimes reduced to conidiogenous cells, when present 1-septate, hyaline to pale yellowish-brown, cylindrical, 10–23 × 1–2.5 μm. Conidiogenous cells cylindrical to flexuous, tapered towards apex, hyaline, 8–18 × 1.5–3 μm. Alpha conidia abundant, fusiform, narrowed towards apex and base, mostly biguttulate, hyaline, 4.5–9 × 2–3.5 μm. Beta conidia scarce to abundant, flexuous to J-shaped, hyaline, 17.5–32 × 1–2 μm. Perithecia not seen.

Habitat and distribution.

Isolated from leaves of Millettia reticulata in China

Etymology.

Species epithet millettiae, referring to the host, Millettia reticulata from which the strain was isolated.

Notes.

Phylogenetic analysis combining four gene loci showed that Diaporthe millettiae (strain GUCC 9167) displayed a close relationship with D. arengae, D. pseudomangiferae and D. perseae with high bootstrap values (Figure 1). We compared the DNA base pair differences of the four gene regions, the main differences being in the β-tubulin and tef1 genes, especially tef1. Diaporthe millettiae produced two types of conidia (α, β), whereas D. pseudomangiferae only produced alpha conidia and D. perseae produced three types of conidia (α, β, γ). The β-conidia of D. arengae were smaller (20–25 × 1.5 μm) than those of Diaporthe millettiae (17.5–32 × 1–2 μm). The shape of β-conidia was also different. Conidiophores of D. arengae (10–60 μm) with more septa (0–6), were longer than those of D. millettiae (10–23 × 1–2.5 μm; 0-1-septate) (Gomes et al. 2013).

Diaporthe osmanthi

H. Long, K.D. Hyde & Yong Wang bis sp. nov.

AB53D9B53F1B5833915C42D14348045C

MB829564

Figure 4

Figure 4.

Figure 4.

Diaporthe osmanthi (GUCC9165). a–b upper (a) and lower (b) surface of colony on PDAc–d conidiomata e conidiophores, conidiogenous loci and conidia f α-conidia g two types of conidia h β-conidia. Scale bars: 10 µm (e, f, g, h).

Diagnosis.

Characterised by size of α-conidia and β-conidia.

Type.

China, Guangxi province, Nanning City, from leaves of Osmanthus fragrans, 20 September, 2016, Y. Wang, HGUP 9165, holotype, ex-type living culture GUCC 9165.

Description.

Colonies on PDA attaining 9 cm diam. after 10 days; coralloid with feathery branches at margin, adpressed, without aerial mycelium, with numerous irregularly zonated dark stromata, isabelline becoming lighter towards the margin; reverse similar to the surface with zonations more apparent. Conidiomata pycnidial and multilocular, scattered, abundant on PDA after 3 wks, globose, subglobose or irregular, up to 1–1.5 mm diam., ostiolate, necks absent or up to 1 mm. Conidiophores formed from the inner layer of the locular wall, reduced to conidiogenous cells or 1-septate, hyaline to pale yellowish-brown, cylindrical, 20.5–61 × 1–3 μm. Conidiogenous cells cylindrical to flexuous, tapered towards apex, hyaline, 10–15 × 1.5–3 μm. Alpha conidia abundant, fusiform, narrowed towards the apex and base, apparently biguttulate, hyaline, 5.5–8.5 × 2–3 μm. Beta conidia scarce to abundant, flexuous to J-shaped, hyaline, 20–31.5 × 1–2.5 μm. Perithecia not seen.

Habitat and distribution.

Isolated from leaves of Osmanthus fragrans in China.

Etymology.

Species epithet osmanthi, referring to the host, Osmanthus fragrans from which our strain was isolated.

Notes.

Diaporthe osmanthi (strain GUCC9165) formed an independent lineage, but was also related to D. arecae and D. hongkongensis (Figure 1). The sequences of β-tubulin and tef1 included about two-three differences between D. osmanthi (GUCC9165) and D. arecae (42) and D. hongkongensis (78) and thus they were different species according to the guidelines of Jeewon and Hyde (2016). Additionally, Diaporthe hongkongensis produced three types of conidia, but Diaporthe osmanthi did not produce γ-conidia. In addition, β-conidia of D. hongkongensis (18–22 μm) were shorter than those of Diaporthe osmanthi (Gomes et al. 2013). According to original description Srivastava et al. (1962), D. arecae also produced two types of conidia. The α-conidia (7.2–9.6 × 2.4 μm) were longer than in Diaporthe osmanthi, but its β-conidia (14.4–24 × 1.2 μm) were shorter and their shape also had some differences.

Diaporthe longicicola

Y.H. Gao & L. Cai, Fungal Biology 119(5): 303 (2015)

6147DDC1497558DB8A559F58761A6EC6

Figure 5

Figure 5.

Figure 5.

Diaporthe longicicola (GUCC9146). a–b upper (a) and lower (b) surface of colony on PDAc–d conidiomata e two types of conidia f conidiophores, conidiogenous loci and conidia g α-conidia h β-conidia. Scale bars: 10 µm (e, f, g, h).

