11urn:lsid:arphahub.com:pub:C004A564-9D6A-5F9F-B058-6A3815DFE9C3MycoKeysMC1314-40571314-4049Pensoft Publishers10.3897/mycokeys.66.4657546575Research ArticleAncylistaceaeEntomophthoromycotinaMolecular systematicsTaxonomyWorldA taxonomic revision of the genus Conidiobolus (Ancylistaceae, Entomophthorales): four clades including three new generaNieYong12YuDe-Shui1WangCheng-Fang1LiuXiao-Yongliuxiaoyong@im.ac.cn3HuangBobhuang@ahau.edu.cn1Anhui Provincial Key Laboratory for Microbial Pest Control, Anhui Agricultural University, Hefei 230036, ChinaAnhui Agricultural UniversityHefeiChinaSchool of Civil Engineering and Architecture, Anhui University of Technology, Ma’anshan 243002, ChinaAnhui University of TechnologyMa’anshanChinaState Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaInstitute of Microbiology, Chinese Academy of SciencesBeijingChina
Corresponding author: Xiao-Yong Liu (liuxiaoyong@im.ac.cn), Bo Huang (bhuang@ahau.edu.cn)
Academic editor: M. Stadler
202030032020665581A633A7E0-4ED7-52E9-A612-3DA654ED24D737425791409201913032020Yong Nie, De-Shui Yu, Cheng-Fang Wang, Xiao-Yong Liu, Bo HuangThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The genus Conidiobolus is an important group in entomophthoroid fungi and is considered to be polyphyletic in recent molecular phylogenies. To re-evaluate and delimit this genus, multi-locus phylogenetic analyses were performed using the large and small subunits of nuclear ribosomal DNA (nucLSU and nucSSU), the small subunit of the mitochondrial ribosomal DNA (mtSSU) and the translation elongation factor 1-alpha (EF-1α). The results indicated that the Conidiobolus is not monophyletic, being grouped into a paraphyletic grade with four clades. Consequently, the well-known Conidiobolus is revised and three new genera Capillidium, Microconidiobolus and Neoconidiobolus are proposed along with one new record and 22 new combinations. In addition, the genus Basidiobolus is found to be basal to the other entomophthoroid taxa and the genus Batkoa locates in the Entomophthoraceae clade.
ZygomycetesEntomophthoralesMorphologyPhylogenyNew taxaNational Natural Science Foundation of China501100001809http://doi.org/10.13039/501100001809Citation
Nie Y, Yu D-S, Wang C-F, Liu X-Y, Huang B (2020) A taxonomic revision of the genus Conidiobolus (Ancylistaceae, Entomophthorales): four clades including three new genera. MycoKeys 66: 55–81. https://doi.org/10.3897/mycokeys.66.46575
Introduction
More than 250 species of entomophthoroid fungi were isolated from insects, soil and litter throughout the world (Gryganskyi et al. 2013). For a long time, this group has been considered to be polyphyletic (Nagahama et al. 1995; Jensen et al. 1998; James et al. 2006; Liu and Voigt 2010) and was classified into a subphylum Entomophthoromycotina and a pending taxon Basidiobolus (Hibbett et al. 2007). However, a recent phylogeny using the multi-gene dataset, 18S rDNA, 28S rDNA, mtSSU and RPB2, indicated that this group formed a monophyletic lineage including Basidiobolus and it was consequently reclassified as a new fungal phylum Entomophthoromycota. More recently, a phylogenomic analysis (192 clusters of orthologous proteins) has divided traditional zygomycotan into two phyla Mucoromycota and Zoopagomycota and the entomophthoroid fungi have been re-assigned into the subphylum Entomophthoromycotina under the latter phylum (Spatafora et al. 2016). This taxonomic scheme was supported by the phylogeny of mitochondrial genomes (Nie et al. 2019).
Together with other two genera Ancylistes and Macrobiotophthora, the genus Conidiobolus belongs to Ancylistaceae, Entomophthorales, Entomophthoromycetes, Entomophthoromycotina (Humber 2012). There are six and two accepted species within the Ancylistes and Macrobiotophthora, respectively, while Conidiobolus, one of the largest groups in the entomophthoroid fungi, contains 76 names (http://www.indexfungorum.org/). The genus Conidiobolus is typified by C.utriculosus Bref. 1884 and characterised morphologically by simple sporophores, globose to pyriform multinucleate primary conidia, various types of secondary conidia and resting spores (Brefeld 1884; Humber 1997). Up to the 1940s, for half a century, only three more species were reported, C.minor Bref., C.villosus Martin and C.brefeldianus Couch (Brefeld 1884; Martin 1925; Couch 1939). In the 1950s–1960s, 38 Conidiobolus species and a variety were isolated from the United States and India (Drechsler 1952, 1953a, b, 1954, 1955a, b, c, 1956, 1957a, b, c, 1960, 1961, 1962, 1965; Srinivasan and Thirumalachar 1961, 1962a, b, 1965, 1967, 1968a, b). Based on a numerical taxonomy, King (1976a, b, 1977) recognised 27 definitive species. Since then, along with some new combinations, 10 more species have been added to Conidiobolus (Bałazy et al. 1987; Waters and Callaghan 1989; Bałazy 1993; Huang et al. 2007; Waingankar et al. 2008; Nie et al. 2012, 2016, 2017, 2018). A total of 37 species are currently accepted in this genus (Nie et al. 2018).
Three subgenera – Capillidium, Conidiobolus and Delacroixia – were proposed within the Conidiobolus, based on shape of the secondary conidia and, amongst them, the subgenus Delacroixia was reduced from generic rank (Ben-Ze’ev and Kenneth 1982). This subgeneric criterion provided a valuable contribution for the taxonomy of the genus Conidiobolus (Humber 1989). Since the 1990s, molecular analysis has become an increasingly important tool for fungal taxonomy (Bruns et al. 1991; Taylor et al. 2000). The nucLSU rDNA and EF-1α regions proved to be distinguishable amongst Conidiobolus species (Nie et al. 2012), while nucSSU rDNA indicated the genus Conidiobolus might be a polyphyletic group (Jensen et al. 1998). The subgeneric circumscription was not defined because of instability to form a certain type of secondary conidia for each phylogenetic clade (Callaghan et al. 2000; Gryganskyi et al. 2013; Nie et al. 2018). Besides, the phylogenetic relationships amongst species of Conidiobolus have not been fully resolved due to the absence of types. The genus Batkoa, morphologically similar to Conidiobolus, was phylogenetically closely related to Entomophthoraceae rather than Ancylistaceae (Gryganskyi et al. 2012, 2013).
