Research Article
Print
Research Article
Morphological and phylogenetic characterisations reveal three new species of Samsoniella (Cordycipitaceae, Hypocreales) from Guizhou, China
expand article infoWan-Hao Chen, Yan-Feng Han§, Jian-Dong Liang, Wei-Yi Tian, Zong-Qi Liang§
‡ Guizhou University of Traditional Chinese Medicine, Guiyang, China
§ Guizhou University, Guiyang, China
Open Access

Abstract

Samsoniella species have been found on lepidopteran larvae or pupae buried in soil or leaf litter. Three new species, Samsoniella hymenopterorum, S. coleopterorum and S. lepidopterorum, parasitic on hymenopteran larvae, coleopteran larvae and lepidopteran pupae, respectively, are reported. Morphological comparisons with extant species and DNA-based phylogenies from analysis of a multigene (ITS, RPB1, RPB2 and TEF) dataset supported the establishment of the new species. Unusually, all three new species have mononematous conidiophores. The new species are clearly distinct from other species in Samsoniella occurring in separate subclades.

Keywords

Isaria-like, morphology, nutritional preference, phylogeny

Introduction

The genus Isaria Pers. was introduced for entomogenous fungi with mononematous or synnematous conidiophores, usually consisting of several verticillate branches, each bearing a dense whorl of phialides characters. The phialides consist of a cylindrical or swollen basal portion, terminating in a thin, often long neck and produce divergent conidial chains (Samson 1974). However, entomogenous species, morphologically similar to Isaria, can be found distributed throughout the Hypocreales (Luangsa-ard et al. 2004).

Kepler et al. (2017) proposed the rejection of Isaria in favour of Cordyceps, owing to the confusion surrounding the application of Isaria and combined 11 species into Cordyceps. Mongkolsamrit et al. (2018) described some Isaria-like species and proposed the new genus Samsoniella Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard. The typical characteristics of Samsoniella are oval to fusiform conidia and bright red-orange teleomorphic stromata and anamorphic synnemata. Samsoniella species inhabit lepidopteran larvae and pupae in leaf litter or soil. Currently, Samsoniella consists of three species, S. alboaurantia (G. Sm.) Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard, S. aurantia Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard and S. inthanonensis Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard.

Three infected insect specimens were collected during a survey of entomogenous fungi in south-western China. Morphological and molecular phylogenetic analyses suggested that these isolates represented three new species, which are described here as Samsoniella hymenopterorum sp. nov., S. coleopterorum sp. nov. and S. lepidopterorum sp. nov.

Materials and methods

Specimen collection and identification

Three fungus-infected insect specimens were collected from Xishui County (28°29'56.70"N, 106°24'31.04"E) (A1950 and A1952) and Dali, Rongjiang County (26°01'58.70"N, 108°24'48.06"E) (DL1007), Guizhou Province, on 20 July and 1 October 2018, respectively. Isolation of the fungi was done as described by Chen et al. (2019). The surface of the specimens was rinsed with sterile water, followed by surface sterilisation with 75% ethanol for 3–5 sec. A part of the insect body was cut off and inoculated with haemocoel on potato dextrose agar (PDA) and PDA, to which 1% w/v peptone (PDAP) had been added. Fungal colonies emerging from specimens were isolated and cultured at 22 °C for 14 d under 12 h light/12 h dark conditions following protocols described by Zou et al. (2010). Accordingly, strains A19501, A19502, A19521, A19522, DL10071 and DL10072 were obtained. The specimens and the isolated strains were deposited in the Institute of Fungus Resources, Guizhou University (formally Herbarium of Guizhou Agricultural College; code, GZAC), Guiyang City, Guizhou, China.

Macroscopic and microscopic morphological characteristics of the fungi were examined and growth rates determined from PDA cultures incubated at 25 °C for 14 d. Hyphae and conidiogenous structures were mounted in lactophenol cotton blue or 20% lactate solution and observed with an optical microscope (OM, DM4 B, Leica, Germany).

DNA extraction, PCR amplification and nucleotide sequencing

DNA extraction was carried out in accordance with Liang et al. (2011). The extracted DNA was stored at −20 °C. Translation elongation factor 1 alpha (TEF) and RNA polymerase II largest subunit 2 (RPB2) genes were amplified using 983F/2218R and RPB2-5F/RPB2-7Cr primers, according to van den Brink et al. (2012). The RNA polymerase II largest subunit 1 (RPB1) gene was amplified with the primer pair CRPB1 and RPB1-Cr (Castlebury et al. 2004). The internal transcribed spacer (ITS) region was amplified by PCR using ITS4/ITS5, which was described by White et al. (1990). PCR products were purified using the UNIQ-10 column PCR products purification kit (no. SK1141; Sangon Biotech (Shanghai) Co., Shanghai, China) in accordance with the manufacturer’s protocol and sequenced at Sangon Biotech (Shanghai) Co. The resulting sequences were submitted to GenBank.

