683 MycoKeys MycoKeys 98: 19-35 (2023) DOI: 10.3897/mycokeys.98.103484 Research Article Morphological and molecular analyses reveal two new species of Microcera (Nectriaceae, Hypocreales) associated with scale insects on walnut in China Feng Liu'’?®, Yu Deng'”, Fei-Hu Wang'2, Rajesh Jeewon*®, Qian Zeng’, Xiu-Lan Xu*®, Ying-Gao Liu'?, Chun-Lin Yang"? 1 College of Forestry, Sichuan Agricultural University, Chengdu, Sichuan, 611130, China 2 National Forestry and Grassland Administration Key Laboratory of Forest Resources Conservation and Ecological Safety on the Upper Reaches of the Yangtze River and Forestry Ecological Engineering in the Upper Reaches of the Yangtze River Key Laboratory of Sichuan Province, College of Forestry, Sichuan Agricultural University, Chengdu, Sichuan, China 3 Department of Health Sciences, Faculty of Medicine and Health Sciences, University of Mauritius, Reduit, Mauritius 4 Forestry Research Institute, Chengdu Academy of Agricultural and Forestry Sciences, Chengdu 611130, Sichuan Province, China Corresponding author: Chun-Lin Yang (yangcl0121@163.com) OPEN Qaceess Academic editor: Sajeewa Maharachchikumbura Received: 14 March 2023 Accepted: 10 May 2023 Published: 29 May 2023 Citation: Liu F, Deng Y, Wang F-H, Jeewon R, Zeng Q, Xu X-L, Liu Y-G, Yang C-L (2023) Morphological and molecular analyses reveal two new species of Microcera (Nectriaceae, Hypocreales) associated with scale insects on walnut in China. MycoKeys 98: 19-35, https://doi.org/10.3897/ mycokeys.98.103484 Copyright: © Feng Liu et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract The fungal genus Microcera consists of species mostly occurring as parasites of scale insects, but are also commonly isolated from soil or lichens. In the present study, we surveyed the diversity and assess the taxonomy of entomopathogenic fungi in Sichuan Province, China. Two new species of Microcera, viz. M. chrysomphaludis and M. pseudaulacaspidis, were isolated from scale insects colonising walnut (Juglans regia). Maximum Likelihood and Bayesian Inference analyses of ITS, LSU, tefl-a, rpb1, rpb2, ac/1, act, tub2, cmdA and his3 sequence data provide evidence for the validity of the two species and their placement in Nectriaceae (Hypocreales). Microcera pseudaulacaspidis primarily differs from similar species by having more septate and smaller cylindrical macroconidia, as well as DNA sequence data. Meanwhile, Microcera chrysomphaludis has elliptical, one-septate ascospores with acute ends and cylindrical, slightly curved with 4-6 septate macroconidia up to 78 um long. Morphological descriptions with illustrations of the novel species and DNA-based phylogeny generated from analyses of multigene dataset are also provided to better understand species relationships. Key words: Two new taxa, entomopathogenic fungi, morphology, phylogenetic analyses Introduction The genus Microcera Desm. (Nectriaceae, Hypocreales) was introduced in the 19" century and was typified by M. coccophila Desm., commonly known as the “red-headed fungus”. Microcera has been considered to be a synonym of the Fusarium Link in some major taxonomic revisions (Booth 1971; Nelson et al. 1983; Leslie and Summerell 2006). The genus is characterised by superficial, flame-like conidiomata, forming a fusarium-like asexual stage (Grafenhan et al. 2011; Herrera et al. 2013). Microcera species exhibit diverse ecological charac- teristics and are typically regarded as entomogenous fungi that are associated Feng Liu et al.: Isolation and identification of two new species of Microcera with scale insects, although they can occasionally be isolated from other sub- strates, such as aphids, adelgids, lichens and soil (Grafenhan et al. 2011; Crous et al. 2021a, b, 2022a). Currently, there are eight accepted species within the genus Microcera (Bills et al. 2009; Grafenhan et al. 2011; O'Donnell et al. 2012; Herrera et al. 2013; Dao et al. 2015, 2016; Lombard et al. 2015; Crous et al. 2021b, 2022a; Xu et al. 2021). Based