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Asexual-sexual morph connection in the type species of Berkleasmium
IMA Fungus volume 8, pages99–105(2017)
Berkleasmium is a polyphyletic genus comprising 37 dematiaceous hyphomycetous species. In this study, independent collections of the type species, B. concinnum, were made from Eastern North America. Nuclear internal transcribed spacer rDNA (ITS) and partial nuc 28S large subunit rDNA (LSU) sequences obtained from collections and subsequent cultures showed that Berkleasmium concinnum is the asexual morph of Neoacanthostigma septoconstrictum (Tubeufiaceae, Tubeufiales). Phylogenies inferred from Bayesian inference and maximum likelihood analyses of ITS-LSU sequence data confirmed this asexual-sexual morph connection and a re-examination of fungarium reference specimens also revealed the co-occurrence of N. septoconstrictum ascomata and B. concinnum sporodochia. Neoacanthostigma septoconstrictum is therefore synonymized under B. concinnum on the basis of priority. A specimen identified as N. septoconstrictum from Thailand is described as N. thailandicum sp. nov., based on morphological and genetic distinctiveness.
Naturalist Thomas Gibson Lea botanized extensively in Ohio until his death in 1844, after which his fungal specimens were sent to his correspondent Miles J. Berkeley (Lea 1849). Berkeley (1845) described Sporidesmium concinnum (“A very pretty object under the microscope”) from Lea’s specimen growing on dead wood from a rotten tree trunk (host unknown) in Ohio. Zobel (in Corda 1854) later described the new genus Berkleasmium to accommodate S. concinnum, illegitimately devising the name Berkleasmium cordeanum. More than one century later, Moore (1958) re-established the generic name Berkleasmium to accommodate sporodochial species previously placed in Sporidesmium, later accepting ten species (Moore 1959). Berkleasmium currently comprises 37 species characterized by sporodochial conidiomata bearing macronematous conidiophores and monoblastic conidiogenous cells that give rise to brown or black, dry, rhexolytically-seceding, dictyoconidia (Ellis 1971, Bussaban et al. 2001, Seifert et al. 2011, http://www.speciesfungorum.org/). Berkleasmium species are associated with a variety of decaying above-ground tissues of monocot and dicot plants from terrestrial and aquatic habitats. No known sexual morph has yet been connected to Berkleasmium.
Available sequences in GenBank are restricted to species described from Thailand or Micronesia, including Berkleasmium crunisia, B. micronesicum, B. nigroapicale, and B. typhae. Previous phylogenetic analyses based on SSU and LSU sequences of these species indicate the polyphyly of Berkleasmium, with B. micronesicum and B. nigroapicale placed incertae sedis sister to Sporormiaceae, and B. crunisia and B. typhae placed incertae sedis sister to Mycopepon smithii (Pinnoi et al. 2007, Wang et al. 2007, Hyde et al. 2016). This polyphyly is not surprising given the treatment of Berkleasmium as a genus characterised by the sporodochia bearing dark brown dictyoconidia. For example, Monodictys monilicellularis was transferred to Berkleasmium because of sporodochia formation, despite the presence of distinctive sterile moniliform appendages, while B. papillatum was placed within the genus despite proliferating conidiophores; the taxonomic informativeness of these characters remains to be tested by molecular phylogenetic analyses (Raghuveer Rao & Rao 1964, Whitton et al. 2012).
Determining the phylogenetic placement of the type species, B. concinnum, is crucial to delineate taxonomic boundaries within this polyphyletic and morphologically heterogeneous genus. In this study, ITS and LSU sequences were analyzed with other taxa in Tubeufiales to estimate phylogenetic relationships.