Sequence alignment and phylogenetic analyses

The DNA sequences, generated in this study, were assembled and edited using Lasergene software (version 6.0 DNASTAR). Generated ITS, RPB1, RPB2 and TEF sequences were aligned with those published by Mongkolsamrit et al. (2018) and others selected on the basis of BLAST algorithm-based searches in GenBank (Table 1). Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson (isolates CBS 284.36 and CBS 431.87) and Beauveria bassiana (Bals.-Criv.) Vuill. (ARSEF 1564) were chosen as outgroup taxa for the analysis of Samsoniella in Cordycipitaceae and Samsoniella species and closely-related species, respectively. Multiple datasets of ITS, RPB1, RPB2 and TEF were aligned using MAFFT v7.037b (Katoh and Standley 2013) and alignments were edited with MEGA6 (Tamura et al. 2013). Sequences were concatenated with SequenceMatrix v.1.7.8 (Vaidya et al. 2011). The partition homogeneity test in PAUP4.0b10 (Swofford 2002) was undertaken by using the command ‘hompart’.

Table 1.

Taxa included in the phylogenetic analyses.

Species Strain No. GenBank Accession No.
ITS RPB1 RPB2 TEF
Akanthomyces aculeatus HUA 772 KC519371 KC519366
А. attenuatus CBS 402.78 AJ292434 EF468888 EF468935 EF468782
А. coccidioperitheciatus NHJ 6709 JN049865 EU369067 EU369086
А. farinosa CBS 541.81 AY624180 JQ425686
А. kanyawimiae TBRC 7242 MF140751 MF140784 MF140808 MF140838
TBRC 7243 MF140750 MF140783 MF140807 MF140837
TBRC 7244 MF140752 MF140836
А. lecanii CBS 101247 JN049836 DQ522407 DQ522466 DQ522359
А. sulphureus TBRC 7247 MF140756 MF140785 MF140811 MF140841
TBRC 7248 MF140758 MF140787 MF140812 MF140843
TBRC 7249 MF140757 MF140786 MF140734 MF140842
А. thailandicus TBRC 7245 MF140754 MF140809 MF140839
TBRC 7246 MF140755 MF140810 MF140840
А. tuberculatus BCC 16819 GQ250012 GQ250037
OSC111002 JN049830 DQ522384 DQ522435 DQ522338
А. waltergamsii TBRC 7250 MF140749 MF140835
TBRC 7251 MF140747 MF140781 MF140805 MF140833
TBRC 7252 MF140748 MF140782 MF140806
Ascopolyporus polychrous P.C. 546 DQ127236 DQ118745
Beauveria acridophila HUA 179219 JX003857 JX003841 JQ958613
B. acridophila QCNE 186726 JQ958605 JX003855 JQ958618
B. bassiana ARSEF 1564 HQ880761 HQ880833 HQ880905 HQ880974
B. brongniartii ARSEF 617 HQ880782 HQ880854 HQ880926 HQ880991
BCC 16585 JN049867 JN049885 JF415991 JF416009
B. caledonica ARSEF 2567 HQ880817 EF469086 HQ880961 EF469057
B. diapheromeriphila MCA 1557 JQ958608 JX003851 JQ958612
QCNE 186272 JQ958599 JX003848 JQ958610
QCNE 186714 JQ958603 JX003850 JQ958611
B. locustiphila HUA 179217 JQ958609 JX003847
HUA 179218 JQ958606 JX003846 JX003845 JQ958619
B. malawiensis ARSEF 7760 HQ880897 HQ880969 DQ376246
B. pseudobassiana ARSEF 3405 AY532022 HQ880864 HQ880936 AY531931
B. scarabaeidicola ARSEF 5689 JN049827 DQ522380 DQ522431 DQ522335
B. staphylinidicola ARSEF 5718 EF468881 EF468776
Blackwellomyces cardinalis OSC 93609 DQ522370 DQ522422 DQ522325
B. cardinalis OSC 93610 JN049843 EF469088 EF469106 EF469059
B. pseudomilitaris NBRC 101409 JN943305 JN992482
NBRC 101410 JN943307 JN992481
Cordyceps amoene-rosea CBS 729.73 MG665235 HM161732
C. amoene-rosea CBS 107.73 AY624168
C. bifusispora EFCC 5690 EF468854 EF468909
EFCC 8260 EF468855 EF468910 EF468747
C. blackwelliae TBRC 7253 MF140739 MF140774 MF140798 MF140825
TBRC 7254 MF140738 MF140773 MF140797 MF140824
TBRC 7255 MF140737 MF140772 MF140796 MF140823
TBRC 7256 MF140736 MF140771 MF140795
TBRC 7257 MF140735 MF140770 MF140794 MF140821
C. cateniannulatus CBS 152.83 AY624172 JQ425687
TBRC 7258 MF140753 MF140767 MF140850
C. cateniobliqua CBS 153.83 AY624173 MG665236 JQ425688
C. cf. farinosa OSC 111004 EF468886 EF468780