on DNA sequence data and ecological association, Grafenhan et al. (2011) revised many anamorph- and teleomorph-typified genera of the Nectriaceae, resurrected Microcera and accepted four Microcera species, viz., M. coccophila, M. diploa (Berk. & M.A. Curtis) Grafenhan & Seifert, M. rubra Grafenhan & Seifert and M. larvarum (Fuckel) Grafenhan, Seifert & Schroers. Lombard et al. (2015) supported Microcera as a monophyletic group distantly related to Fusarium, based on further phylogenetic inferences from DNA se- quence data. Xu et al. (2021) isolated M. kuwanaspidis X.L. Xu & C.L. Yang from armoured scale insects Kuwanaspis howardi on Phyllostachys heteroclada in China. Two additional species, M. lichenicola and M. physciae Crous & Boers have been described from lichens (Crous et al. 2021b, 2022a). During a survey of entomopathogenic fungi in Sichuan Province, China, two Microcera species, in association with the two scale insects Pseudaulacaspis pentagona and Chrysomphalus aonidum on walnut, were isolated. Microcera pseudaulacaspidis sp. nov. and M. chrysomphaludis sp. nov. are introduced here based on the morphological characteristics and multi-locus analyses (DNA based). They were compared morphologically with existing taxa. In this study, comprehensive descriptions, micrographs of macroscopic and micro- scopic morphological characteristics, as well as DNA sequence data, are pro- vided to support the establishment of the new species. Materials and methods Specimen collection and isolation Three specimens of scale insects (SICAU 22-0161, SICAU 22-0162 and SICAU 22-0163) that were infected, were collected from Neijiang City (29°48'15'N, 105°06'44"E) and Liangshan Yi Autonomous Prefecture (26°56'43'N, 102°16'16"E), Sichuan Province, on 16 April and 8 October 2022. The specimens were placed in sterilised tubes or plastic boxes and returned to the laboratory as described by Senanayake et al (2020). The fungi were isolated, based on the single spore isolation technique described by Chomnunti et al. (2014). Cultures were grown on PDA for 20-40 days, at 25 °C, under 12 h light/12 h dark for re- cording growth rates, shape, texture and colour of the colonies. Ascomata and sporodochia were observed and photographed using a dissecting microscope NVT-GG (Shanghai Advanced Photoelectric Technology Co. Ltd., Shanghai, Chi- na). We observed microscopic characteristics, such as asci, ascospores, pseu- doparaphyses, ascomata wall, conidia, conidiophores, number of septa, metulae and conidiophores using an Olympus BX43. No fewer than 20 measurements of the two species were made for each feature using the Image Frame Work (IFW 0.9.0.7). The type specimens were deposited at the Herbarium of Sichuan Agricultural University, Chengdu, China (SICAU). The ex-type cultures were de- posited at the Culture Collection in Sichuan Agricultural University (SICAUCC). Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 20 Feng Liu et al.: Isolation and identification of two new species of Microcera DNA extraction, PCR amplification and nucleotide sequencing The New Plant Genomic DNA Kit (Beijing Aidlab Biotechnologies Co., Ltd, Beijing, China) was used to extract genomic DNA from fresh fungal mycelium. The extract- ed DNA to be used was stored at -20 °C. Amplified gene markers and their cor- responding primers are shown in Table 1. Polymerase chain reaction (PCR) was performed in 25 ul reaction mixture containing 22 ul Master Mix (Beijing LABLEAD Biotech Co., Ltd., Beijing, China), 1 pl DNA template and 1 ul each of forward and reverse (10 uM) primers. The amplification reactions were performed as described by Grafenhan et al. (2011), Lombard et al. (2015), Dai et al. (2016) and Wanasing- he et al. (2021). PCR products were sequenced at Hangzhou Youkang Biotech Co., Ltd., Chengdu, China. The newly-generated sequences were deposited in Gen- Bank. New species are established as recommended by Jeewon and Hyde (2016). Sequence alignment and phylogenetic analyses