Materials and Methods
Sampling and isolation of fungi
Field collections of Berkleasmium concinnum were made independently in Gatineau, Quebec, Canada and the Great Smoky Mountains National Park, North Carolina and Tennessee, USA. Single conidium cultures were generated by transferring individual conidia to 6 cm diam Petri dishes containing MEA using an electrolytically sharpened tungsten needle (Brady 1965). Conidial germination was visually verified and cultures were incubated at 16 °C under 12:12 h dark:light conditions. Specimens were accessioned in the Canadian National Mycological Herbarium (Ottawa, Canada; DAOM) or the Illinois Natural History Survey Fungarium (Champaign, USA; ILLS) and living cultures were deposited in the Canadian Collection of Fungal Cultures (Ottawa, Canada; DAOMC).
Conidiomata and ascomata from fresh and dried specimens were mounted in deionized water, 5% KOH, or lactic acid. Observations were made using an Olympus BX50 light microscope (Olympus, Tokyo) and an Olympus SZX12 stereomicroscope and micrographs were captured using an InfinityX-32 camera (Lumenera, Ottawa) and Infinity Analyze (Lumenera) software. Photographic plates were assembled using Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA).
DNA extraction, PCR amplification, and sequencing
Total genomic DNA was extracted from 12-wk-old cultures or directly from conidiomata of B. concinnum using the Ultraclean Microbial DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) or NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany) following the manufacturers’ protocol. The entire ITS and the first two domains of LSU were amplified and sequenced following the methods of Promputtha & Miller (2010) and Tanney et al. (2015).
Sequence contigs were assembled and trimmed using Geneious R8 v. 8.1.5 (Biomatters, Auckland, New Zealand). ITS and LSU sequences were concatenated to create a dataset containing 37 sequences that was aligned using MAFFT v. 7 (Katoh & Standley 2013) and visually inspected in Geneious. The most suitable sequence evolution model (GTR+I+G) was determined based on the optimal Akaike information criterion scores in MrModeltest v. 2.2.6 (Nylander 2004). The ex-type culture of Botryosphaeria corticis (CBS119047; NR 111213) was selected as outgroup based on previous analyses (Sanchez et al. 2012). Bayesian inference (BI) phylogenetic reconstruction was performed with MrBayes v. 3.2 (Ronquist et al. 2012). Three independent Markov Chain Monte Carlo (MCMC) samplings were performed with 12 chains (11 heated and one cold) with sampling every 500 generations until the standard deviation of split frequencies reached a value < 0.01. The first 25% of trees were discarded as burn-in and the remaining trees kept and combined into one 50% majority rule consensus tree. Convergence was assessed from the three independent runs using Tracer v. 1.6 (Rambaut et al. 2014). Maximum likelihood (ML) analysis was performed using RAxML v. 8.2.4 (Stamatakis 2014) in PAUP v. 4.0b10 (Swofford 2003) starting from a random starting tree with 1000 bootstrap replicates. Consensus trees were visualized in FigTree 1.4.2 (available at http://tree.bio.ed.ac.uk/software/figtree/) and exported as SVG vector graphics for assembly in Adobe Illustrator v10 (Adobe Systems, San Jose, CA). All novel sequences used in this study were accessioned in GenBank (Table 1) and taxonomic novelties and associated metadata were deposited in MycoBank (www.mycobank.org).
The concatenated ITS-LSU alignment consisted of 37 sequences and 1277 positions. All Berkleasmium concinnum specimens shared identical ITS sequences except for a single A-to-G transition (position 441 in alignment) in specimen ILLS 80804. Berkleasmium concinnum ITS sequences were identical to the ITS sequence of the holotype of Neoacanthostigma septoconstrictum (ILLS 59356), except for the single bp difference in ILLS 80804. LSU sequences for all Berkleasmium concinnum isolates were identical and 99% similar to the N. septoconstrictum type (ILLS 59356; NR_119758), with one A-to-T transversion (position 758) and two C-to-T transitions (positions 806 and 1213).