C. cf. ochraceostromata ARSEF 5691 EF468867 EF468921 EF468759
C. cf. takaomontana NHJ 12623 EF468884 EF468932 EF468778
C. chiangdaoensis TBRC 7274 KT261393 KT261403
C. coleopterorum CBS 110.73 AY624177 JN049903 JF416006 JF416028
C. farinosa CBS 111113 AY624181 GU979973 GQ250022
C. fumosorosea CBS 107.10 AY624184 MG665237 HM161735
CBS 244.31 AY624182 JQ425690
CBS 375.70 AY624183 MG665238 HM161736
CBS 337.52 EF411219 MG665233
C. javanica CBS 134.22 AY624186 JQ425683
TBRC 7259 MF140745 MF140780 MF140804 MF140831
TBRC 7260 MF140744 MF140779 MF140803 MF140830
TBRC 7261 MF140743 MF140778 MF140802 MF140829
TBRC 7262 MF140746 MF140832
C. kintrischica ARSEF 7218 EU553278 GU734751
ARSEF 8058 GU734764 GU734750
C. kyusyuensis EFCC 5886 EF468863 EF468917
C. lepidopterorum TBRC 7263 MF140765 MF140768 MF140792 MF140819
TBRC 7264 MF140766 MF140769 MF140793 MF140820
C. militaris OSC 93623
C. morakotii TBRC 7275 KT261388 KT261398
TBRC 7276 KT261390 KT261400
C. ninchukispora EFCC 5197 EF468868 EF468760
EFCC 5693 EF468869 EF468762
EGS 38.165 EF468900 EF468795
EGS 38.166 EF468901 EF468794
NHJ 10627 EF468870 EF468763
NHJ 10684 EF468871 EF468761
C. oncoperae ARSEF 4358 EF468891 EF468936 EF468785
C. piperis CBS 116719 DQ127240 EU369083 DQ118749
C. pruinosa ARSEF 5413 JN049826 DQ522397 DQ522451 DQ522351
Cordyceps sp. CBS 102184 EF468907 EF468948 EF468803
C. takaomontana BCC 28612 FJ765285 FJ765268
C. tenuipes ARSEF 5135 AY624196 JN049896 JF416000 JF416020
OSC 111007 DQ522395 DQ522449 DQ522349
TBRC 7265 MF140741 MF140776 MF140827
TBRC 7266 MF140742 MF140777 MF140801 MF140828
TBRC 7267 MF140740 MF140775 MF140799 MF140826
Engyodontium aranearum CBS 309.85 DQ522387 DQ522439 DQ522341
Gibellula longispora NHJ 12014 EU369055 EU369075 EU369017
G. ratticaudata ARSEF 1915 DQ522408 DQ522467 DQ522360
Gibellula sp. NHJ 10788 EU369058 EU369078 EU369019
NHJ 13158 EU369057 EU369077 EU369020
NHJ 10808 EU369056 EU369076 EU369018
NHJ 5401 EU369059 EU369079
NHJ 7859 EU369064 EU369085
Hevansia cinerea NHJ 3510 EU369048 EU369070 EU369009
H. nelumboides BCC 41864 JN201871 JN201867
H. novoguineensis NHJ 4314 EU369051 EU369071 EU369012
NHJ 10469 EU369047 EU369008
NHJ 11923 EU369052 EU369072 EU369013
NHJ 13117 EU369049 EU369073 EU369010
NHJ 13161 EU369050 EU369011
Hyperdermium pulvinatum P.C. 602 DQ127237 DQ118746
Lecanicillium aranearum CBS 350.85 DQ522396 DQ522450 DQ522350
L. aranearum CBS 726.73a EF468887 EF468934 EF468781
L. fusisporum CBS 164.70 EF468889 EF468783
L. psalliotae CBS 101270 EF469095 EF469113 EF469066
CBS 363.86 EF468890 EF468784
CBS 532.81 EF469096 EF469112 EF469067
Purpureocillium lilacinum CBS 284.36 AY624189 EF468792 EF468898 EF468941
P. lilacinum CBS 431.87 AY624188 EF468897 EF468940 EF468791
Samsoniella alboaurantium CBS 240.32 AY624178 JN049895 JF415999 JF416019
S. alboaurantium CBS 262.58 AY624179 JQ425685
S. aurantia TBRC 7271 MF140764 MF140791 MF140818 MF140846
TBRC 7272 MF140763 MF140817 MF140845
TBRC 7273 MF140762 MF140816 MF140844
S. coleopterorum A19501 MT626376 MT642600 MN101585 MN101586
A19502 MT626625 MT642603 MN101587 MT642602
S. hymenopterorum A19521 MN128224 MT642601 MT642604 MN101588
A19522 MN128081 MN101589 MN101590 MN101591
S. inthanonensis TBRC 7915 MF140761 MF140790 MF140815 MF140849
TBRC 7916 MF140760 MF140789 MF140814 MF140848
TBRC 7270 MF140759 MF140788 MF140813 MF140847
S. lepidopterorum DL10071 MN128076 MN101592 MN101593 MN101594
DL10072 MN128084 MT642605 MT642606
Simplicillium lamellicola CBS 116.25 AJ292393 DQ522404 DQ522462 DQ522356
S. lanosoniveum CBS 101267 AJ292395 DQ522405 DQ522463 DQ522357
CBS 704.86 DQ522406 DQ522464 DQ522358
Torrubiella wallacei CBS 101237 EF469102 EF469119 EF469073