In the present study, a reclassification of the entomophthoroid fungi, including as many as available Conidiobolus types, was constructed based on four loci (nucSSU, nucLSU, EF-1α and mtSSU) to present the taxonomic delimitation of the genus Conidiobolus and to re-evaluate the phylogenetic relationship between Basidiobolus and Batkoa.
Materials and methodsIsolates and morphology
A total of 26 ex-types of Conidiobolus were purchased from the American Type Culture Collection, Manassas, USA (ATCC) and collected from the China General Microbiological Culture Collection Center, Beijing, China (CGMCC) and the Research Center for Entomogenous Fungi of Anhui Agricultural University, Anhui Province, China (RCEF). Dried cultures were deposited in the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS). Morphology was observed with an Olympus BX51 research microscope and photographed by an Olympus DP25 microscope-camera system. Growth diameter on PDA (potato 200 g, dextrose 20 g, agar 20 g, H2O 1 l), Mycelia, primary conidiophores, primary conidia, microconidia, capilliconidia and resting spores were measured and described with the method of King (1976a).
DNA extraction, PCR amplification and sequencing
Fungal strains were incubated on PDA for 7 d at 21 °C. Total genomic DNA was extracted from the fresh fungal mycelia by using modified CTAB method (Watanabe et al. 2010). Four gene portions from cell nuclei and mitochondria and one protein coding gene were used in this study: the large subunit of nuclear ribosomal RNA genes (nucLSU), the small subunit of nuclear ribosomal RNA genes (nucSSU), the small subunit of mitochondrial ribosomal RNA genes (mtSSU) and the translation elongation factor 1-alpha gene (EF-1α). The nucLSU region was amplified with the primers LR0R and LR5 (Vilgalys and Hester 1990), the nucSSU region with nucSSU-0021-5’ (Gargas and DePriest 1996) and nucSSU-1780-3’ (DePriest 1993) and EF-1α region with the primers EF983 and EF1aZ-1R (http://www.aftol.org/primers.php). These PCR reactions have been described by Liu et al. (2005), Jensen et al. (1998) and Nie et al. (2012). The primers used for the mtSSU region were mtSSU1 and mtSSU2R and the PCR reaction was performed using the following cycling parameters: denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, extension at 72 °C for 1 min and finalised with an extra extension at 72 °C for 7 min (Zoller et al. 1999). PCR products were purified and sequenced by Shanghai Genecore Biotechnologies Company (Shanghai, China) with the same primers as relative PCR. The nucleotide sequence data have been deposited in the GenBank (Table 1).
The species used in phylogenetic analyses.
Species
Strains*
GenBank accession numbers
nucSSU
nucLSU
EF-1α
mtSSU
Allomycesarbusculus
AFTOL 300
AY552524
DQ273806
DQ275334
–
Basidiobolushaptosporus
ARSEF 261
JX242606
JX242586
–
JX242626
B.heterosporus
CBS 311.66
JX242607
JX242587
–
JX242627
B.magnus
CBS 205.64
JX242608
JX242588
–
JX242628
B.meristosporus
CBS 931.73
JX242609
JX242589
–
JX242629
B.microsporus
CBS 130.62 (T)
JX242610
JX242590
–
JX242630
B.ranarum
NRRL 34594
AY635841
DQ273807
DQ275340
EF392490
Batkoaapiculata
ARSEF 3130
DQ177437
EF392404
–
EF392513
B.gigantea
ARSEF 214
JX242611
JX242591
–
JX242631
B.major
ARSEF 2936
EF392559
EF392401
–
EF392511
B.obscurus**
CBS 182.60
JX242614
JX242595
–
JX242635
B.pseudapiculata**
ARSEF 395
EF392557
EF392378
–
EF392508
Coemansiareversa
AFTOL 140
AY546685
AY546689
DQ282615
–
Conidiobolusadiaeretus
ARSEF 451 (T)
–
KC461182
–
–
C.adiaeretus
CGMCC 3.15888
–
MN061284
MN061481
MN061287
C.antarcticus
ARSEF 6913 (T)
–
DQ364207
–
DQ364227
C.bangalorensis
ARSEF 449 (T)
–
DQ364204
–
DQ364225
C.brefeldianus
ARSEF 452 (T)
AF368506
EF392382
–
EF392495
C.chlamydosporus
ATCC 12242 (T)
–
JF816212
JF816234
MK301178
C.coronatus
NRRL 28638
AF113418
AY546691
DQ275337
–
C.coronatus
RCEF 4518
–
JN131537
JN131543
–
C.couchii
ATCC 18152 (T)
–
JN131538
JN131544
MK301179
C.dabieshanensis
CGMCC 3.15763 (T)
–
KY398125
KY402206
MK301180
C.denaeosporus
ATCC 12940 (T)
–
JF816215
JF816228
MK301181
C.firmipilleus
ARSEF 6384
JX242612
JX242592
–
JX242632
C.gonimodes
ATCC 14445 (T)
–
JF816221
JF816226
MK301182
C.heterosporus
RCEF 4430
–
JF816225
JF816239
MK301183
C.humicolus
ATCC 28849 (T)
–
JF816220
JF816231
MK301184
C.incongruus
NRRL 28636
AF113419
AF113457
–
–
C.iuxtagenitus
ARSEF 6378 (T)
–
KC788410
