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Metarhizium baoshanense sp. nov., a New Entomopathogen Fungus from Southwestern China

PJZ_50_5_1739-1746

 

 

Metarhizium baoshanense sp. nov., a New Entomopathogen Fungus from Southwestern China

Zihong Chen1, Ling Xu2, Xiaona Yang2, Yaguan Zhang3* and Yuming Yang4

1Institute of Biological Resources of Gaoligong Mountains, Baoshan University, Baoshan 678000 Yunnan, China

2College of Resources and Environment, Baoshan University, Baoshan 678000, Yunnan, China

3College of Chemistry and Environmental Science, Qujing Normal University, Qujing 655011Yunnan, China

4Yunnan Province Key Laboratory of Forest Plant Cultivation and Utilization, Yunnan Academy of Forestry, Kunming 650201 Yunnan, China

ABSTRACT

Metarhizium is an entomopathogenic fungal genus relevant in biological protection. A new species of the genus, collected from Taibao mountain, Baoshan City, Yunnan Province, southwestern China, was described here as Metarhizium baoshanense. It was proposed and determined based on morphological characters combined with a multigene phylogenetics analysis involving 5.8S–ITS, nrSSU, nrLSU, EF–1α, RPB1 and RPB2. In multilocus phylogeny, M. baoshanense was grouped as a sister clade to Metarhizium majus, Metarhizium guizhouense, Metarhizium lepidiotae and Metarhizium indigoticum, but conclusively separated from these allied species with high support value. Morphological character differentiated M. baoshanense from its relatives on that its conidial size was obviously smaller than M. majus and M. indigoticum, a little shorter than M. guizhouense and thicker than M. lepidiotae. Still the new species was characterized by that its colony colour became slightly shallow with culture time on PDA plates at 25°C, being grayish green at 12 d and turning gray black at 30 d of cultivation.


Article Information

Received 23 May 2018

Revised 30 June 2018

Accepted 12 July 2018

Available online 03 August 2018

Authors’ Contribution

ZC designed the study, performed experimental work and analyzed the data. LX performed the experiment, analyzed the data and wrote the manuscript. XY helped in DNA extraction and PCR. YZ helped in morphological evaluation. YY helped in paper writing.

Key words

Metarhizium, Morphology character, Multigene phylogenetics, Taxonomy.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.5.1739.1746

* Corresponding author: zhangyaguan2013@163.com

0030-9923/2018/0005-1739 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan



Introduction

 

Metarhizium is one of the ubiquitous anamorphic genera of entomopathogenic fungi (Driver et al., 2000). Entomophthoro anisopliae Metsch was described from Russia as a pathogen of wheat cockchafer (Anisolia austriaca Hbs.) and turned to be a type species of a new genus Metarhizium (Metsch.) Sorokin. Teleomorph of Metarhizium was firstly confirmed as Cordyceps s. l. by Liang et al. (1991) and then assigned to a new genus Metacordyceps by Sung et al. (2007).

A high number of species and varieties of Metarhizium had been discovered in recent decades and their convergent morphologies hampered the taxonomic studies. Molecular evidence enabled great resolution of fungi taxonomy than allowed by morphology alone, and mutigene phylogenetic approaches particularly urged/forced the clarification of the genetic relationship at species rank (Kepler et al., 2012; Nonaka et al., 2013). Based on 4-gene (EF–1α, RPB1, RPB2 and β–tubulin) dataset, Bischoff et al. (2009) revealed that the Metarhizium anisopliae complex included 9 species. Based on 5–gene (nrSSU, nrLSU, EF–1α, RPB1 and RPB2) dataset, Kepler et al. (2012) identified that Metacordyceps contained 14 species and 1 varietas, and then Kepler et al. (2014) revised the boundaries of Metarhizium as including the majority of species recognized in Metacordyceps, green–spored species in Nomuraea, Chamaeleomyces and several species formerly in Paecilomyces.

During a survey of entomogenous fungi at Taibao mountain in Baoshan City, Yunnan Province, China in 2015, a new Metarhizium species, M. baoshanense was discovered. The objective of this study was to confirm its taxonomy position based on morphological characteristics and 6–locus phylogenetic analysis. The anamorph of the related species, M. indigoticum (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber, was firstly described.