Maximum Likelihood (ML) analyses were constructed with RAxMLGUI (Silvestro and Michalak 2012). The GTRGAMMA model was used for all partitions, in accordance with recommendations in the RAxML manual against the use of invariant sites. For Bayesian Inference (BI), a Markov Chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities using MrBayes v.3.2 (Ronquist et al. 2012) for the combined sequence datasets. The selection of the best-fit nucleotide substitution model for each locus was calculated by the Akaike Information Criterion (AIC) with jModelTest 2 (Darriba et al. 2012). The TIM+I+G model was selected for the concatenated ITS+RPB1+RPB2+TEF sequences. The Bayesian analysis resulted in 20,001 trees after 10,000,000 generations. The first 4,000 trees, representing the burn-in phase of the analyses, were discarded, while the remaining 16,001 trees were used for calculating posterior probabilities in the majority rule consensus tree. After the analysis was finished, each run was examined using the programme Tracer v1.5 (Drummond and Rambaut 2007) to determine burn-in and confirm that both runs had converged. The final alignment is available from TreeBASE under submission ID: 24710 (http://www.treebase.org).

Results

Phylogenetic analyses

The phylogenetic tree of Samsoniella in Cordycipitaceae (Fig. 1) and Samsoniella species and closely related species (Fig. 2) were generated from the ML and BI analysis, based on a combined data set of ITS, RPB1, RPB2 and TEF sequence data. Statistical support (≥ 50%/0.5) is shown at the nodes for ML bootstrap support/BI posterior probabilities (Figs 1, 2). The strain numbers are noted after each species’ name. The concatenated sequences of analysis 1 and analysis 2 included 67 and 17 taxa, and consisted of 2,152 (ITS: 528, RPB1: 488, RPB2: 442 and TEF: 694) and 2,194 (ITS: 477, RPB1: 565, RPB2: 473 and TEF: 679) characters with gaps, respectively.

Figure 1. 

Phylogenetic relationships of the genus Samsoniella in Cordycipitaceae, based on multigene dataset (ITS, RPB1, RPB2 and TEF). Statistical support values (≥ 50%/0.5) are shown at the nodes for ML bootstrap support/BI posterior probabilities.

