- Email: [email protected]
A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/myc
Full paper
A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae Ying Chu a, Siti Aisyah Alias b, Mohammed Rizman-Idid b, Sheng-Yu Guo b, Ka-Lai Pang c,* a
Department of Life Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC Institute of Ocean and Earth Sciences, University Malaya, 50603 Kuala Lumpur, Malaysia c Institute of Marine Biology and Center of Excellence for the Oceans, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC b
article info
abstract
Article history:
Nimbospora is a genus in the Halosphaeriaceae with three species: N. effusa (the type species), N.
Received 2 March 2013
bipolaris and N. octonae. All species have two-celled ascospores with a prominent sheath. A
Received in revised form
second type of appendage is present in two species, a single tuft of fibrillar appendages is
25 February 2014
present in N. effusa and two occur in N. bipolaris. Nimbospora effusa and N. bipolaris are
Accepted 25 February 2014
morphologically similar, but there are major morphological differences in ascomatal
Available online xxx
morphology between N. effusa/N. bipolaris and N. octonae. In this study, we investigated the morphology of N. effusa and N. octonae and the phylogenetic relationships of the three Nimbo-
Keywords:
spora species based on partial sequences of 18S and 28S rRNA genes. Bayesian analysis sug-
Ascomycota
gested that Nimbospora is not monophyletic. Nimbospora effusa groups with N. bipolaris in a well-
Marine fungi
supported clade, with Naufragella spinibarbata forming a sister group. Nimbospora octonae,
Sordariomycetes
however, clusters with Haligena elaterophora in a separate, well-supported clade. The ascomata
Taxonomy
of N. octonae differ from those of N. effusa and N. bipolaris by their thick peridium, and ascospores that lack equatorial tufts of appendages but possess polar and equatorial subulate appendages after the sheath is dissolved. Based on these characters and the correlating phylogenetic distance, the new genus Ebullia is established to accommodate N. octonae. ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
1.
Introduction
Nimbospora Jørg. Koch is a genus in the Halosphaeriaceae, Microascales (Sordariomycetes, Ascomycota) with N. effusa Jørg. Koch as the type species (Koch 1982). Currently, there are three lignicolous species in Nimbospora, namely N. effusa, N.
bipolaris K.D. Hyde & E.B.G. Jones (Hyde and Jones 1985) and N. octonae Kohlm. (Kohlmeyer 1985). Originally described from driftwood collected from a coastal area of Sri Lanka in the Indian Ocean, N. effusa is a typical member of the Halosphaeriaceae, with globose ascomata, catenophyses, thinwalled, deliquescing asci and hyaline ascospores with
* Corresponding author. Tel.: þ886 2 24622192x5319; fax: þ886 2 24633152. E-mail address: [email protected] (K.-L. Pang). http://dx.doi.org/10.1016/j.myc.2014.02.003 1340-3540/ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
2
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
appendages. Ascospores of N. effusa are surrounded by a thick sheath, and a tuft of fibrillar appendages is attached to one side of the ascospore septum, which extends after the asci are dissolved (Koch 1982). Nimbospora bipolaris is similar to N. effusa in morphology. Instead of having one tuft of fibrillar appendages at the spore equator, N. bipolaris has two tufts of appendages, one on each side of the central septum, and the sheath is constricted at the equator and less eccentric than in N. effusa (Read et al. 1993). Although an exosporic sheath is present around the ascospores of N. octonae, no tuft-like equatorial appendages occur. Instead, subulate appendages are present at the equatorial and polar positions of the ascospores after the sheath has deliquesced (Kohlmeyer 1985). During our ongoing study of the diversity of marine fungi in Taiwan, all three known Nimbospora species were collected. A morphological study was initiated to re-examine the features of these species, especially the peridial wall structure based on paraffin sections. Because partial 18S and 28S rRNA genes were available for most species of the Halosphaeriaceae, these genes were consequently sequenced and analyzed by Bayesian inference or maximum likelihood to determine (1) if Nimbospora as currently delimited is monophyletic; and (2) the phylogenetic relationships between Nimbospora and other genera in the family.
2.
Materials and methods
2.1.