Description.

Colonies on PDA attaining 9 cm diam. in 10 days; coralloid with feathery branches at margin, adpressed, without aerial mycelium, without numerous irregularly zonated dark stromata, isabelline becoming lighter towards the margin; reverse similar to the surface with zonations more apparent. Conidiomata pycnidial and multilocular, scattered, abundant on PDA after 20 d, subglobose or irregular, 1.5–1.8 mm diam., ostiolate and up to 1 mm long. Conidiophores formed from the inner layer of the locular wall, densely aggregated, hyaline to pale yellowish-brown, cylindrical, tapering towards the apex, 15–25 × 1.5–2 μm. Alpha conidia abundant, ellipsoid to fusiform, apparently biguttulate, hyaline, 6–9 × 2–3 μm. Beta conidia scarce to abundant, flexuous to J-shaped, hyaline, 25.5–35.5 × 1–2.5 μm.

Habitat and distribution.

Isolated from leaves of Camellia sinensis in Duyun, Guizhou Province, China

Notes.

Phylogenetic analyses (Figures 1, 2) indicated that GUCC 9146 has a close relationship with D. longicicola, D. rosicola, D. eres and D. cotoneastri. Morphological comparison indicated that this strain was most similar to D. longicicola but not a related species by the width of alpha conidia and length of beta conidia (Udayanga et al. 2014; Gao et al. 2015).

Discussion

Phylogenetic analysis and morphology provide evidence for the introduction of Diaporthe millettiae and D. osmanthi as new species. In order to support the validity of these new species, we followed the guidelines of Jeewon and Hyde (2016) in comparing base pair differences (Suppl. material 1: Table S1). In accordance with Udayanga et al. (2014), we also believed that the ITS fragment was problematic for the D. eres species-complex. When not considering ITS, integration with morphological comparison was helpful and we concluded that GUCC 9146 is D. longicicola. Diaporthe longicicola was firstly reported on Lithocarpus glabra in Zhejiang Province, but our strain (GUCC 9146) was recovered from Camellia sinensis in Guizhou Province. Thus, this is the report of a new host and new location in China for D. longicicola.

Supplementary Material

XML Treatment for Diaporthe millettiae
XML Treatment for Diaporthe osmanthi
XML Treatment for Diaporthe longicicola

Acknowledgements

This research is supported by the project funding of National Natural Science Foundation of China (No. 31560489), Genetically Modified Organisms Breeding Major Projects of China [2016ZX08010-003-009], Agriculture Animal and Plant Breeding Projects of Guizhou Province [QNYZZ2013-009], Key Laboratory of Superior Forage Germplasm in the Qinghai-Tibetan Plateau (2017-ZJ-Y12), Talent project of Guizhou science and technology cooperation platform ([2017]5788-5 and [2019]5641) and Guizhou science, technology department international cooperation base project ([2018]5806) and postgraduate education innovation programme of Guizhou Province (ZYRC[2014]004). Dr Kevin D. Hyde would like to thank “the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (DBG6080013)” and “Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (RDG6130001)”.

Citation

Long H, Zhang Q, Hao Y-Y, Shao X-Q, Wei X-X, Hyde KD, Wang Y, Zhao D-G (2019) Diaporthe species in south-western China. MycoKeys 57: 113–127. https://doi.org/10.3897/mycokeys.57.35448

Funding Statement

The research is supported by the project of National Natural Science Foundation of China (No. 31560489), Genetically Modified Organisms Breeding Major Projects of China [2016ZX08010-003-009], Agriculture Animal and Plant Breeding Projects of Guizhou Province [QNYZZ2013-009], Key Laborary of Superior Forage Germplasm in the Qinghai-Tibetan Plateau (2017-ZJ-Y12), Talent project of Guizhou science and technology cooperation platform ([2017]5788-5) and Guizhou science, technology department international cooperation base project ([2018]5806) and postgraduate education innovation program of Guizhou Province (ZYRC[2014]004). Dr Kevin D. Hyde also would like to thank “the future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (DBG6080013)” and “Impact of climate change on fungal diversity and biogeography in the Greater Mekong Subregion (RDG6130001)”.

Supplementary materials

Supplementary material 1

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Hui Long, Qian Zhang, Yuan-Yuan Hao, Xian-Qiang Shao, Xiao-Xing Wei, Kevin D. Hyde, Yong Wang, De-Gang Zhao

The DNA bases difference between our strains and related taxa on four gene regions

Data type: molecular data

mycokeys-57-113-s001.docx (16.7KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Diaporthe millettiae
XML Treatment for Diaporthe osmanthi
XML Treatment for Diaporthe longicicola
Supplementary material 1

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Hui Long, Qian Zhang, Yuan-Yuan Hao, Xian-Qiang Shao, Xiao-Xing Wei, Kevin D. Hyde, Yong Wang, De-Gang Zhao

The DNA bases difference between our strains and related taxa on four gene regions

Data type: molecular data

mycokeys-57-113-s001.docx (16.7KB, docx)

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