 

Materials and methods

 

Fungal isolation and morphological evaluation

Soil specimens were collected from plant rhizosphere at mid–montane of humid evergreen broad–leaved forest in Taibao Mountain, Baoshan city, Yunnan Province in China. Fungi were isolated from soil by insect baiting method according to Keyser et al. (2015). Soil samples (~100 mL) were moistened to slightly damp in sterile glass canned bottles (150 mL). Ten healthy 6–7th instar Tenebrio molitor L. larvae were cultured in each bottle and checked every two days. Dead insects were transferred to a sterile petri dish with moist filter paper and maintained at 25°C for about 10 d. Metarhizium conidia were transferred to PDA plates and cultured at 25°C. Pure (axenic) strains were indentified morphologically and evaluated microscopically under a motic BA410 microscope. Morphological characteristics of the new species were compared with its related species among M. anisopliae complex. Anamorphic strains of the related species, M. indigoticum, were isolated from Gaoligong mountains and morphologically described.

DNA extraction and PCR

Genomic DNA was extracted from mycelia and conidia with the Plant Genomic DNA Purification Kit (Qiagen) following the manufacturer instructions. The partial sequences of 6 nuclear loci, including 5.8S–ITS, nrSSU, nrLSU, EF–1α, RPB1 and RPB2, were amplified. Primer pairs of the 6–locus were referred to Chen et al. (2013). PCR reactions were carried out in 20 μL reaction mixture containing 10 μL 2×EasyTaq PCR Supermix (TransGen Biotech, Beijing, China), 1 μL of each primer (10 µM), 1 μL of template DNA (1–2 ng) and 7μL sterile water. PCR programs of ITS–5.8S, nrSSU and nrLSU were referred to Chen et al. (2013) and that of EF–1α, RPB1 and RPB2 were referred to Bischoff et al. (2006).

Cloning and sequencing

PCR products were purified with Gel Purification Kit (Bioteke, Beijing, China) and the fragments were cloned with TaKaRa PMDTM18–T vector system (TaKaRa Bio, Dalian, China). DNA sequencing was performed at SinoGenoMax Co. Ltd., and the resulting datasets were submitted to GenBank.

Phylogenetic analysis

DNA sequences of 6–locus included 34 species, M. baoshanense, all dataset (30 species) in Metarhizium used by Kepler et al. (2014), Metarhizium martialis (Speg.) Kepler, G.H. Sung & Spatafora used by Kepler et al. (2012), Metarhizium shibinensis T.C. Wen, J.C. Kang & K.D. Hyde used by Wen et al. (2015) and Beauveria bassiana (Bals.) Vuill. as the outgroup. All sequences were retrieved from the GenBank with accession number detailed in Table I.

The combined dataset of 6–locus were aligned with Clustal X2.0 (Larkin et al., 2007). Ambiguous regions in both sides were excluded and gaps were treated as missing data in the subsequent phylogenetic analyses. Phylogenic consensus tree was analyzed with the program MEGA6 (Tamura et al., 2013). Maximum Likelihood (ML) estimation was performed with 1000 replicates. T92+G+I (Tamura 3–parameter + Gamma distribution (+G) with invariant sites (+I) nucleotide substitution model was taken as suggested by looking for a best–fit substitution model (ML). Clades supported with ML values of ≥70% were considered significantly supported by the data.

 

Table I.- Taxon information and GenBank accession numbers of the sequences used for phylogenetic analysis.