Collection, identification and isolation
Driftwood/trapped wood was collected in northeastern Taiwan. Wood samples were placed in large Zip-lock plastic bags and incubated at room temperature in the laboratory. Ascomata of Nimbospora species on wood were cut open with a razor blade under a SZ61 stereomicroscope (Olympus, Tokyo). Centrum material was transferred to a drop of sterile, natural seawater on a glass slide. The morphology of asci and ascospores was observed under a BX51 microscope (Olympus) and photographs made on a DP20 Microscope Camera (Olympus). For isolation, a spore suspension of Nimbospora species was made by transferring centrum material to a drop of sterile, natural seawater on a sterilized glass slide. The spore mass was dispersed evenly in the drop of seawater with sterilized tweezers and identifications were confirmed by observation under a compound microscope. More sterile, natural seawater was added and dispensed onto the surface of a cornmeal seawater agar (CMAS) plate (Difco, Sparks, MD, USA) supplemented with 0.5 g/L each of penicillin G and streptomycin sulfate (BioShop, Burlington, ON, Canada). The plate was incubated at 25 C for 1e3 days. Germinated single spores were picked up and transferred to fresh CMAS plates. The plates were incubated at 25 C and cultures are deposited at the Bioresource Collection and Research Centre (BCRC), Hsinchu, Taiwan (ROC).
2.2.
Section of ascomata
For sections of ascomata, pieces of wood with ascomata were cut out and fixed in FAA solution (5% formaldehyde and 5%
glacial acetic acid in 50% ethanol) overnight at 4 C. The fixed samples were washed three times in 50% ethanol. Samples were then dehydrated in a graduated t-butanol/ethanol/ water series (10/40/50, 20/50/30, 35/50/15, 55/45/0, 75/25/0, 100/0/0, 100/0/0, in percentage by volume), and infiltrated gradually and embedded in paraffin. Paraffin sections (7 mm) were cut on a FRM-200P rotary microtome (Physitemp Instruments, Taipei), floated on water at 42 C to relax compression and mounted on microscope slides. Dried sections were de-paraffinised and rehydrated through a graded series of ethanol. The sections were stained with 1% safranin O in 50% ethanol (10 s) and 0.5% Orange G in 95% ethanol (30 s). After washing and dehydration, each stained section was permanently mounted with a cover slip and Histokitt (Assistent, Sonheim/Rho¨n, Germany). Specimens were observed on the Olympus BX51 microscope and light micrographs were taken.
2.3.
Molecular analysis
The isolates were grown on potato dextrose seawater agar plates (Difco) for at least 2 weeks at 25 C. Mycelium was scraped off the agar surface and ground into powder in a mortar and pestle in liquid nitrogen. The DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used for genomic DNA extraction following the manufacturer’s instructions. Extracted DNA was used directly for PCR reactions with the following ingredients: 0.2 mM of each primer (NS1/NS4, White et al. 1990; LROR/LR6, Bunyard et al. 1994), 0.2 mM of each dNTP, 2.5 mM MgCl2 and 1 U of Taq Polymerase (Invitrogen, Sa˜o Paulo, Brazil). The amplification cycle consisted of an initial denaturation step of 94 C for 5 min followed by 35 cycles of (i) denaturation (94 C for 0.5 min), (ii) annealing (55 C for 0.5 min) and (iii) elongation (72 C for 0.5 min) and a final 11 min elongation step at 72 C. The PCR products were analyzed by agarose gel electrophoresis and shipped to Tri-I Biotech, Inc., Taiwan, for purification and direct sequencing with the same primers. Returned sequences were checked for ambiguity, assembled and deposited in GenBank (Table 1). These sequences and those from the GenBank were manually aligned in Se-Al v1.0a1 (Rambaut 1999). The alignments (TreeBASE accession no. 14728) were entered into BEAUti v1.7.2 for generation of XML files for Bayesian analysis in BEASTv.1.7.2 (Drummond and Rambaut 2007). Two data sets were analyzed (28S, 18S þ 28S) with the following analytical settings: GTR, estimated base frequency, gamma þ invariant sites, number of gamma categories set at 4, a strict clock with estimated evolutionary rate and normal rate distribution, the Yule process as the speciation model, with 20 million for 28S and 50 million generations for the combined 18S þ 28S data sets, with parameters and trees sampled every 1000 generations. Convergence of the analyses was checked in Tracer v1.5 (Drummond and Rambaut 2007) and the effective sample size (ESS) of the parameter statistics >200 was ensured. The first 10% of the trees were treated as burn-in and discarded. A summary tree was produced using TreeAnnotator v1.7.2 (Drummond and Rambaut 2007) and viewed and edited in FigTree v1.3.1 (Rambaut 2009). For the combined 18S þ 28S data set, a maximum likelihood bootstrap analysis was performed in Mega 5.2.2 (Tamura et al. 2011) with the following settings: 1000
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
3
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Table 1 e Sequences used in the phylogenetic analysis.