Phylogenies inferred from BI and ML analyses were generally concordant. However, the backbone topology was polytomous in the BI analysis versus dichotomous in the ML analysis, albeit with low-supported branch nodes in both analyses. The placement of Acanthostigma filiforme was also discordant among both analyses, probably a result of the poorly-supported backbone, and PP values were generally higher than BS values. Berkleasmium concinnum and N. septoconstrictum, including the type species N. fusiforme (MFLUCC 110510; KF301529) and a specimen identified as N. septoconstrictum (MFLUCC 151248; KX454176) but with a distinct ITS sequence, formed a well-supported clade in both analyses (BS = 93; PP = 1) (Fig. 1). The Berkleasmium-Neoacanthostigma clade was weakly-supported (BS = 37; PP = 0.57) sister to a clade comprising Helicoma conicodentatum, Helicosporium linderi, Tubeufia khunkornensis, and a strain identified as Chlamydotubeufia cf. huaikangplaensis.
Sequence similarity and ITS-LSU phylogenetic analyses reveal that B. concinnum is the asexual morph of N. septoconstrictum (Tubeufiaceae, Tubeufiales) and that the type species of both genera occur in a well-supported clade (BS = 100; PP = 1). Although not originally reported with the description of N. septoconstrictum (Promputtha & Miller 2010), re-examination of the type specimen revealed numerous conidiomata of B. concinnum covering the woody substrate along with ascomata of N. septoconstrictum. Examining additional reference specimens of B. concinnum confirmed the co-occurrence of the ascomatal sexual morph (i.e. N. septoconstrictum) among the asexual B. concinnum conidiomata (DAOM 24916, DAOM 29377, DAOM 41029, DAOM 75764, DAOM 155883, DAOM 155884, DAOM 210103, ILLS 80802; Fig. 2A–C). Since Berkleasmium Zobel 1854 is an earlier name than Neoacanthostigma Boonmee et al. 2014, N. septoconstrictum must be synonymized under B. concinnum, on the assumption that all priority currently accorded to generic names typified by a sexual morph is ended in July 2017. In addition, the specimen previously identified by Hyde et al. (2016) as N. septoconstrictum (MFLUCC 15–1248; KX454176) is not conspecific with the type specimen of this species (ILLS59356; NR119758) based on asexual morph dissimilarity (hyaline helicoid conidia) and genetic (13% ITS sequence divergence) differences and is therefore described as a new species, N. thailandicum.
Berkleasmium concinnum (Berk.) S. Hughes, Canad. J. Bot. 36: 740 (1958).
Basionym: Sporidesmium concinnum Berk., London J. Bot. 4: 309 (1845).
Synonyms: Berkleasmium cordeanum Zobel, in Corda, Icon. Fung. 6: 4 (1854); nom. illegit. (Art. 52.1).
Neoacanthostigma septoconstrictum (Promp. & A.N. Mill.) S. Boonmee & K.D. Hyde, Fungal Diversity 68: 279 (2014).
Acanthostigma septoconstrictum Promp. & A.N. Mill., Mycologia 102: 579 (2010).
Type: USA: Ohio: on dead wood, T.G. Lea 168 (K(M) — holotype, n.v.; K(M)-IMI94327 — slide ex-holotype).