Analysis 1: Samsoniella in Cordycipitaceae. The RAxML analysis of the combined dataset (ITS+RPB1+RPB2+TEF) yielded a best scoring tree (Fig. 1) with a final ML optimisation likelihood value of –28,809.222105. Parameters for the GTR model of the concatenated dataset was as follows: estimated base frequencies; A = 0.234094, C = 0.301291, G = 0.260521, T = 0.204093; substitution rates AC = 1.111784, AG = 3.130020, AT = 0.930972, CG = 0.886915, CT = 6.300092, GT = 1.000000; gamma distribution shape parameter α = 0.390179. In the phylogenetic tree (Fig. 1), Samsoniella species were clustered in a clade and resolved into two obvious clades. Samsoniella species have a close relationship with Akanthomyces species.

Analysis 2: Samsoniella species and closely-related species. The RAxML analysis of the combined dataset (ITS+RPB1+RPB2+TEF) yielded a best scoring tree (Fig. 2) with a final ML optimisation likelihood value of –9,722.503130. Parameters for the GTR model of the concatenated data set were as follows: estimated base frequencies; A = 0.233473, C = 0.298686, G = 0.261629, T = 0.206212; substitution rates AC = 1.250081, AG = 2.534760, AT = 0.891128, CG = 0.827805, CT = 5.916085, GT = 1.000000; gamma distribution shape parameter α = 0.674468. In the phylogenetic tree (Fig. 2), Samsoniella species were clustered in a clade and easily distinguished with Akanthomyces species. S. coleopterorum and S. lepidopterorum clustered in a clade (Fig. 2) and formed two independent branches. S. hymenopterorum was phylogenetically close to S. inthanonensis and S. aurantia.

Figure 2. 

Phylogenetic relationships between the genus Samsoniella and closely-related species, based on multigene dataset (ITS, RPB1, RPB2 and TEF). Statistical support values (≥ 50%/0.5) are shown at the nodes for ML bootstrap support/BI posterior probabilities.

Taxonomy

Samsoniella coleopterorum W.H. Chen, Y.F. Han & Z.Q. Liang, sp. nov.

MycoBank No: 831735
Fig. 3

Diagnosis

Differs from Samsoniella aurantia by having smaller conidia and snout beetle host in the family Curculionidae. Differs from S. lepidopterorum by having cylindrical to ellipsoidal phialides, smaller fusiform to ellipsoidal conidia and a different host.

Figure 3. 

Samsoniella coleopterorum A infected insect (Coleoptera) B, C top (B) and underside (C) of a colony cultured on PDA medium at 14 d D, E, F, G, I phialides and conidia in chains J conidiophore and phialides H conidia. Scale bars: 10 mm (B, C); 10 μm (D–J).

Type

China, Guizhou Province, Xishui County (28°29'56.70"N, 106°24'31.04"E), July 2018, Jiandong Liang, holotype GZAC A1950, ex-type culture GZAC A19501. Sequences from isolated strain A19501 have been deposited in GenBank with accession numbers: ITS = MT626376, RPB1 = MT642600, RPB2 = MN101585 and TEF = MN101586.

Description

Colonies on PDA, 3.6–4.0 cm diam. in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose hyphal overgrowth, reverse yellowish. Prostrate hyphae smooth, septate, hyaline, 1.1–1.8 μm diam. Erect conidiophores usually arising from aerial hyphae, Isaria-like with phialides in whorls of two to four. Phialides 5.4–9.7 × 1.2–1.8 μm, with a cylindrical to ellipsoidal basal portion, tapering into a short distinct neck. Conidia in chains, hyaline, fusiform, ellipsoidal or subglobose, one-celled, 1.7–2.5 × 1.2–1.8 μm. Chlamydospores and synnemata not observed. Size and shape of phialides and conidia similar in culture and on natural substratum. Sexual state not observed.

Host

Snout beetle, family Curculionidae.

Distribution

Xishui County, Guizhou Province, China.

Etymology

Referring to its insect host, order Coleoptera.

Remarks

Samsoniella coleopterorum was easily identified as belonging to Samsoniella based on the phylogenetic analyses (Fig. 1). Comparing with the typical characteristics of three species (Table 2), S. coleopterorum has a close relationship with S. aurantia by having cylindrical to ellipsoidal phialides and similar in size. However, it differs from S. aurantia by having shorter conidia and snout beetle host in the family Curculionidae. Based on the combined dataset of ITS, RPB1, RPB2 and TEF sequences, S. coleopterorum has a close relationship with S. lepidopterorum (Fig. 2). However, S. coleopterorum has cylindrical to ellipsoidal phialides, smaller fusiform to ellipsoidal conidia and a different host.

Table 2.