Species

GenBank accession number

nrSSU

nrLSU

EF-1α

RPB1

RPB2

5.8S-ITS

Metarhizium acridum

Data missing

Data missing

EU248844.1

EU248896.1

EU248924.1

HM055449.1

M. acridum

Data missing

Data missing

EU248845.1

EU248897.1

EU248925.1

NR132019.1

M. album

DQ518775.1

DQ522560.1

DQ522352.1

DQ522398.1

DQ522452.1

Data missing

M. album

Data missing

Data missing

KJ398807.1

KJ398618.1

Data missing

HM055452.1

M. anisopliae

Data missing

Data missing

EU248852.1

EU248904.1

EU248932.1

HQ331464.1

M. anisopliae

Data missing

Data missing

DQ463996.2

DQ468355.1

DQ468370.1

NR132017.1

M. baosha nense

KY264177

KY264174

KY264169

KY264180

KY264183

KY264172

M. baosha nense

KY264178

KY264175

KY264170

KY264181

KY264184

KY264173

M. baosha nense

KY264179

KY264176

KY264171

KY264182

KY264185

Data missing

M. brasiliense

Data missing

Data missing

KJ398809.1

KJ398620.1

Data missing

Data missing

M. brunneum

Data missing

Data missing

EU248855.1

EU248907.1

EU248935.1

NR132023.1

M. brunneum

Data missing

Data missing

EU248854.1

EU248906.1

EU248934.1

HQ331451.1

M. carneum

EF468843.1

EF468988.1

EF468789.1

EF468894.1

EF468938.1

NR131993.1

M. cylindros porum

Data missing

Data missing

KJ398814.1

KJ398625.1

Data missing

AF368270.1

Species

GenBank accession number

nrSSU

nrLSU

EF-1α

RPB1

RPB2

5.8S-ITS

Metarhizium flavoviride

Data missing

Data missing

DQ463999.1

DQ468358.1

DQ468373.1

Data missing

M. flavoviride

Data missing

AF138269.1

KJ398804.1

KJ398614.1

DQ468374.1

AF138269.1

M. frigidum

Data missing

Data missing

KJ398818.1

KJ398628.1

Data missing

Data missing

M. frigidum

Data missing

Data missing

DQ464002.1

DQ468361.1

DQ468376.1

NR132012.1

M. globosum

Data missing

Data missing

EU248846.1

EU248898.1

EU248926.1

NR132020.1

M. granulomatis

HM635078.1

Data missing

KJ398782.1

KJ398593.1

Data missing

HM195306.1

M. granulomatis

HM195304.1

HM635076.1

KJ398781.1

Data missing

Data missing

NR132013.1

M. guizhouense

Data missing

Data missing

EU248862.1

EU248914.1

EU248942.1

HQ331448.1

M. guizhouense

Data missing

Data missing

EU248857.1

EU248909.1

EU248937.1

HQ331447.1

M. indigoticum

JF415968.1

JF415952.1

JF416010.1

JN049886.1

JF415992.1

JN049874.1

M. indigoticum

JF415952.1

JF415969.1

JF416011.1

JN049887.1

JF415993.1

JN049875.1

M. indigoticum

KY264192

KY264190

KY264186

KY264194

KY264196

KY264188

M. indigoticum

KY264193

KY264191

KY264187

KY264195

KY264197

KY264189

M. khaoyaiense

JF415971.1

Data missing

KJ398796.1

JN049889.1

Data missing

Data missing

M. khaoyaiense

JF415970.1

Data missing

KJ398797.1

JN049888.1

Data missing

JN049869.1

M. koreanum

Data missing

Data missing

KJ398806.1

KJ398616.1

Data missing

Data missing

M. koreanum

Data missing

Data missing

KJ398805.1

KJ398615.1

Data missing

Data missing

M. kusanagi ensis

JF415972.1

JF415954.1

JF416014.1

JN049890.1

Data missing

JN049873.1

M. lepidiotae

Data missing

Data missing

EU248864.1

EU248916.1

EU248944.1

HQ331455.1

M. majus

Data missing

Data missing

EU248867.1

EU248919.1

EU248947.1

HM055450.1

M. majus

Data missing

Data missing

KJ398801.1

KJ398610.1

Data missing

HQ331445.1

M. marquandii

EF468845.1

EF468990.1

EF468793.1

EF468899.1

EF468942.1

NR131994.1

M. martialis

JF415956.1

JF415975.1

JF416016.1

JN049892.1

JF415995.1

JN049881.1

M. minus

AF280635.1

AF280632.1

DQ464006.1

KJ398609.1

DQ468380.1

HM055453.1

M. minus

AF339531.1

AF339580.1

DQ464007.1

DQ468366.1

DQ468381.1

AF138271.1

M. novozeala ndicum

Data missing

Data missing

KJ398811.1

KJ398622.1

Data missing

Data missing

M. novozeala ndicum

Data missing

Data missing

KJ398812.1

KJ398623.1

Data missing

Data missing

M. owariense

HQ165730.1

HQ165669.1