Based on BLAST searches in GenBank and recent publications (Bills et al. 2009; Grafenhan et al. 2011; O’Donnell et al. 2012; Herrera et al. 2013; Dao et al. 2015, 2016; Lombard et al. 2015; Xu et al. 2021), using the large subunit of the ATP citrate lyase (ac/1), actin (act) regions, calmodulin (cmdA), histone H3 (his3), the internal transcribed spacer (ITS), the partial large subunit nuclear rDNA (LSU), the RNA polymerase II largest subunit (rpb1), the RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1-a), B-tubulin (tub2) and sequence data, reference sequences were downloaded and separate phylogenet- ic analyses, based on single gene datasets were carried out to initially determine the placement of the two species. Information on the taxa used and GenBank Table 1. Gene markers and primer pairs used in this study. Gene markers acl act cmdA his3 ITS LSU rpb1 rpb2 tefl tub2 Primers Sequences of Primers 5’-3’ References acl1-230up AGCCCGATCAGCTCATCAAG Grafenhan et al. (2011) acl1-1220low CCTGGCAGCAAGATCVAGGAAGT ACT-512F ATGTGCAAGGCCGGTTTCGC Carbone and Kohn (1999) ACT1Rd CRICGTACTCCTGCTTBGAGATCCAC Groenewald et al. (2013) CAL-228F GAGTTCAAGGAGGCCTTCTCCC Carbone and Kohn 1999) CAL2Rd TGRICNGCCTCDCGGATCATCTC Groenewald et al. (2013) CYLH3F AGGTCCACTGGTGGCAAG Crous et al. (2006) CYLH3R AGCTGGATG TCCTTGGAC ITSS GGAAGTAAAAGTCGTAACAAGG White et al. (1990) ITS4 TCCTCCGCTTATTGATATGC LROR ACCCGCTGAACT TAAGC Rehner and Samuels (1994) LR5 ATCCTGAGGGAAACTTC Vilgalys and Hester (1990) RPB1-Ac CAYCCWGGYTTYATCAAGAA Castlebury et al. (2004) RPB1-Cr CCNGCDATNTCRTITRICCATRTIA RPB2-5F2 GGGGWGAYCAGAAGAAGGC O’Donnell et al. (2007) RPB2-7cR CCCATRGCTTGYTTRCCCAT EF1-728F CATCGAGAAGT TCGAGAAGG Carbone and Kohn (1999) ER2 GGARGTACCAGTSATCATG O’Donnell et al. (1998) T1 AACATGCGTGAGATTGTAAGT O’Donnell and Cigelnik (1997) CYLTUB1R AGTTGTCGG GACGGAAGAG Crous et al. (2006) Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 1 Feng Liu et al.: Isolation and identification of two new species of Microcera accession numbers of our novel species are listed in Table 2. Alignments for the individual locus matrices were generated with the online version of MAFFT version 7.429 (Katoh and Standley 2013) and ambiguous regions were excluded using BioEdit version 7.0.5.3 (Hall 1999). Combined sequences of ITS, LSU, tef1-a, rpb1, rpb2, acl1, act, tub2, cmdA and his3 were performed by SequenceMatrix v.1.7.8 (Vaidya et al. 2011). Maximum Likelihood (ML) and Bayesian Inference (BI) were constructed as described in Xu et al (2020). The phylogenetic tree constructed was viewed and edited using FigTree version 1.4.2 and Adobe Illustrator CS6. Genealogical concordance phylogenetic species recognition analysis Phylogenetically closely-related species were analysed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model by performing a pairwise homoplasy index (PHI) test as described by Quaedvlieg et al. (2014). The PHI test was performed in SplitsTree v.4.17.1 (Huson 1998; Huson and Bryant 2006) in order to determine the recombination level within phylogenet- ically closely-related species using a 6-locus concatenated dataset (ITS, LSU, tefl-a, acl1, cmdA and his3). The results can be visualised by constructing a split graph using LogDet conversion and the Splits options. Pairwise homo- plasy index below a 0.05 threshold (©, < 0.05) indicates significant recombi- nation present in the dataset. The relationship between closely-related species was visualised by constructing a Splits graph. Results Phylogenetic analyses The ML and BI analyses resulted in trees with similar topologies. Multi-locus phylogenetic analyses of species of Nectriaceae (Hypocreales) include sequences from 25 taxa and Tilachlidium brachiatum (Batsch) Petch (CBS 363.97, CBS 505.67) were used as outgroup (Fig. 1). The alignment contained 11882 characters (ITS = 1213, LSU = 1456, tefl-a = 1246, rpb1 = 1634, rpb2 = 2053, aci1 = 1060, act = 1206, tub2 = 707, cmdA = 779, his3 = 530), including gaps. The matrix had 