–
–
C.iuxtagenitus
RCEF 4445
–
JX946695
JX946700
MK333391
C.khandalensis
ATCC 15162 (T)
–
KX686994
KY402204
MK301185
C.lachnodes
ARSEF 700
–
KC788408
–
–
C.lamprauges
ARSEF 2338
AF296754
DQ364206
–
DQ364226
C.lichenicolus
ATCC 16200 (T)
–
JF816216
JF816232
MK301186
C.lobatus
ATCC 18153 (T)
–
JF816218
JF816233
MK301187
C.marcosporus
ATCC 16578 (T)
–
KY398124
KY402209
MK301188
C.megalotocus
ATCC 28854 (T)
–
MF616383
MF616385
MK301189
C.mirabilis
CGMCC 3.17763 (T)
–
MH282852
MH282853
MK333389
C.mycophagus
ATCC 16201 (T)
–
JX946694
JX946698
MK301190
C.mycophilus
ATCC 16199 (T)
–
KX686995
KY402205
MK301191
C.nodosus
ATCC 16577 (T)
–
JF816217
JF816235
MK333388
C.osmodes
ARSEF 79
AF368510
EF392371
–
DQ364219
C.osmodes
RCEF4447
–
JN131539
JN131545
MK333392
C.pachyzygosporus
CGMCC 3.17764 (T)
–
KP218521
KP218524
MK333390
C.paulus
ARSEF 450 (T)
–
vv
–
–
C.polyspermus
ATCC 14444 (T)
–
MF616382
MF616384
MK301193
C.polytocus
ATCC 12244 (T)
–
JF816213
JF816227
MK301194
C.pumilus
ARSEF 453 (T)
JX242615
EF392383
–
EF392496
C.rhysosporus
ATCC 12588 (T)
–
JN131540
JN131546
MK301195
C.sinensis
RCEF 4952 (T)
–
JF816224
JF816238
MK301196
C.stilbeus
RCEF 5584 (T)
–
KP218522
KP218525
MK301197
C.stromoideus
ATCC 15430 (T)
–
JF816219
JF816229
MK301198
C.terrestris
ATCC 16198 (T)
–
KX752050
KY402208
MK301199
C.thromboides
ATCC 12587 (T)
–
JF816214
JF816230
MK301200
C.thromboides
FSU 785
JX242616
JX242597
–
JX242637
C.thromboides
RCEF 4492
–
JF816223
JF816236
MK333393
C.undulatus
ATCC 12943 (T)
–
JX946693
JX946699
MK301201
Dimargarisbacillispora
AFTOL 136
AB016020
DQ273791
DQ282609
–
Endogonepisiformis
AFTOL 539
DQ322628
DQ273811
DQ282618
–
Entomophagaaulicae
ARSEF 172
EF392542
EF392372
–
EF392487
E.conglomerata
ARSEF 2273
AF368509
–
–
–
E.maimaga
ARSEF 1400
EF392556
EF392395
–
EF392505
Eryniopsiscaroloniana
ARSEF 640
EF392552
EF392387
–
EF392500
Entomophthorachromaphidis
ARSEF 1860
AF353725
–
–
–
E.culicis
ARSEF 387
AF368516
–
–
–
E.grandis
ARSEF 6701
–
DQ481229
–
–
E.scatophaga
ARSEF 6704
–
DQ481226
–
–
E.muscae
ARSEF 3074
AY635820
DQ273772
DQ275343
–
E.planconiana
ARSEF 6252
AF353723
GQ285878
–
–
E.schizophorae
ARSEF 5348
AF052402
GQ285883
–
–
E.syrphi
ARSEF 5595
–
DQ481230
–
–
E.tripidium
ARSEF 6518
AF296755
–
–
–
Eryniaconica
ARSEF 1439
AF368513
EF392396
–
EF392506
E.ovispora
ARSEF 400
JX242620
JX242601
–
JX242641
E.rhizospora
ARSEF 1441
AF368514
EF392397
–
EF392507
E.sciarae
ARSEF 1870
AF368515
EF392399
–
EF392509
Furiaamericana
ARSEF 742
EF392554
EF392389
–
–
F.gastropachae
ARSEF 5541
EF392562
EF392407
–
EF392516
F.ithacensis
ARSEF 663
EF392553
EF392388
–
EF392501
F.neopyralidarum
ARSEF 1145
AF368518
EF392394
–
EF392504
F.pieris
ARSEF 781
AF368519
EF392390
–
EF392502
F.virescens
ARSEF 1129
EF392555
EF392393
–
EF392503
Gaertneriomycessemiglobiferus
AFTOL 34
AF164247
DQ273778
DQ275338
–
Macrobiotophthoravermicola
ARSEF 650
AF052400
–
–
–
Massosporacicadina
ARSEF 374
EF392548
EF392377
–
EF392492
Mortierellaverticillata
AFTOL 141
AF157145
DQ273794
–
–
Pandorablunckii
ARSEF 217 (T)
JX242621
JX242602
–
–
P.delphacis
ARSEF 459
AF368521
EF392384
–
EF392497
P.dipterigena
ARSEF 397
AF368522
EF392380
–
EF392565
P.kondoiensis
CBS 642.92
JX242622
JX242603
–
JX242642
P.neoaphidis
ARSEF 3240
EF392560
EF392405
–
EF392514
Piptocephaliscorymbifera
AFTOL 145
AB016023
AY546690
DQ282619
–
Rhizophagusintraradices
AFTOL 845
DQ322630
FJ461839
DQ282611
–
Rozellaallomycis
AFTOL 297
AY635838
DQ273803
DQ275342
–
Schizangiellaserpentis
ARSEF 2237
AF368523
EF392428
–
EF392488
Strongwellseacastrans
–
AF052406
–
–
–
Zancudomycesculisetae
AFTOL 29
AF277007
DQ273773
–
–
Zoophthoraanglica
ARSEF 396
–
EF392379
–
EF392493
Z.lanceolata
ARSEF 469
EF392550
EF392385
–
EF392498
Z.phalloides
ARSEF 2281
EF392558
EF392400
–
EF392510
Z.radicans
ARSEF 388
JX242624
JX242605
–
JX242644
* AFTOL, Assembling the Fungal Tree of Life; ARSEF, ARS Entomopathogenic Fungus Collection (Ithaca, U.S.A.); ATCC, American Type Culture Collection (Manassas, U.S.A); CGMCC, China General Microbiological Culture Collection Center (Beijing, China); FSU, Jena Microbial Resource Collection (Friedrich-Schiller-University of Jena, Germany); NRRL, ARS Culture Collection (Peoria, U.S.A); RCEF, Research Center for Entomogenous Fungi (Hefei, China). T = ex-type. **Batkoa sp. CBS 182.60 was received as Conidiobolusobscurus, while B.pseudapiculataARSEF 395 was received as C.pseudapiculatus.