HQ165689.1

HQ1665747.1

Data missing

HQ165712.1

M. pemphigi

Data missing

Data missing

KJ398819.1

KJ398629.1

DQ468379.1

Data missing

M. pemphigi

Data missing

Data missing

KJ398813.1

KJ398624.1

DQ468378.1

Data missing

M. pinghaense

Data missing

Data missing

EU248850.1

EU248902.1

EU248930.1

NR077205.1

M. pinghaense

Data missing

Data missing

EU248851.1

EU248903.1

EU248931.1

HQ331454.1

M. pseudoatr ovirens

JF415977.1

Data missing

Data missing

JN049893.1

JF415997.1

JN049870.1

M. rileyi

Data missing

Data missing

KJ398803.1

KJ398613.1

Data missing

Data missing

M. rileyi

Data missing

AY526491.2

EF468787.1

EF468893.1

EF468937.1

NR119513.1

M. robertsii

Data missing

Data missing

DQ463994.1

DQ468353.1

DQ468368.1

HQ331453.1

M. viride

Data missing

Data missing

KJ398808.1

KJ398619.1

Data missing

EU553291.1

M. viride

HQ165735.1

HQ165673.1

HQ165692.1

Data missing

HQ165652.1

HQ165714.1

M. viridulum

Data missing

Data missing

KJ398815.1

KJ398626.1

Data missing

Data missing

M. yongmu nense

Data missing

EF468979

EF468834

EF468769

EF468877

Data missing

M. yongmu nense

EF468833.1

EF468977.1

EF468770.1

EF468876.1

Data missing

JN049856.1

M. shibinensis

KR153588.1

Data missing

KR153589.1

KR153590.1

Data missing

KR153585.1

Beauveria bassiana

HQ880975.1

HQ880975.1

HQ880975.1

HQ880834.1

HQ880834.1

HQ880762.1


 

Results

 

Phylogenetic analyses

BLAST analysis revealed that 6 genes of M. baoshanense respectively shared sequences identities with different species of Metarhizium and Metacordyceps, ITS 98% with M. anisopliae M3419 (FJ609313.1), EF–1α 99% with M. majus ARSEF 7505 (EU248870.1), RPB1 99% with M. indigoticum NBRC 100684 (KJ398595.1), RPB2 99% with Metacordyceps brittlebankisoides (KC429019.1), nrLSU and nrSSU 98% with Metacordyceps indigotica TNS–F 18554 (JF415953.1). It was hard to determine the species position with a single gene. Further multilocus analysis was employed to confirm the species taxonomic status.

The combined alignment of 6–locus dataset of Metarhizium comprised 5049 base pairs (5.8S–ITS: 717 bp; nrSSU: 1018 bp; nrLSU: 846 bp; EF–1α: 917 bp; RPB1: 713 bp; RPB2: 838 bp). Phylogenetic tree of combined 6–locus for 34 species revealed similar affiliation inferred from 5 nuclear loci by Kepler et al. (2014) (Fig. 1). M. baoshanense was clustered in M. anisopliae complex which contained 11 individuals. The 3 strains (CCTCC M 2016589, BUM 63.4 and BUM 1700) of M. baoshanense were clustered in the same clade with M. majus, M. indigoticum, M. guizhouense Q.T. Chen & H.L. Guo and M. lepidiotae (Driver & Milner) J.F. Bisch., Rehner & Humber, but formed a distinct clade and was clearly separated from the allied species with high support value.

The position of the strains BUM 2600 and BUM 1512.8 isolated from Gaoligong mountains was also confirmed. Their 6–locus dataset was clustered in the same clade with the reported sequences (TNS–F18553 and NBRC 100684) of M. indigoticum, illustrating that they were the anamorph stage of M. indigoticum and their morphological data in this study was credible.

Morphological comparison of M. Baoshanense and its related species

M. baoshanense was obviously distinguished from M. majus and M. indigoticum for the conidial feature, M. majus possessing the biggest conidia (8.5–13.0×3.0–5.0 µm) in M. anisopliae complex and M. indigoticum (8.2–12.3×2.6–4.2 µm) being next to it, much greater than M. baoshanense (6.7–8.5×2.6–3.3 µm) (Table II). The conidia of M. baoshanense were close to M. lepidiotae (6.0–7.5×2.0–3.0 µm) for the length and a little shorter than M. guizhouense (7.0–10.0×2.0–3.0 µm), while being a little thicker than M. guizhouense and M. lepidiotae (Table II; Fig. 2I-L).

 

Table II.- Morphological characterscomparison among Metarhiziumanisopliae complex.