4402 distinct alignment patterns, with 51.12% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.236233, C = 0.270063, G = 0.255057, T = 0.238647, with substitution rates AC = 1.239837, AG = 3.452130, AT = 1.264349, CG = 0.971857, CT = 5.853200 and GT = 1.000000. The gamma distribution shape parameter a = 0.347827 and the Tree-Length = 2.637211. The best scoring RAXML tree with a final likelihood value of -68,438.855836 is presented in Fig. 1 where the isolates from this study formed two distinct, well-supported lineages (MLBS = 100%, BIPP = 1.00) and, thus, were considered to represent two previously-unknown species. Pairwise homoplasy index (PHI) test The pairwise homoplasy index (PHI) test revealed that there was no significant recombination (, = 1) between Microcera pseudaulacaspidis (SICAUCC 22- 0163), M. coccophila (CBS 310.34), M. diploa (CBS 735.79) and M. kuwanaspidis (SICAUCC 21-0006) (Fig. 2). 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UaWIDadS *Z ajqeL 23 MycoKeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 Feng Liu et al.: Isolation and identification of two new species of Microcera osii.oor 96/1.00} ! 00/1.00. 100/100 190/100 100)1.00 92/100) a pora i Pseudocosm 97/1,00 Pseudocosmospora eut A tte 00/100 100/1.00) 100/1.00 Cosmospora coccin Tilachlidium brachiatum CBS 363.97 00/106 Tilachlidiaceae(Outgroup) Tilachlidium brachiatum CBS 505.67 J 0.07 Figure 1. Phylogram generated from RAxML analysis, based on combined ITS, LSU, tef1-a, rpb1, rpb2, acl1, act, tub2, cmdA and his3 sequence data of Microcera isolates. Bootstrap support values from Maximum Likelihood (MLBS, left) higher than 75% and Bayesian posterior probabilities (BIPP right) equal to or greater than 0.95 are indicated at the nodes, respectively. The sequences from ex-type strains are in bold. The newly-generated sequence is in red. Taxonomy Microcera pseudaulacaspidis Feng Liu & C.L. Yang, sp. nov. Index Fungorum No: 555034 Fig..3 Etymology. In reference to the generic name of scale insect from which it was isolated. Holotype. SICAU 22-0161. Host. Pseudaulacaspis pentagona (Diaspididae, Homoptera) Habitat. On the trunk of Jug/ans regia. Sexual state. Undetermined. Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 24 Feng Liu et al.: Isolation and identification of two new species of Microcera M. diploa CBS 735.79 M. coccaphila CBS 310.34° M. pseudaulacaspidis SICAUCC 22-0163" 0.1 M. kuwanaspidis dw = 1.0 SICAUCC 21-0006' Figure 2. The result of the pairwise homoplasy index (PHI) test of closely-related species using both LogDet transforma- tion and Splits decomposition. PHI test results (®,) < 0.05 indicate significant recombination within the dataset. Asexual state. Stromata byssoid, well-developed, bright orange to or- ange-red, formed directly on the margin of host scales or their covers with 1-7 sporodochia. Sporodochia 250-900 um long, 400-860 um wide, (x= 620 x 570 um, n = 50), conical, orange-red, upright masses on margin of host scales. Macroconidia 70-120 um long x 4.2—10.5 um wide (x= 95.7 x 6.5 um, n = 50), hyaline or jasmine, cylindrical, slightly curved, slender towards each end, 3-10 septate, mostly 7-9 septate, difficult to distinguish apical cell and basal cell. Microconidia and chlamydospores were not observed. Material examined. CHINA, Sichuan Province, Neijiang City, Dongxing District, Paifang Village walnut industrial base (29°48'15"N, 105°06'44"E, alt. 340 m), on scale insect Pseudaulacaspis pentagona, 16 April 2022, Feng Liu, LF202204001, (SICAU 22-0161, holotype), ex-type culture SICAUCC 22-0163. Culture characters. Colonies from a single macroconidium on PDA grow slow- ly and reach approximately 2 cm in diameter after 12 days at 25 °C, circular, flat, producing masses of macroconidia in the centre of the colony, measuring 76- 125 um long x 5.3-7.6 um wide (x= 91.2 x 6.3 um, n = 50), smaller than those in nature, white mycelium on the surface and the back of colonies is dark orange. Notes. Based on multi-gene