Morphological comparison of three new species with other Samsoniella species.

Species Morphological characteristics Reference
Phialide (μm) Conidia (μm) Hosts/substrates
Samsoniella alboaurantium 5–8 × 2 ovate to lemon-shaped soil, lepidopterous pupa Smith 1957
2.3–2.5(–3) × 1.5–1.8
S. aurantia cylindrical to ellipsoidal fusiform lepidopterous larvae Mongkolsamrit et al. 2018
(5–)5.5–8.5(–13) × 2–3 (2–)2.5–3.5(–4) × (1–)1.5(–2)
S. inthanonensis cylindrical short fusiform lepidopterous larvae Mongkolsamrit et al. 2018
(4–)6.5–10(–12) × (1–)1.5–2(–3) (2–)3(–3.5) × 1.5–2
S. coleopterorum cylindrical to ellipsoidal fusiform, ellipsoidal or subglobose snout beetle this study
5.4–9.7 × 1.2–1.8 1.7–2.5 × 1.2–1.8
S. hymenopterorum cylindrical fusiform to ovoid bee this study
6.5–10.6 × 1.2–2.0 1.9–2.5 × 1.5–2.1
S. lepidopterorum ellipsoidal fusiform to subglobose lepidopterous pupa this study
5.2–8.5(–13.1) × 1.1–1.7 2.0–2.5 × 1.2–2.0

Samsoniella hymenopterorum W.H. Chen, Y.F. Han & Z.Q. Liang, sp. nov.

MycoBank No: 831736
Fig. 4

Diagnosis

Differs from Samsoniella inthanonensis and S. aurantia by having smaller, fusiform to ovoid conidia and a host in the family Vespidae.

Type

China, Guizhou Province, Xishui County, at 28°29'56.70"N, 106°24'31.04"E, July 2018, Jiandong Liang, holotype GZAC A1952, ex-type culture GZAC A19522. Sequences from isolated strain A19522 have been deposited in GenBank with accession numbers: ITS = MN128224, RPB1 = MT642603, RPB2 = MT642604 and TEF = MN101588.

Figure 4. 

Samsoniella hymenopterorum A infected insect (Hymenoptera) B, C top (B) and underside (C) of a colony cultured on PDA medium at 14 d D–H, J phialides and conidia in chains K conidiophore and phialides I conidia. Scale bars: 10 mm (B, C); 10 μm (D–K).

Description

Colonies on PDA, 6.2–6.4 cm diam. in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose hyphal overgrowth, reverse yellowish. Prostrate hyphae smooth, septate, hyaline, 1.1–1.6 μm diam. Erect conidiophores usually arising from aerial hyphae, Isaria-like with phialides in whorls of three to four. Phialides 6.5–10.6 × 1.2–2.0 μm, with a cylindrical basal portion, tapering to a distinct neck. Conidia in chains, hyaline, fusiform to ovoid, 1-celled, 1.9–2.5 × 1.5–2.1 μm. Chlamydospores and synnemata not observed. Size and shape of phialides and conidia similar in culture and on natural substratum. Sexual state not observed.

Host

Bee, family Vespidae.

Distribution

Xishui County, Guizhou Province, China.

Etymology

Referring to its insect host, order Hymenoptera.

Remarks

Samsoniella hymenopterorum was identified as belonging to Samsoniella, based on the phylogenetic analyses (Fig. 1). Comparing with the typical characteristics of the three species (Table 2), S. hymenopterorum has a close relationship with S. inthanonensis by a having cylindrical basal in phialide and similar in size. However, it is distinguished from S. inthanonensis by having smaller, fusiform to ovoid conidia and a host in the family Vespidae. Based on combined dataset of ITS, RPB1, RPB2 and TEF sequences, S. hymenopterorum is phylogenetically close to S. aurantia and S. inthanonensis (Fig. 2). However, S. hymenopterorum has smaller fusiform to ovoid conidia and a different host.

Samsoniella lepidopterorum W.H. Chen, Y.F. Han & Z.Q. Liang, sp. nov.

MycoBank No: 831737
Fig. 5

Diagnosis

Differs from Samsoniella coleopterorum by having larger, ellipsoidal phialide conidia and a host in the order Lepidoptera.

Type

China, Guizhou Province, Rongjiang County (26°01'56.13"N, 108°24'48.06"E), October 2018, Wanhao Chen, holotype GZAC DL1007 = RJ1807, ex-type culture GZAC DL10071 = RJ18071. Sequences from isolated strain DL10071 have been deposited in GenBank with accession numbers: ITS = MN128076, RPB1 = MN101592, RPB2 = MN101593 and TEF = MN101594.