Phylogenetic analyses
More available nucLSU, nucSSU, mtSSU and EF-1α sequences of 14 Conidiobolus species and 47 other entomophthoroid fungi were obtained from GenBank. Ten species of Glomeromycotina, Mortierellomycotina, Mucoromycotina, Kickxellomycotina, Zoopagomycotina, Blastocladiomycota, Chytridiomycota and Cryptomycota, were chosen as outgroups. Alignments were constructed separately for each locus with MUSCLE 3.8.31 (Edgar 2004) and the concatenated matrices were assembled by SequenceMatrix 1.7.8 (Vaidya et al. 2011). The best model for the phylogenetic analysis was selected with Akaike Information Criterion (AIC) by using Modeltest 3.7 (Posada and Crandall 1998). Phylogenetic relationships were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI). The best-scoring ML tree analysis was performed using raxmlGUI 1.5b1 with GTRGAMMA model and 1000 replicates (Silvestro and Michalak 2012). The BI analysis was performed using MrBayes 3.2.2 (Ronquist and Huelsenbeck 2003). Markov Chain Monte Carlo (MCMC) chains ran until the convergences met and the standard deviation fell below 0.01. The first 25% of trees were discarded as burn-in. The combined dataset was deposited at TreeBase (No. S25064). Phylogenetic trees were checked and modified in FigTree 1.4 (Rambaut 2012).
ResultsPhylogenetic analyses
The combined dataset contained 4521 characters of nucLSU (1–1326), nucSSU (1327–3424), EF-1α (3425–4062) and mtSSU (4063–4521) after alignment. With the optimal model GTR+I+G and random starting trees, four Markov chains were run for 7 million generations and every 100th generation was sampled once. ML and BI analyses of the combined dataset resulted in phylogenetic reconstructions with almost similar topologies and the average standard deviation of split frequencies was 0.006721 (BI).
In the ML phylogenetic tree (Figure 1), the Basidiobolaceae lineage (88/0.94) is located at the base of the entomophthoroid fungi and is closely related to the Ancylistaceae group (56/0.91). The Batkoa lineage is grouped within the Entomophthoraceae Clade (60/0.89). All Conidiobolus lineages are clustered into a paraphyletic grade and therefore cannot be considered congeneric. Moreover, the Conidiobolus grade consists of four well supported clades. In detail, there are 7, 10, 16 and 3 species in Clade I (100/1.00), II (77/1.00), III (100/1.00) and IV (99/1.00), respectively.
Phylogenetic tree constructed by maximum likelihood analyses of nucLSU, nucSSU, EF-1α and mtSSU sequences for Entomophthoromycotina, with some chytrid and mucoralean fungi as outgroups. Three new genera and one Chinese new record are shown in red. Maximum likelihood bootstrap values (≥ 50%) / Bayesian posterior probabilities (≥ 0.50) of main clades are indicated along branches. Scale bar indicates substitutions per site.
https://binary.pensoft.net/fig/395592Taxonomy
In order to provide a more natural taxonomic classification, four genera (Capillidium, Conidiobolus, Microconidiobolus and Neoconidiobolus) and their type species (Ca.heterosporum, C.utriculosus, M.paulus and N.thromboides) are described here in this paper. Additionally, a new record Ca.adiaeretum, C.coronatus and C.iuxtagenitus with new isolates from China and C.khandalensis being first reported to produce microconidia are illustrated herein.
FungiEntomophthoralesAncylistaceaeD46F46A3-E0E1-5C9B-AD08-2A5C10757790Capillidium831596B. Huang & Y. Niegen. nov.Etymology.
Referring to unique ellipsoidal secondary conidia (capilliconidia).
Type species.
Capillidiumheterosporum (Drechsler) B. Huang & Y. Nie.
Description.
Mycelia colourless. Primary conidiophores simple, bearing a single primary conidia. Primary conidia forcibly discharged multinucleate, colourless, globose, pyriform to obovoid. Two kinds of replicative conidia, the first one is similar and smaller than primary conidia, the second one (capilliconidia) arises from elongate and slender conidiophores. Zygospores present or absent, formed in axial alignment with conjugating segments, globose to subglobose, often smooth, sometimes rough, colourless or yellowish.
Notes.
Conidiobolussubgen.Capillidium Ben-Ze’ev & Kenneth was firstly established to include species with capilliconidia (Ben-Ze’ev and Kenneth 1982). In this phylogenetic analysis, all members of the subgenus Capillidium grouped with good support (100/1.00) and, therefore, it was raised from subgenus to genus status based on the monophyly, as well as the stability to form ellipsoidal secondary conidia (capilliconidia). In addition to capilliconidia, C.adiaeretus also produces microconidia.
FungiEntomophthoralesAncylistaceae033D89FA-FE07-5B76-ABF5-D805C655AA30Capillidiumheterosporum831601(Drechsler) B. Huang & Y. Niecomb. nov.Figure 2ConidiobolusheterosporusDrechsler, Am. J. Bot. 40: 107 (1953). Basionym.=Conidiobolusrugosus Drechsler, Am. J. Bot. 42: 437 (1955). Specimens examined.
Colonies on PDA at 25 °C after 3 d, white, reaching ca. 21 mm in diameter. Mycelia colourless, 5–9 μm wide. Primary conidiophores, colourless, unbranched and producing a single globose conidium with widening upwards, extending to a length of 30–245 μm into the air, 8–17 μm wide. Primary conidia forcibly discharged, colourless, globose to subglobose, measuring 12–37 μm in greatest length and 11–31 μm in total width, including a basal papilla 1.5–5 μm high and 5–12 μm wide. After discharging on to 2% water-agar, similar and smaller secondary conidia arise from primary conidia, 1–6 ellipsoidal secondary conidia (capilliconidia, 10–20 × 12–38 μm) arise from slender conidiophores (50–250 × 2.5–4 μm). Resting spores not observed.
Notes.
The ex-type living culture is ATCC 12941 (United States, Maryland, 18 Mar 1952, Drechsler).
Capillidiumheterosporuma colony on PDA after 3 d at 25 °C b primary conidiophores bearing primary conidia c primary conidia d, e, f ellipsoidal secondary conidia arising from slender conidiophores g, h production of secondary conidia. Scale bars: 10 mm (a); 20 μm (b, c, d, g, h); 100 μm (e, f).
https://binary.pensoft.net/fig/395593FungiEntomophthoralesAncylistaceae348681A3-0E38-5892-8481-DEEAF76FC24ACapillidiumadiaeretum831602(Drechsler) B. Huang & Y. Niecomb. nov.Figure 3ConidiobolusadiaeretusDrechsler, J. Wash. Acad. Sci. 43: 42 (1953). Basionym.Specimens examined.