phylogenetic analyses, Microcera pseudaulac- aspidis is closely related to M. kuwanaspidis (Fig. 1). However, we observed significant differences in the DNA sequence data, including base-pair differ- ences and gaps, with values of 1.45% (0 gaps), 17.67% (17 gaps), 3.22% (2 gaps), 1.53% (2 gaps), 1.70% (1 gap) and 3.82% (1 gap) in the ITS, LSU, tef1-a, tub2, cmdA and his3 genes, respectively. The PHI test also showed that no significant recombination events between M. pseudaulacaspidis and closely phylogenetically-related species occurred (Fig. 2). Based on a comparison of their morphological characteristics, M. pseudaulacaspidis can be distinguished Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 25 Feng Liu et al.: Isolation and identification of two new species of Microcera | \ | ; \ i \ , et | | \, / | X , i \ | j i \ i / | ‘ ee \ / \ \ be \ / | \ y j ‘ 4 (| an / } \ Pa yp 4 \ \ \ Ye | | [| | | ty f EA} 3] hee 1A Mea oe oO ‘ | \ | i qr \ | ik \\ ff / ae fee 1 | by ia fay i ms f ) | { i, ‘4 y “ i r ” Aa VAN AN igs) [os 2 \\ i | Walt - | he a \ \e \ fr } ff | \ NY \ \ { f yi ae \ N/a) Lid Wi f tt j 1 i | | | [ ; j | | | | | | | : | | i | | Fo | | f ’ \ ; | | ee | \ | \ NX \. Figure 3. Microcera pseudaulacaspidis (SICAU 22-0161) a, b stromata and sporodochia on host substrate c-e conidio- phore with developing macroconidia f germinated conidium g—o Macroconidia 0, p colonies on PDA after 30 days. Scale bars: 200 um (a, b); 20 um (ce); 10 um (f-n). from M. kuwanaspidis by shorter macroconidia (95.7 x 6.5 um vs. 107 x 7.3 um) with more septa (7—9-septate vs. 5-7-septate) (Xu et al. 2021). Given the morphological dissimilarities, distinct nucleotides at various sites and the well-supported lineage in our phylogeny, we have sufficient evidence to estab- lish M. pseudaulacaspidis as a new species. Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 6 Feng Liu et al.: Isolation and identification of two new species of Microcera Microcera chrysomphaludis Feng Liu & C.L. Yang, sp. nov. Index Fungorum No: 559445 Figs 4,5 Etymology. In reference to the generic name of scale insect from which it was isolated. Holotype. SICAU 22-0162. Host. Chrysomphalus aonidum (Diaspididae, Homoptera) Habitat. On the trunk of Jug/ans regia. Sexual state. Perithecia 285-429 um high, 216-386 um _ diam. (x= 350 x 290 um, n = 50), scattered, gregarious, formed directly on margin of host scales, bright red to dark red, subglobose, ellipsoidal in section, a central, rounded, papillate ostiole, lined internally with periphyses. Peridium 62-95 um thick, comprising two layers, outer stratum 32-55 um thick, composed of small, hyaline to light brown cells of textura angularis; inner stratum 35-45 um thick, composed of thinner, orange cells of textura angularis; thicker at sides to- wards apex, thinner at base. Hamathecium 8.5-19.2 um diameter (x= 12.3 um, n = 30), longer than asci, septate, unbranched, paraphyses. Asci 83.3-128.5 x 7.5-15.2 um (x= 109.2 x 10.2 um, n = 50), 8-spored, bitunicate, cylindrical, straight or curved, rounded at apex. Ascospores 16.8-27.5 x 7.8-10.8 um (x= 20.9 x 9.6 um, n = 50), uniseriate, elliptical, with rounded ends, one-septate, slightly constricted at septum, hyaline, smooth-walled, with many guttules. Asexual state. Stromata byssoid, pale yellow, formed directly on margin of host scales with 1-6 sporodochia. Sporodochia conical, erupted, yellowish, scattered or aggregated. Macroconidia 73-89 long, 6.9-10.6 um wide (x= 78.8 x 8.5 um, n = 50), hyaline, cylindrical, slightly curved, slender towards each end, 2-7 septa, mostly 4-6 septa, slightly constricted at septum, difficult to distinguish apical cell and basal cell. Microconidia and chlamydospores were not observed. Material examined. CHINA, Sichuan Province, Liangshan Yi Autonomous Pre- fecture, Huili County (26°56'43"N, 107°16'16’E, alt. 1780 m), on scale insect Chrysomphalus aonidum, 8 October 2022, Feng Liu, LF202208001, (SICAU 22- 0162, holotype), ex-type culture SICAUCC 