Other specimens examined: Canada: Quebec: Gatineau, Aylmer, Boucher Forest, on large, rotten, decorticated hardwood log, 17 Jul. 2015, J.B. Tanney (DAOM 696473); ibid., 29 Aug. 2015, J.B. Tanney (DAOM 696486); ibid., 10 Jul. 2015, J.B. Tanney (DAOM 696499, DAOMC 251512, DAOMC 251513); ibid., fallen hardwood log, 5 Sep. 2016, J.B. Tanney (NB-789). — Russia: Primorsky Krai, Khasansky District, Kedrovaya Pad Nature Preserve, log of an unknown deciduous species, 3 Oct. 1987, I.M. Bacuebeba (DAOM 210103). — USA: Louisiana: St. Tammany Parish, Honey Island Swamp near Pearl River, rotten wood, 6 Jun. 1976, S.J. Hughes (DAOM 155883); ibid., 6 Jun. 1976, W.B. Cooke (DAOM 155884). Massachusetts: Salem, on Salix wood, 1834 (DAOM 43686). Missouri: St Louis County, Benbush, on dead wood, 15 Nov. 1941, G.D. Darker (DAOM 75764). New York: Flatbush, 1890, I.L. Zabriskie (DAOM 34307); Lloyd-Cornell Preserve, Ringwood, on rotten wood, 6 Sep. 1952, S.J. Hughes (DAOM 29052, DAOM 29377); ibid., on decorticated wood (old), 6 Sep. 1952, W.I. Illman (DAOM 239154); Buffalo, G.W. Clinton (DAOM 34362). North Carolina: Haywood Co., Great Smoky Mountains National Park, Big Creek, Baxter Creek Trail, 35.7485° N, -83.1119° W, 610 m elev., decorticated 3 cm diam branch on ground, 16 Jun. 2007, A.N. Miller et al. (ANM 1141, ILLS 80802); Cullowhee, on rotten wood, Jun. 1887, R. Thaxter (DAOM 29026). South Carolina: on wood, (DAOM 34361). Tennessee: Blount County, Bote Mountain, Great Smoky Mountains National Park, on rotten wood and bark, 23 Aug. 1977, S.J. Hughes (DAOM 188987, DAOM 169302); Cocke County, Great Smoky Mountains National Park, Cosby, Low Gap Trail, 35.7539° N, 83.2071° W, 716 m elev., decorticated wood on ground, 15 Jul. 2005, A.N. Miller & A.M. Stchigel (ANM536.1, ILLS 59356 - holotype of Acanthostigma septoconstrictum); ibid., decorticated 5 cm diam. branch on ground, 3 Nov. 2007, A.N. Miller et al. (ANM 1407, ILLS 80805); ibid., Cosby Nature Trail, 35.7538o° N, 83.2072° W, 716 m elev., 50 cm diam log on ground, 20 May 2008, A.N. Miller et al. (ANM 1701, ILLS 80806); Sevier Co., Great Smoky Mountains National Park, 5 miles east of Gatlinburg, Greenbrier, Old Settlers Trail, 35.7076° N, 83.3804° W, 457 m elev., decorticated 6 cm diam branch on ground, 19 Jun. 2007, A.N. Miller et al. (ANM 1171, ILLS 80803); Twin Creeks, Twin Creeks Nature Trail, near ATBI plot, 35.6881° N, 83.4999° W, 549 m elev., decorticated 5 cm diam branch on ground, 18 Jun. 2007, A.N. Miller et al. (ANM 1227, ILLS 80804). West Virginia: Giles County, Mountain Lake, on well decayed log of deciduous tree, 2 Sep. 1936, D.H. Linder (DAOM 24916); Nuttallburg, Nov. 1893, on rotten wood, L.W. Nuttall (DAOM 34290). Wisconsin: Devil’s Lake near Madison, 4 Sep. 1953, R.F. Cain (DAOM 41029).
Neoacanthostigma thailandicum Tanney & A.N. Mill. sp. nov.
Etymology: Named for the country where the type specimen was collected.
Diagnosis: Neoacanthostigma thailandicum is distinguished from all other species in the genus by macronematous conidiophores and larger, pale brown to brown, multi-septate conidia (to 920 µm long).
Description: For a complete description of this taxon see Hyde et al. (2016: 125; N. septoconstrictum MFLU 16–1134).
Type: Thailand: Prachuap Khiri Khan, Bang Sapan, Ron Thai, on decaying wood in flowing freshwater stream, 30 Jul. 2015, K.D. Hyde KH02 (MFLU 16–1134 — holotype, n.v.; BBH 41051 — isotype, n.v.; MFLUCC 15–1248, TBRC — cultures ex-type).