China, Jiangsu Province, Nanjing City, Laoshan Forest Park, 32°5'58"N, 118°35'53"E, Plant detritus, 1 Dec 2018, Y. Nie and Y. Gao, HMAS 248358, culture CGMCC 3.15888 (=RCEF 6550).
Description.
Colonies on PDA at 25 °C after 3 d, white, reaching ca. 7–10 mm in diameter. Mycelia colourless, 3–4.5 μm wide. Primary conidiophores, colourless, unbranched and producing a single globose conidium with widening upwards; they offer a pronounced dimensional contrast with the mycelial filaments, extending to a length of 50–210 μm into the air, 3–25 μm wide. Primary conidia forcibly discharged, colourless, globose, measuring 15–45 μm in greatest length and 13–42 μm in total width, including a basal papilla 2–6 μm high and 5–17 μm wide. After discharging on to 2% water-agar, similar and smaller secondary conidia arise from primary conidia, two generations of multiple spherical units forming on the parent globose conidia Microconidia only formed from the second set, 5–12 × 9–10 μm. Capilliconidia formed readily from discharged microconidia, 16–24 × 5–6 μm. Chlamydospores formed within the substratum, colourless, globose to ellipsoidal, 13–40 × 15–45 μm.
Notes.
The species was firstly reported from America (Drechsler 1953a). The ex-type living culture is ATCC 12589 isolated by Drechsler (1953a). It is mainly characterised and differs from other Capillidium species by its ability to form both microconidia and capilliconidia (Callaghan et al. 2000). The Chinese specimen CGMCC 3.15888 clusters completely (100/1.00) with an isotype ARSEF 451 (98% sequence similarity in nucLSU) and fits well with its morphological descriptions. It is reported in China for the first time.
Capillidiumadiaeretuma colony on PDA after 3 d at 25 °C b mycelia c, d primary conidiophores bearing primary conidia e, f primary conidia g Production of secondary conidia h first stage of forming microconidia i second stage of forming microconidia j, k ellipsoidal secondary conidia arising from slender conidiophores l chlamydospores. Scale bars: 10 mm (a); 100 μm (b); 20 μm (c–l).
Mycelia colourless. Primary conidiophores simple or branched dichotomously, positively phototropic, bearing a single or 2–4 primary conidia. Primary conidia forcibly discharged, multinucleate, colourless, pyriform, obovoid, globose to subglobose. Secondary conidia usually with shape of primary conidia but smaller, formed singly on short secondary conidiophores. Microspores arising from primary or secondary conidia. Villose appendaged globose conidia and formed villose conidia. Chlamydospores formed intercalarily within assimilative hyphae. Zygospores formed in axial alignment with one or two (homothallic or heterothallic) conjugating segments.
Notes.
C.utriculosus, the type species of the genus Conidiobolus, has not been re-collected since Brefeld isolated it in 1884 and most taxonomists working on entomophthoroid fungi now universally recognised it as C.coronatus (Gryganskyi et al. 2013). However, the smaller pear-shaped conidia of C.utriculosus are different from the larger globose conidia of C.coronatus and villose spores in C.coronatus are not observed in C.utriculosus (Brefeld 1884; King 1977). Consequently, C.coronatus is not synonymised with C.utriculosus in this study. Instead, this study agrees with Srinivasan and Thirumalachar (1967) and King (1977) to place C.minor in synonymy with C.utriculosus because the small conidia of C.minor were probably replicative conidia of C.utriculosus. Nevertheless, neither C.utriculosus nor C.minor has available living cultures. Therefore, we have not yet designated an epitype and thus no DNA sequences for explaining this type. Fortunately, we are able to recognise clade III (Fig. 1) as Conidiobolus on the basis of its synapomorph, namely microspores.
Due to the lack of ex-type, plates 3, 4, and 5 in Brefeld, Mykol. Untersuch. 6(2): 35 (1884) are designated here as the lectotype for Conidiobolusutriculosus.
FungiEntomophthoralesAncylistaceaeC501E77E-6761-514E-8E81-5DC66636ED0FConidioboluscoronatus283037(Costantin) A. Batko, Entomophaga, Mémoires hors série 2: 129 (1964)Figure 4BoudierellacoronataCostantin, Bull. Soc. mycol. Fr. 13: 40 (1897). Basionym.Delacroixiacoronata(Costantin) Sacc. & P. Syd., Syll. fung. (Abellini) 14(1): 457 (1899).Entomophthoracoronata(Costantin) Kevorkian, J. Agric. Univ. Puerto Rico 21(2): 191 (1937).= Conidiobolusvillosus G.W. Martin, Bot. Gaz. 80(3): 317 (1925). Specimens examined.
Colonies grown on PDA for 3 d at 21 °C, reaching ca. 65 mm in diameter. Mycelia colourless, 8–20 μm wide. Primary conidiophores, positively phototropic, colourless, unbranched and producing a single globose conidium, extending to a length of 53–287 μm into the air, 7.5–20.5 μm wide. Primary conidia forcibly discharged, colourless, globose, measuring 36–52 μm in greatest width and 42–65 μm in total length, including a basal papilla 12–18 μm high and 6.5–14 μm wide. After discharging on to 2% water-agar, similar and smaller secondary conidia arise from primary conidia. Microconidia produced readily from primary conidia, globose or almond-shaped, 13–19 × 11–15 μm. Villose spores formed after 4–5 d, globose, 20–42 μm.
Notes.
The ex-type living culture is ATCC 28691 (United States, Louisiana, Plant detritus, 3 January 1972). Due to the absence of molecular data of ex-type strain ATCC 28691, the molecular data of the authentic strain NRRL 28638, which has been applied in many other phylogenetic analysis (James et al. 2006; Liu and Voigt 2011; Gryganskyi et al. 2012; Tretter et al. 2014; Spatafora et al. 2016), was used in this study instead. The monotypic genus Delacroixia was typified by D.coronata which was transferred from an ascomycete Boudierellacoronata Costantin (Costantin 1897; Saccardo and Sydow 1899). After that, it was reclassified as a subgenus of Conidiobolus, namely Conidiobolus sub. Delacroixia (Sacc. & P. Syd.) Tyrrell & MacLeod to define all those Conidiobolus species capable of forming microspores and, consequently, D.coronata was recombined as C.coronatus (Tyrrell and MacLeod 1972; Ben-Ze’ev and Kenneth 1982).