Figure 5. 

Samsoniella lepidopterorum A infected insect pupa (Lepidoptera) B, C top (B) and underside (C) of a colony cultured on PDA medium at 14 d D–G, I phialides and conidia in chains H conidia J conidiophore and phialides. Scale bars: 10 mm (B, C); 10 μm (D–J).

Description

Colonies on PDA, 3.7–3.8 cm diam. in 14 d at 25 °C, white, consisting of a basal felt and cottony, floccose hyphal overgrowth, reverse yellowish. Prostrate hyphae smooth, septate, hyaline, 1.1–2.2 μm diam. Erect conidiophores usually arising from aerial hyphae, Isaria-like with phialides in whorls of two to four. Phialides 5.2–8.5 (–13.1) × 1.1–1.7 μm, with an ellipsoidal basal portion, tapering into a distinct neck. Conidia in chains, hyaline, fusiform to subglobose, 1-celled, 2.0–2.5 × 1.2–2.0 μm. Chlamydospores and synnemata not observed. Size and shape of phialides and conidia similar in culture and on natural substratum. Sexual state not observed.

Host

Pupa, order Lepidoptera

Distribution

Rongjiang County, Guizhou Province, China

Etymology

Referring to its insect host, order Lepidoptera

Remarks

Samsoniella lepidopterorum was easily identified as belonging to Samsoniella, based on the phylogenetic analyses (Fig. 1). Based on the combined dataset of ITS, RPB1, RPB2 and TEF sequences (Fig. 2) and the typical characteristics of Samsoniella species (Table 2), S. lepidopterorum has a close relationship with S. coleopterorum. However, S. lepidopterorum has larger, ellipsoidal phialide conidia and its pupa host is in the order Lepidoptera.

Discussion

Phylogenetic analyses, based on the combined datasets of (ITS+RPB1+RPB2+TEF), suggest that the three new species are members of the Cordycipitaceae and belong to the genus Samsoniella (Fig. 1). Mongkolsamrit et al. (2018) noted that the typical characteristics of Samsoniella were oval to fusiform conidia, bright red-orange stromata of the sexual morphs and synnemata of the asexual morphs. The phialides in this genus range from cylindrical to possessing a swollen basal portion. S. coleopterorum, S. hymenopterorum and S. lepidopterorum all have cylindrical phialides and fusiform conidia. However, the three new species had mononematous conidiophores rather than synnemata. Synnematous entomopathogenic fungi (such as Gibellula spp.) can be found on abaxial leaf surfaces of shrubbery, forest floors and shallow soil layers (Hywel-Jones 1996). As air flow under the forest canopy is slow and humid, the dispersal of conidia through airflow diffusion may be difficult. Therefore, these entomopathogenic fungi may employ a particular strategy, such as producing synnemata and sticky conidia, to accommodate various arthropod activities and facilitate conidium spread (Abbott 2002). The three new species were located in the more open portion of the forest and this may favour the dispersal of dry conidia. Thus, we could speculate that the mononematous conidiophores of the three new species may be the result of a convergent evolution to adapt to the ecological environment.

The evolutionary dynamics of fungi and their hosts are usually described either by co-evolution or by host shifts. Shifts often occur to new hosts that are evolutionarily distant, but which occupy a common ecological niche (Vega et al. 2009). Nutrient requirements often determine whether host shifts occur (Vega et al. 2009). Relationships between insects and fungi have been described as biotrophy, necrotrophy and hemibiotrophy, inter alia. The common ancestor of Hypocreaceae and Clavicipitaceae corresponds to a departure from plant-based nutrition to one that specialises on animals and fungi (Spatafora et al. 2007). Prediction of the characteristics and evolutionary placement of any given member should be based on the correlation between molecular-phylogenetic genealogy and nutritional preferences (Spatafora et al. 2007; Vega et al. 2009). Species of Samsoniella were originally found on lepidopteran larvae or pupae buried in soil or leaf litter (Mongkolsamrit et al. 2018). Mongkolsamrit et al. (2018) also reported that the true range of host affiliations of Samsoniella in nature may not be currently represented. Here, we report Samsoniella spp. from coleopteran, hymenopteran larvae and lepidopteran pupae. The presence of different hosts indicates that the nutrient requirements of Samsoniella spp. can change with the environment (Spatafora et al. 2007).