Species

Strain

Geography

Conidia(µm)

Phialides(µm)

Reference

Metarhizium majus

ARSEF 1914

Philippines

10.5-13.0 ×2.5-4.0

10.0-18.0 ×2.5-3.5

Bischoof et al. (2009)

M. majus

ARSEF 7505

Australia

8.5-13.0 ×3.0-5.0

9.0-18.5 ×2.5-4.5

Bischoof et al. (2009)

M. indigoticum

BUM 1512.8

China

8.2-12.3 ×2.6-4.2

(9.7±1.0 ×3.1±0.6)

8.5-20.5 ×1.7-3.5

(13.7±3.7 ×2.5±0.6)

This study

M. guizhouense

ARSEF 4321

Australia

5.5-8.0 ×2.5-3.5

8.0-12.0 ×2.5-3.5

Bischoof et al. (2009)

M. guizhouense

CBS 258.90

China

7.0-10.0 ×2.0-3.0

7.0-12.0 ×2.0-3.0

Bischoof et al. (2009)

M. guizhouense

ARSEF 4321

Australia

5.5-8.0 ×2.5-3.5

8.0-12.0 × 2.5-3.5

Bischoof et al. (2009)

M. baoshanense

CCTCC M 2016589

China

6.7-8.5 ×2.6-3.3

(7.4±0.5× 2.9±0.3)

8.2-19.2 × 2.1-3.5

(11.9±3.3× 2.5±0.4)

This study

M. lepidiotae

ARSEF 4154

Australia

6.0-7.5× 2.0-3.0

6.5-12.0× 2.0-3.0

Bischoof et al. (2009)

M. lepidiotae

ARSEF 7488

Australia

5.0-7.0× 3.0-4.0

8.0-12.0× 2.5-3.5

Bischoof et al. (2009)

M. pingshaense

CBS 257.90

China

6.0-8.0 ×2.5-3.5

7.0-17.0× 2.5-3.5

Bischoof et al. (2009)

M. pingshaense

ARSEF 4342

Solomon islands

4.5-7.0 ×2.0-3.0

7.0-13.0× 2.0-3.0

Bischoof et al. (2009)

M. robertsii

ARSEF 7501

Australia

5.0-7.5 ×2.0-3.0

6.0-12.0× 2.0-3.0

Bischoof et al. (2009)

M. robertsii

ARSEF 727

Brazil

5.0-7.0 ×2.0-3.5

7.0-14.5× 2.5-3.5

Bischoof et al. (2009)

M. anisopliae

ARSEF 7487

Eritrea

5.0-7.0 ×2.0-3.5

8.0-11.5 ×2.0-3.0

Bischoof et al. (2009)

M. brunneum

ARSEF 4179

Australia

5.5-8.0 ×2.0-3.0

6.0-13.0 ×2.0-3.0

Bischoof et al. (2009)

M. brunneum

ARSEF 2107

USA

4.5-7.5 ×2.0-3.0

9.5-18.0 ×2.0-5.0

Bischoof et al. (2009)

M. brunneum

ARSEF 4152

Australia

5.0-7.0 ×2.5-3.5

7.5-13.5 ×2.5-4.0

Bischoof et al. (2009)

M. acridum

ARSEF 5748

Mexico

4.0-5.0 ×2.5-3.5

6.0-13.0 ×2.0-3.5

Bischoof et al. (2009)

M. acridum

ARSEF 7486

Niger

4.0-5.5 ×3.0-4.0

4.5-12.5 ×2.5-4.5

Bischoof et al. (2009)

M. globosum

ARSEF 2596

India

4.0-5.0 ×4.0-5.0

5.0-12.0 ×3.0-4.0

Bischoof et al. (2009)


 

The culture character was further conducted among M. baoshanense, M. lepidiotae, M. guizhouense and M. indigoticum (Fig. 2 A-D). The conidia layers of all the four species were white in early day, while showed apparent discrepancy with culture time. After 12 d of cultivation on PDA plates at 25°C, M. baoshanense was grayish green (Fig. 2A), M. guizhouense was blackish green (Fig. 2B), M. lepidiotae was light black (Fig. 2C) and M. indigoticum was dark black (Fig. 2D). After one month of cultivation, M. baoshanense and M. lepidiotae were gray black, M. guizhouense was dark green, and M. indigoticum was black.

Taxonomy

MycoBank number

MB 819373.