Conidioboluscoronatusa colony on PDA after 3 d at 21 °C b primary conidia c production of secondary conidia d, e primary conidiophores bearing primary conidia f, g microconidia h villose spores. Scale bars: 10 mm (a); 20 μm (b–h).
Colonies on PDA at 21 °C after 3 d white, flat, slow-growing, reaching ca. 13 mm in diameter. Mycelia colourless, 5.5–11 μm wide. Primary conidiophores, positively phototropic, arising from hyphal segments, colourless, 28–75 × 7.5–10 μm, unbranched and producing a single globose conidium. Primary conidia forcibly discharged, globose, 27–37 × 21–28 μm, with a basal papilla 6–10 μm wide. Secondary conidia arising from primary conidia, similar to, but smaller than the primary ones, forcibly discharged. Tertiary conidium fusiform arising from primary conidia, 30–45 × 16–22 μm. Zygospores in a position separated by a short beak near a lateral conjugation, globose to subglobose, smooth, 21–25 × 17–24 μm, with a 1–2 μm thick wall.
Notes.
The ex-type living culture is ARSEF 6378 (United Kingdom, Staffordshire, Plant detritus, 31 October 1983, M. F. Smith).
a–gConidiobolusiuxtagenitushConidioboluskhandalensisa colony on PDA after 3 d at 21 °C b primary conidiophores bearing primary conidia c primary conidia d tertiary fusiform conidium from a globose spore e zygospore formation with the beak almost emptied of protoplasm f production of secondary conidia g zygospores h microconidia produced from global conidia. Scale bars: 10 mm (a); 20 μm (b–h).
India, Khandala, Dec. 1961, Srinivasan and Thirumalachar, ATCC 15162.
Description.
Refer to Srinivasan and Thirumalachar (1962b). Microconidia produced from global conidia on the 2% water-agar at 16 °C (Fig. 5h).
Notes.
According to the original morphological description (Srinivasan and Thirumalachar 1962b) and the re-examination by King (1977), microconidia have not been reported. However, we observed the microconidia produced from global conidia on 2% water-agar at 16 °C. Moreover, this specimen was located in the Conidiobolus lineage (Figure 1) which was supported by our morphological analyses.
FungiEntomophthoralesAncylistaceae744CDD7E-6E4B-56B4-BF10-59FE98E3F107Microconidiobolus831597B. Huang & Y. Niegen. nov.Etymology.
Referring to small discharged primary conidia.
Type species.
Microconidioboluspaulus (Drechsler) B. Huang & Y. Nie.
Description.
Mycelia colourless. Primary conidiophores simple and short, bearing a single primary conidia. Primary conidia forcibly discharged, multinucleate, colourless, globose to obovoid, usually small, mostly less than 20 μm. Only globose replicative conidia produced, similar and smaller than primary conidia. Chlamydospores globose, formed terminally on hyphae or from globose cells by thickening of the wall. Zygospores formed in axial alignment with two conjugating segments, globose to ellipsoidal, smooth and yellowish.
Notes.
This genus includes three species producing smaller primary conidia (mostly less than 20 μm) without microspores or capilliconidia compared to other Conidiobolus spp. These three species are C.nodosus, C.paulus and C.terrestris. According to the taxonomic scheme of Conidiobolus by King (1977), C.undulatus is a synonym of C.paulus, which is supported herein by molecular evidence (Figure 1). However, the phylogeny does not support C.nodosus and C.terrestris as synonyms of C.lachnodes, since the former two were located in clade IV and the latter in clade II (Figure 1). Therefore, we accept the taxonomic status at species level for C.nodosus and C.terrestris, based on the morphological and phylogenetic analyses.
The ex-type living culture is ATCC 12942 (United States, Wisconsin, 18 November 1954, Drechsler).
FungiEntomophthoralesAncylistaceae8A004282-B8EF-516F-A99D-77EBD48BE926Neoconidiobolus831598B. Huang & Y. Niegen. nov.Etymology.
Referring to the subgenus Conidiobolus raised to generic rank.
Type species.
Neoconidiobolusthromboides (Drechsler) B. Huang & Y. Nie.
Description.
Mycelia colourless. Primary conidiophores simple, sometimes branched from hyphal knots or differentiated from aerial hyphae, positively phototropic, bearing a single primary conidium. Primary conidia forcibly discharged, multinucleate, colourless, globose, pyriform to obovoid. Replicative conidia similar and smaller than primary conidia. Chlamydospores globose, formed terminally on hyphae or from globose cells by thickening of the wall. Zygospores formed in axial alignment with two conjugating segments, globose to ellipsoidal, smooth, colourless, rarely pale yellowish.
Notes.
The genus Neoconidiobolus is strikingly similar to the subgenus Conidiobolus which produces neither microconidia nor capilliconidia. All members in the clade of Neoconidiobolus share the following characteristics: forcibly discharged, colourless, globose, pyriform to obovoid primary conidia. Two kinds of replicative conidia produced. One is discharged, similar and smaller than primary conidia and the other is elongate and forcibly discharged. Two types of resting spores produced: zygospores and chlamydospores.
FungiEntomophthoralesAncylistaceae672EE869-86FB-5516-BDC0-D34E25189680Neoconidiobolusthromboides831606(Drechsler) B. Huang & Y. Niecomb. nov.Figure 6ConidiobolusthromboidesDrechsler, J. Wash. Acad. Sci. 43: 38 (1953). Basionym.Specimens examined.
Colonies grown on PDA for 3 d at 25 °C, white, reaching ca. 30 mm diameter. Mycelium colourless, filamentous, 5–7.5 µm wide. Primary conidiophores colourless, unbranched and producing a single conidium, 50–122.5 × 6–16.5 µm. Primary conidia forcibly discharged, colourless, globose to subglobose, 20–26.5 µm wide, 26.5–34 µm long, including a basal papilla 6–10 µm wide. Secondary conidia globose, forming from the primary conidia. Zygospores most often formed between segments of separate hyphae. Mature zygospores smooth, globose to subglobose, 25–30 μm in diameter with wall 2–3 μm thick.