22-0164. Ibid. LF202008002 (SICAU 22-0163, paratype), living culture SICAUCC 21-0165. Culture characters. Ascospores germinate on PDA within 12 h and cultures grow slowly on PDA. Colonies reach 2.4 cm in diameter after 20 days. Colonies from single conidia flocculent, clinging to medium, with irregular margin, white to pink mycelium on surface and back of colonies dark orange. Mycelium creamy-white starting at centre, but gradually becoming pale pink after 20 days, forming sparsely distributed mycelial clumps near edge of colony. Conidia germinate on PDA within 12 h, cultures grow slowly on PDA. Colonies 2.5 cm in diameter after 20 days. Colonies from single ascospores cottony and hard, with regular margin; mycelium creamy-white to pale pink, with concentric rings; back of colonies pale yellow. Notes. Multi-gene phylogenetic analyses have revealed that Microcera chrysomphaludis forms a highly robust clade that is closely related to M. coc- cophila and M. diploa. However, it is distinct from these two species with a high level of bootstrap support (ML/BY 100/1.00; Fig. 1). Morphologically, M. chrysomphaludis exhibits similar characteristics to M. coccophila, includ- ing superficial, subglobose, bright red ascomata, cylindrical asci and elliptical ascospores, as well as cylindrical macroconidia. However, M. chrysomphaludis Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 7 Feng Liu et al.: Isolation and identification of two new species of Microcera Figure 4. Microcera chrysomphaludis (SICAU 22-0162) a, b ascomata on host substrate c vertical section through ascos- tromata d peridium e ostiole of locule f paraphyses h ocular chamber g-j asci k-o ascospores p germinated ascospores; q, r colonies on PDA after 30 days. Scale bars: 200 um (a, b); 50 um c, 20 um (d, e); 10 um (f-p). can be differentiated from M. coccophila by its larger ascomata (285-429 x 216-386 um vs. 194-387 x 194-355 um), slightly shorter asci (109.2 x 10.2 um vs. 115 x 15 um), longer ascospores (16.8-27.5 x 7.8-10.8 um vs. 14-19 x 6-10 um) and shorter macroconidia (73-89 x 6.9-10.6 um vs. 90-132 x 6-9 tm) and fewer septa (4-6 vs. 7-9) (Grafenhan et al. 2011; Dao et al. 2015). Hence, we describe our collection as a new species in Microcera. Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 28 Feng Liu et al.: Isolation and identification of two new species of Microcera Figure 5. Microcera chrysomphaludis (SICAU 22-0163) a-c stromata and sporodochia on host substrate d-g conidio- phore with developing macroconidia h-I macroconidia m germinated conidium n, o colonies on PDA after 30 days. Scale bars: 200 um (b, c); 20 um (dg); 10 um (h-m). Discussion In this study, two new species (Microcera chrysomphaludis and M. pseudaulac- aspidis) associated with scale insects from walnut were introduced, based on phylogenetic inferences of a combined ITS, LSU, tef1-a, ac/1, act, cmdA, his3, rpb1, rpb2 and tub2 DNA sequence dataset and morphological evidence. Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 29 Feng Liu et al.: Isolation and identification of two new species of Microcera Ecologically, Microcera species are mainly distributed in tropical regions, but they have also been reported in the subtropical and temperate regions. Most of the Microcera species are pathogens of scale insects (Grafenhan et al. 2011; O’Donnell et al. 2012; Dao et al. 2015, 2016; Crous et al. 20214; Xu et al. 2021), However, two new species have recently been described from lichens (Crous et al. 2021b, 2022a). Most Microcera species infecting scale insects occur in the tree canopy and are more noticeable under moist conditions (Dao et al. 2015, 2016; Xu et al. 2021), consistent with the findings of this study. Morpho- logically, the sexual morph in this genus is characterised by orange to dark red perithecia with a blunt papilla producing cylindrical to narrowly clavate asci and 1(-3)-septate ascospores, while the asexual morph is predominantly fu- sarium-like, with verticillate to penicillate