Species diagnosis

The new species is clustered with M. guizhouense, M. lepidiotae, M. majus and M. indigoticum in the same clade in the multiple gene (5.8S–ITS, nrSSU, nrLSU, EF–1α, RPB1 and RPB2) sequence analyses, but the conidial size of M. baoshanense was obviously smaller than M. majus and M. indigoticum, a little shorter than M. guizhouense and thicker than M. lepidiotae.

Species description

Colonies on PDA medium being 61 mm in diameter after 16 d at 25 °C, white at first, turning grayish green after 16 d of cultivation. Hyphae hyaline, septate, branched, smooth–walled, 2.0–3.2 (= 2.5±0.6) µm wide. Conidiophores solitary or branched. Phialides cylindrical, 8.2–19.2 × 2.1–3.5 (= 11.9±3.3 × 2.5±0.4) µm. Conidia forming columns in culture and hyaline, aseptate, smooth, long oval or cylindrical, grayish green en masse, 6.7–8.5 × 2.6–3.3 (= 7.4±0.5 × 2.9±0.3) µm.

Type: China. Yunnan Province: Taibao mountain, alt. 1740 m, 3 May 2015, Zihong Chen (Holotype, CCTCC M 2016589; ex–type culture, BUM 63.4)

Sexual state: Unknown.

Host: Unknown.

Habitat and distribution: The soil at mid–montane of humid evergreen broad–leaved forest in Taibao mountain, Baoshan city, Yunnan Province, China.

 

Discussion

 

Most species in Metarhizium are useful biological control agents and often act as regulators of insect populations (Kepler et al., 2014; Pattemore et al., 2014; Shoukat et al., 2018). Further understanding their diversity can provide new genetic resource for development the entomopathogen as a biopesticide. In this study, M. baoshanense was determined to be a new species in Metarhizium based on morphological study and molecular evidence.

Muti–locus phylogeny is routinely used to delimit species of Ascomycota (Wang et al., 2015). Six loci including 5.8S-ITS, nrSSU, nrLSU, EF-1α, RPB1 and RPB2 were performed analysis in this study for their wide usage in phylogenetic reconstructions of fungi (Kepler et al., 2012; Sanjuan et al., 2014; Wen et al., 2015). Multi-genes analyses involved almost all currently recognized species of Metarhizium and the resulted affiliation was consistent with the works of Kepler et al. (2014), being able to provide strong evidence for the taxonomic status of M. baoshanense. The 2 strains BUM 2600 and BUM 1512.8 were placed as M. indigoticum and the 3 strains of M. baoshanense were resolved as a novel clade in M. anisopliae complex, clearly separated from its allied species, M. majus, M. guizhouense, M. lepidiotae and M. indigoticum.

Cordyceps indigotica Kobayasi & Shimiz was firstly reported by Kobayasi and Shimizu (1978) and determined to be a member of Metacordyceps by Kepler et al. (2012). However, so far the asexual morphological description of M. indigoticum had been still missing. To better clarify the relationship of M. baoshanense and its related species, 2 strains from Gaoligong Mountains were confirmed as M. indigoticum by molecular analysis and morphologically compared with M. baoshanense (Table II). The conidial size of M. baoshanense was obviously smaller than M. majus and M. indigoticum, a little shorter than M. guizhouense, and a little thicker than Metarhizium lepidiotae. The colony color could well clarify the interspecific relationship, that of M. baoshanense being grayish green, obviously shallow than M. lepidiotae, M. guizhouense and M. indigoticum after 12 d of cultivation on PDA plates at 25 °C.

 

Conclusion

 

In this study, a new species of Metarhizium, M. baoshanense was determined based on morphological characters and 6-locus (5.8S–ITS, nrSSU, nrLSU, EF–1α, RPB1 and RPB2) molecular data. M. baoshanense was obviously separated from its sister species, M. majus, M. guizhouense, M. lepidiotae and M. indigoticum in phylogenetic tree and was also clearly morphologically distinguished from its relatives.

 

Acknowledgement

 

The authors wish to thank Dr. Yong–Dong Dai (Institute of Herbal Biotic Resources, Yunnan University, Yunnan, China) for his kindly help to analyse the multigene sequences. This work was funded by the National Natural Science Area Fund Projects of China (31460153), Key Project of Universities Joint Foundation in Yunnan province (2017FH001-126), Surface Project of Universities Joint Foundation OF Yunnan province (2017FH001-029).

 

Statement of conflict of interest

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

 

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Pakistan Journal of Zoology

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Vol. 50, Iss. 4, Pages 1199-1600

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