Notes.
The ex-type living culture is ATCC 12587 (United States, New Hampshire, September 1957, Drechsler).
Neoconidiobolusthromboidesa colony on PDA after 3 d at 25 °C b, c primary conidiophores bearing primary conidia d production of secondary conidia e zygospores f primary conidia. Scale bars: 10 mm (a); 20 μm (b–d, f); 40 μm (e).
https://binary.pensoft.net/fig/395597More new combinations
In addition to previously described taxa, more new combinations were proposed herein and their descriptions refer to relevant protologues.
FungiEntomophthoralesAncylistaceae49E94C4C-B2DA-5D26-98C6-CCA98B7576B6Capillidiumbangalorense831607(Sriniv. & Thirum.) B. Huang & Y. Niecomb. nov.ConidiobolusbangalorensisSriniv. & Thirum., Mycologia 59(4): 702 (1967). Basionym.FungiEntomophthoralesAncylistaceaeA9419A69-2648-5350-A992-B432913ECAD1Capillidiumdenaeosporum831608(Drechsler) B. Huang & Y. Niecomb. nov.ConidiobolusdenaeosporusDrechsler, J. Wash. Acad. Sci. 47: 309 (1957). Basionym.FungiEntomophthoralesAncylistaceae63DB8CDB-AF6B-5281-8373-450ED3B2764BCapillidiumlobatum831609(Sriniv. & Thirum.) B. Huang & Y. Niecomb. nov.ConidioboluslobatusSriniv. & Thirum., J. Elisha Mitchell scient. Soc. 84: 212 (1968). Basionym.FungiEntomophthoralesAncylistaceae098895D0-4C8A-590C-A70A-E436E6E987D9Capillidiumpumilum831610(Drechsler) B. Huang & Y. Niecomb. nov.ConidioboluspumilusDrechsler, J. Wash. Acad. Sci. 45: 115 (1955). Basionym.= Conidiobolusglobuliferus Drechsler, Am. J. Bot. 43: 783 (1957) [1956]. = Conidiobolusinordinatus Drechsler, J. Wash. Acad. Sci. 47: 312 (1957). FungiEntomophthoralesAncylistaceaeCDF80202-031E-5743-956C-E7B685CB859FCapillidiumrhysosporum831611(Drechsler) B. Huang & Y. Niecomb. nov.ConidiobolusrhysosporusDrechsler, Am. J. Bot. 41: 567 (1954). Basionym.FungiEntomophthoralesAncylistaceae0645FC92-B51F-5002-A8B6-0B825ACBE86DMicroconidiobolusnodosus831624(Sriniv. & Thirum.) B. Huang & Y. Niecomb. nov.ConidiobolusnodosusSriniv. & Thirum., Mycologia 59(4): 705 (1967). Basionym.FungiEntomophthoralesAncylistaceae2F620080-2CCB-5A7C-897C-E4D1D3C2DCDEMicroconidiobolusterrestris831625(Sriniv. & Thirum.) B. Huang & Y. Niecomb. nov.ConidiobolusterrestrisSriniv. & Thirum., Mycopathol. Mycol. appl. 36(3–4): 344 (1968). Basionym.FungiEntomophthoralesAncylistaceae7C8ECD45-9291-5244-9F60-A808D89F7ECANeoconidioboluscouchii831626(Sriniv. & Thirum.) B. Huang & Y. Niecomb. nov.ConidioboluscouchiiSriniv. & Thirum., J. Elisha Mitchell scient. Soc. 84: 211 (1968). Basionym.FungiEntomophthoralesAncylistaceaeA8105E3E-8740-529B-AF8F-B699C93DBD05Neoconidioboluslachnodes831627(Drechsler) B. Huang & Y. Niecomb. nov.ConidioboluslachnodesDrechsler, Am. J. Bot. 42: 442 (1955). Basionym.FungiEntomophthoralesAncylistaceae6531CB5C-3134-596D-A5BB-EF4FF6757255Neoconidiobolusmirabilis831628(Y. Nie & B. Huang) B. Huang & Y. Niecomb. nov.ConidiobolusmirabilisY. Nie & B. Huang, Mycol. Progr. 17(10): 1204 (2018). Basionym.FungiEntomophthoralesAncylistaceaeFEAA69F5-A749-5BB0-9095-AEF429D1BAFANeoconidiobolusosmodes831629(Drechsler) B. Huang & Y. Niecomb. nov.ConidiobolusosmodesDrechsler, Am. J. Bot. 41: 571 (1954). Basionym.= Conidiobolusantarcticus S. Tosi, Caretta & Humber, Mycotaxon 90(2): 344 (2004). FungiEntomophthoralesAncylistaceae50B684D1-0579-57E4-808E-4B1C906FDFEENeoconidioboluspachyzygosporus831630(Y. Nie & B. Huang) B. Huang & Y. Niecomb. nov.ConidioboluspachyzygosporusY. Nie & B. Huang, Mycol. Progr. 17(10): 1206 (2018). Basionym.FungiEntomophthoralesAncylistaceae278F1289-BCF8-5E30-B7FA-6090B1923C42Neoconidiobolussinensis831631(Y. Nie, X.Y. Liu & B. Huang) B. Huang & Y. Niecomb. nov.ConidiobolussinensisY. Nie, X.Y. Liu & B. Huang, Mycotaxon 120: 432 (2012). Basionym.FungiEntomophthoralesAncylistaceaeA39E4A47-D4E5-532D-934E-D305571D2923Neoconidiobolusstilbeus831632(Y. Nie & B. Huang) B. Huang & Y. Niecomb. nov.ConidiobolusstilbeusY. Nie & B. Huang, Mycosphere 7(6): 804 (2016). Basionym.FungiEntomophthoralesAncylistaceaeEBB4B068-7277-517B-B39B-9E1C0CA6D6FFNeoconidiobolusstromoideus831633(Sriniv. & Thirum.) B. Huang & Y. Niecomb. nov.ConidiobolusstromoideusSriniv. & Thirum., Sydowia 16(1–6): 65 (1963) [1962]. Basionym.FungiEntomophthoralesAncylistaceae0D7F7877-D73B-57D1-9B8E-5C0662E1290CNeoconidiobolusvermicola831634(J.S. McCulloch) B. Huang & Y. Niecomb. nov.EntomophthoravermicolaJ.S. McCulloch, Trans. Br. mycol. Soc. 68(2): 173 (1977). Basionym.Macrobiotophthoravermicola(J.S. McCulloch) B.E. Tucker, Mycotaxon 13(3): 499 (1981).Discussion
The phylogenetic position of Basidiobolus in the Kingdom Fungi has been problematic for a long time. Previous phylogenetic analyses of the rDNA (18S, 28S and 5.8S) sequences grouped Basidiobolus outside or basal in the Entomophthorales (Nagahama et al. 1995; Jensen et al. 1998; White et al. 2006). Combined with the study of other protein-coding molecular markers, Basidiobolus was located inside the Entomophthorales (James et al. 2006). Recently, according to the phylogeny of much more available molecular data of entomophthoroid fungi in three families, Basidiobolus was grouped basal to other entomophthoroid taxa (Gryganskyi et al. 2012) which was also supported by the phylogenomic analyses of zygomycete fungi (Spatafora et al. 2016) and by the multi-gene analyses in this study. Although the morphological characteristics of Batkoa were similar to Conidiobolus, the Batkoa lineage appeared to be most closely related to the other taxa in the Entomophthoraceae Clade and should be distinguished from Conidiobolus lineage by its obligate pathogenicity for invertebrates and by staining readily, while most members of Conidiobolus are saprobic and non-staining.