conidiophores producing small mac- roconidia (Grafenhan et al. 2011; Lombard et al. 2015; Crous et al. 2021a, b). Similar morphs were observed and documented in this study to provide further evidence of a connection between our isolates and other Microcera species (e.g. Figs 4, 5). Grafenhan et al. (2011) analysed an association of Microcera to Fusarium, Cladosterigma Pat., Mycogloea L.S. Olive and Tetracrium Henn. and accepted four species in Microcera. In recent years, numerous newly-discovered species have been described by employing extensive sampling coupled with multigene phylogenies (Sung et al. 2007; Lombard and Crous 2012; Wei et al. 2019; Lucking et al. 2021). Lombard et al. (2015) performed a multi-gene phylogenetic analysis, using combined datasets of ITS, LSU, tef1-a, ac/1, act, cmdA, his3, rpb1, rpb2 and tub2 to clarify intraspecific and intergeneric relationships within Nectriaceae. In this paper, M. pseudaulacaspidis was distinguished from M. kuwanaspidis and established as a new species, based on base-pair differences, particularly in the LSU (17.67%), tef1-a (3.22%) and his3 (3.82%). Additionally, M. chrysomphaludis formed a distinct and well-supported subclade and was found to be morpholog- ically distinct from M. coccophila in terms of the size of asci, ascospores and macroconidia (Grafenhan et al. 2011; O'Donnell et al. 2012; Dao et al. 2015). Through multigene phylogenetic analysis, the connection between the sexual and asexual morphs of M. chrysomphaludis was also confirmed. Entomopathogenic fungi are common on scale insects and have great po- tential in biological control (Zha et al. 2019; Sharma et al. 2020). Based on field trials, Microcera larvarum has been reported to have a significant biolog- ical control effect of Saissetia oleae, an economically important pest of olive and citrus plants (Cozzi et al. 2002). Microcera species have also been ex- ploited for various biopharmaceuticals in recent years due to their secondary metabolites with medicinal properties. For instance, parnafungins, extracted from M. larvarum, have intrinsic antifungal activity (Parish et al. 2008). Isa- ka et al. (2015) isolated two new ascochlorin derivatives from cultures of Microcera sp. BCC 17074 and demonstrated their significant cytotoxic activ- ities against various cancer cells. Furthermore, Cadelis et al. (2020) isolated four new secondary metabolites from M. larvarum isolates, which exhibited potent antimicrobial activity. This paper presents novel findings of two new entomopathogenic fungi, Microcera chrysomphaludis and M. pseudaulacaspidis, which were isolated from scale insects found on walnut trees in China. We conducted surveys in Mycokeys 98: 19-35 (2023), DOI: 10.3897/mycokeys.98.103484 30 Feng Liu et al.: Isolation and identification of two new species of Microcera numerous walnut orchards across Sichuan Province and observed significant infections of scale insects by these two species, resulting in high mortality rates, particularly in wet and humid conditions. Further screening and evalu- ation of these entomopathogenic fungi could facilitate their potential use as commercial biological control agents. Acknowledgements This study was supported by the Sichuan Science and Technology Program (grant number 2022NSFSC1011). The three anonymous reviewers are also acknowledged for their useful comments. Additional information Conflict of interest No conflict of interest was declared. Ethical statement No ethical statement was reported. Funding No funding was reported. Author contributions Funding acquisition: CLY. Investigation: QZ, FHW, YD. Project administration: CLY. Super- vision: XLX, CLY, YGL. Validation: RJ. Writing - review and editing: FL. Author ORCIDs Feng Liu © https://orcid.org/0000-0003-4580-7169 Rajesh Jeewon ® https://orcid.org/0000-0002-8563-957X Xiu-Lan Xu © https://orcid.org/0000-0002-6832-5421 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. 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