Illustration: Hyde et al. (2016: fig. 76).
Notes: Neoacanthostigma thailandicum (MFLU 16–1134) was identified as N. septoconstrictum in Hyde et al. (2016).
Both ITS and LSU sequences confirm that the hyphomycete morph Berkleasmium concinnum and the pyrenomycete morph Neoacanthostigma septoconstrictum are the same species and therefore must be given a single name. Thus, N. septoconstrictum is synonymized under B. concinnum because of the priority of Berkleasmium over Neoacanthostigma.Re-examination of the N. septoconstrictum type specimen and additional B. concinnum reference specimens confirmed the frequent cooccurrence of both the asexual and sexual morphs on the same substrate, providing additional circumstantial support for this connection (Fig. 2A–C).
Neoacanthostigma was proposed to accommodate a new species, N. fusiforme, which was chosen as the type species, along with N. filiforme and N. septoconstrictum, two species previously included in Acanthostigma but demonstrated to be phylogenetically distinct from the type species, A. perpusillum (Boonmee et al. 2014). Based on the ITS-LSU phylogeny presented here, the transfer of Acanthostigma filiforme to Neoacanthostigma appears unwarranted given its phylogenetic distance from N. fusiforme (Fig. 1). However, these two species were weakly clustered together based on the LSU phylogeny from Promputtha & Miller (2010) and the combined ITS and LSU phylogeny from Boonmee et al. (2014). The placement of N. filiforme was not resolved in this study because of low support. Hyde et al. (2016) reported a helicomyces-like asexual morph for N. septoconstrictum based on a specimen from decaying wood in a flowing freshwater stream in Thailand; however, the ITS sequence (KX454176) of this specimen (MFLUCC 15–1248) clearly distinguishes it from the N. septoconstrictum type specimen [identities = 465/537 (87%), gaps = 42/537 (7%)]. The ITS dissimilarity and distinctive asexual morph morphology from B. concinnum resulted in our description of this specimen as a novel species, N. thailandicum.
Tubeufiaceae (Tubeufiales) asexual morphs are morphologically diverse, containing helicosporous genera such as Helicoma, Helicomyces, Helicoön, and Helicosporium, and staurosporous genera including Araneomyces and Tetracrium (Réblová & Barr 2000, Kodsueb et al. 2004, Tsui & Berbee 2006). Reports of dictyosporous conidial morphs in Tubeufiaceae more reminiscent of B. concinnum include Chlamydotubeufia spp., Manoharachariella tectonae, Tubeufia amazonensis, and T. khunkornensis (Rossman & Müller 1979, Boonmee et al. 2011, 2014, Rajeshkumar & Sharma 2013, Doilom et al. 2016). The close phylogenetic relationship between B. concinnum and the helicosporous N. fusiforme and N. thailandicum is unexpected but supported by molecular evidence generated from independently collected and sequenced specimens and isolates. The phylogenetic placement and identification of a sexual morph for B. concinnum presented in this study demonstrates the significance of culturing and sequencing named-but-unsequenced dematiaceous hyphomycetes.
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We are grateful to Scott A. Redhead for nomenclatural advice and to Olga Koppel for Russian translation of the DAOM 210103 specimen label. This study was partially supported by a National Science Foundation grant (DEB-0515558) to ANM and a Natural Sciences and Engineering Research Council of Canada scholarship (PGSD2-459312-2014) to JBT. JBT is grateful to the Microbiology Molecular Technologies Laboratory (MMTL) group of ORDC-AAFC, Ottawa, for processing DNA sequencing, and Keith A. Seifert for his comments on this manuscript.
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Tanney, J., Miller, A.N. Asexual-sexual morph connection in the type species of Berkleasmium. IMA Fungus 8, 99–105 (2017) doi:10.5598/imafungus.2017.08.01.07
- molecular systematics
- new species