In the present study, a four loci phylogenetic analysis showed that S. coleopterorum, S. lepidopterorum and S. hymenopterorum clustered in separate subclades from other Samsoniella species. They represent new taxa, based on morphological characteristics, nutritional preferences and phylogenetic analyses.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31860002), High-level Innovative Talents Training Object in Guizhou Province (No. Qiankehepingtairencai [2020]6005), Science and Technology Foundation of Guizhou Province (No. Qiankehejichu [2020]1Y060), National Survey of Traditional Chinese Medicine Resources (No. Caishe [2017]66, 216) and Engineering Research Center of General Higher Education in Guizhou Province (Qianjiaohe (2015) 337). We also thank Lesley Benyon, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

References

  • Castlebury LA, Rossman AY, Sung GH, Hyten AS, Spatafora JW (2004) Multigene phylogeny reveals new lineage for Stachybotrys chartarum, the indoor air fungus. Mycological Research 108: 864–872. https://doi.org/10.1017/S0953756204000607
  • Chen WH, Liu C, Han YF, Liang JD, Tian WY, Liang ZQ (2019) Three novel insect-associated species of Simplicillium (Cordycipitaceae, Hypocreales) from Southwest China. MycoKeys 58: 83–102. https://doi.org/10.3897/mycokeys.58.37176
  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9(8): 772–772. https://doi.org/10.1038/nmeth.2109
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30(4): 772–780. https://doi.org/10.1093/molbev/mst010
  • Kepler RM, Luangsa-ard JJ, Hywel-Jones NL, Quandt CA, Sung GH, Rehner SA, Aime MC, Henkel TW, Sanjuan T, Zare R, Chen M, Li Z, Rossman AY, Spatafora JW, Shrestha B (2017) A phylogenetically-based nomenclature for Cordycipitaceae (Hypocreales). IMA Fungus 8: 335–353. https://doi.org/10.5598/imafungus.2017.08.02.08
  • Liang JD, Han YF, Zhang JW, Du W, Liang ZQ, Li ZZ (2011) Optimal culture conditions for keratinase production by a novel thermophilic Myceliophthora thermophila strain GZUIFR-H49-1. Journal of Applied Microbiology 110: 871–880. https://doi.org/10.1111/j.1365-2672.2011.04949.x
  • Luangsa-ard JJ, Hywel-Jones NL, Samson RA (2004) The order level polyphyletic nature of Paecilomyces sensu lato as revealed through 18S-generated rRNA phylogeny. Mycologia 96: 773–780. https://doi.org/10.1080/15572536.2005.11832925
  • Mongkolsamrit S, Noisripoom W, Thanakitpipattana D, Wutikhun T, Spatafora JW, Luangsa-ard J (2018) Disentangling cryptic species with Isaria-like morphs in Cordycipitaceae. Mycologia 110: 230–257. https://doi.org/10.1080/00275514.2018.1446651
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. https://doi.org/10.1093/sysbio/sys029
  • Samson RA (1974) Paecilomyces and some allied hyphomycetes. Studies in Mycology 6: 1–119.
  • Swofford DL (2002) PAUP* 4.0b10: phylogenetic analysis using parsimony (*and other methods). Sunderland, MA, Sinauer.
  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725–2729. https://doi.org/10.1093/molbev/mst197
  • Vaidya G, Lohman DJ, Meier R (2011) SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27(2): 171–180. https://doi.org/10.1111/j.1096-0031.2010.00329.x
  • van den Brink J, Samson RA, Hagen F, Boekhout T, de Vries RP (2012) Phylogeny of the industrial relevant, thermophilic genera Myceliophthora and Corynascus. Fungal Diversity 52: 197–207. https://doi.org/10.1007/s13225-011-0107-z
  • Vega FE, Goettel MS, Blackwell M, Chandler D, Jackson MA, Keller KM, Maniania KN, Monzón A, Ownley BH, Pell JK, Rangel DEN, Roy HE (2009) Fungal entomopathogens: new insights on their ecology. Fungal Ecology 2: 149–159. https://doi.org/10.1016/j.funeco.2009.05.001
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds.) PCR protocols: a guide to methods and applications. Academic Press, New York, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Zou X, Liu AY, Liang ZQ, Han YF, Yang M (2010) Hirsutella liboensis, a new entomopathogenic species affecting Cossidae (Lepidoptera) in China. Mycotaxon 111(1): 39–44. https://doi.org/10.5248/111.39