The phylogenetic relationship of the genus Conidiobolus has been unclear for a long time, because of its high heterology (Gryganskyi et al. 2013). This article used more available ex-type strains to revise this genus, based on phylogeny and morphology. According to Figure 1, four main clades were reconstructed and the results showed that Conidiobolus s.l. is not a monophyletic group but paraphyletic with Macrobiotophthoravermicola. The M.vermicola was originally placed in Entomophthora (Mcculloch 1977) and transferred to Macrobiotophthora, based on the morphological characters of primary spores, secondary spores and zygospores (Tucker 1981). The paraphyletic relationship between Macrobiotophthoravermicola and Conidiobolus s.l. was also revealed by Gryganskyi et al. (2012). In this paper, we treated it as a new combination and, therefore, proposed a monophyletic group of the new genus Neoconidiobolus.
In Clade I of the genus Capillidium, seven species grouped in a monophyletic clade with good support (100/1.00) and the synapomorph of producing capilliconidia: Conidiobolusadiaeretus (= Capillidiumadiaeretum), Co.bangalorensis (= Ca.bangalorensis), Co.denaeosporus (= Ca.denaeosporum), Co.heterosporus (= Ca.heterosprum), Co.lobatus (= Ca.lobatum), Co.pumilus (= Ca.pumilum) and Co.rhysosporus (= Ca.rhysosporum). As a note, Co.denaeosporus was synonymised with Co.pumilus (King 1976b), but herein its taxonomic status of species level was accepted according to the phylogeny. Co.adiaeretus forms not only capilliconidia but also microspores (Callaghan et al. 2000).
In Clade II of the genus Neoconidiobolus, all 14 strains comprising 10 species produce neither microspores nor capilliconidia. Amongst these, C.antarcticus was identified as a synonym of C.osmodes (Chen and Huang 2018), which was confirmed here as they grouped into a robust clade.
Considering its long history and significant impact, we kept and emended the genus Conidiobolus and the original illustrations of the type species C.utriculosus (Brefeld 1884) were designated as its lectotype. Thus, we were able to recognise clade III under the genus name Conidiobolus on the basis of its synapomorph, namely microspores. In Clade III of the genus Conidiobolus, all species definitely produce microspores, except Conidiobolusdabieshanensis, C.iuxtagenitus, C.khandalensis and C.lichenicolus. Microspores have never been observed in C.dabieshanensis and C.iuxtagenitus (King 1977; Waters and Callaghan 1989; Nie et al. 2017), but cases for C.khandalensis and C.lichenicolus are somewhat different. For C.khandalensis, the protologue did not document any microspores (Srinivasan and Thirumalachar 1962b; King 1977), but they can be observed on 2% water-agar at 16 °C (Fig. 5h). Although the microspore of C.lichenicolus was not mentioned in the original description, the ability to produce microspores has been exhibited in accordance with original illustrations (Srinivasan and Thirumalachar 1968a). The phylogeny also resulted in the following taxonomic treatments. On the one hand, some previously synonymised taxa recover their specific status, for example, C.gonimodes, C.megalotocus and C.mycophagus should be separated from C.incongruus, C.macrosporus and C.mycophilus, respectively. On the other hand, C.chlamydosporus is synonymised with C.firmipilleus.
In Clade IV of the genus Microconidiobolus, Conidiobolusundulatus was identified as a synonym of C.paulus (= M.paulus) by King (1976b), which is supported by our molecular data. Otherwise, C.nodosus (= M.nodosus) and C.terrestris (= M.terristris) were classified as synonyms of C.lachnodes (= Neoconidioboluslachnodes) in the study of King (1976b). Morphologically, C.lachnodes bears larger primary conidia (9–25 × 10–27 μm) than C.nodosus (13–16 × 17–22 μm) and C.terrestris (8–12 μm in width) (Drechsler 1955b; Srinivasan and Thirumalachar 1967, 1968a). Furthermore, C.lachnodes was located in Clade II and is distantly related to C.nodosus and C.terrestris. Therefore, C.nodosus and C.terrestris are accepted as two distinct species. This clade comprises four ex-type strains, all producing smaller primary conidia (mostly less than 20 μm) and can be morphologically easily distinguished from other Conidiobolus species.
Phylogenetically, Conidioboluslamprauges does group with Clade III and received strong bootstrap support (100/1.00). Morphologically, this species produces small primary conidia (12.5–20 × 15–22 μm) without microconidia or capilliconidia and is similar to species within Clade IV. Its taxonomic status remains unclear in the present study.
Acknowledgements
We thank Dr. Z.F. Yu (Yunnan University) for improving the manuscript. We also thank Y. Gao (Jiangxi Agricultural University) for collecting some Conidiobolus strains. This project was supported by the National Natural Science Foundation of China (Nos. 31900008, 30770008 and 31670019).
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