Table 1 (continued)
Taxa
Taxa
Culture no.
GenBank accession no. 28S
Halosphaeriaceae, Microascales Alisea longicolla CP2464b BF Aniptodera ATCC 32818 chesapeakensis Antennospora GR89/e quadricornuta Arenariomyces JK5409A/e trifurcatus Ascosacculus A444-1D/e aquaticus Ascosacculus A108-7D heteroguttulatus Ceriosporopsis halima JK 5473F Corollospora luteola NBRC 31315 Corollospora maritima JK4834 Corollospora MD832/e portsaidica Haiyanga salina GR85/e Haligena elaterophora PP4705/e Haligena elaterophora JS147/e Halosarpheia fibrosa PP5159/e Halosarpheia japonica IMI 397961 Halosarpheia trullifera PP4268/e Halosarpheia CY2980/e unicellularis Halosphaeriopsis ATCC 16934 mediosetigera Havispora CY5278/e longyearbyenensis Humicola siamensis IT110 Kochiella crispa BCC 33507/e Magnisphaera e/A221-1A spartinae Magnisphaera A409-1B stevemossago Marinospora BBH 28307/e calyptrata Marinospora PP0868/e longissima Morakotiella salina CY3437/e Nais inornata ATCC 200453/J115-5C Natantispora lotica A333-1A Natantispora A231-1D retorquens Naufragella BCC 33482/e spinibarbata Naufragella PP6886/e spinibarbata Nautosphaeria BBH 28308/e cristaminuta Neptunella PP4648/e longirostris Nereiospora comata PP2520/e Nereiospora cristata PP5988/e Nimbospora bipolaris BCRC 34920a Nimbospora effusa JK5104A Nimbospora effusa BCRC 34922a Nimbospora effusa BCRC 34866a Nimbospora octonae BCRC 34925a Nimbospora octonae BCRC 34926a Nohea umiumi JK5103F Oceanitis cincinnatula A318-1C
18S
EU118365 EU118370 U46882 U46870 EF383130
e
U46883
e
AY227136 e AY227121 AF352085 U47844 JN941490 U46884 AB361016
U47843 JN941656 U46871 e
EF383131 AY864845 AY864846 AF396872 HQ009884 AF396875 AF396876
e e e e HQ009885 e e
U46887
U32420
HQ111023 e DQ237875 DQ237874 HQ111019 e AY150221 AF352076 AY227134 AY227140 HQ111035 e AF491266 e
Oceanitis scuticella CP2421/CP2464a Oceanitis unicaudata CY1333/e Oceanitis viscidula ATCC 24310/e Ocostaspora LP53/e apilongissima Okeanomyces LP67/e cucullatus Ondiniella torquata BCC 34303/e Ophiodeira JK5164A monosemeia Panorbis viscosus JK5380A/JK5433C Periconia prolifica e/e Phaeonectriella PP7008/A157-1D lignicola Pseudolignincola IT41 siamensis Remispora maritima BBH 28309 Remispora pilleata BBH 28305/e Remispora BCC 15555/e quadriremis Remispora CY5279/e spitsbergenensis Remispora stellata JS150/e Sagaaromyces abonnis 5304A/K5304A Sagaaromyces glitra PP4672/e Sagaaromyces CY0732/e ratnagiriensis Thalespora IT200 appendiculata Tirispora unicaudata CY2370/e Toriella tubulifera BCC 33511/e Tubakiella galerita BCC 33500 Ceratocystidaceae, Microascales Ceratocystis adiposa CBS 600.74 Ceratocystis fimbriata C89 Hypocreaceae, Hypocreales Hypocrea schweinitzii ATCC 90178/e a b
AY864843 AF539476 AY227124 AY227128
Culture no.
e AF050482 AF352080 AF352086
HQ111034 e
GenBank accession no. 28S
18S
EU118367 AY150222 AY150223 HQ111005
EU118369 e e e
AY490787 e HQ111038 e U46894 U46879 AY227133 AF352083 AY090891 e AY150224 AF050484 DQ237873 DQ237872 HQ111012 HQ111002 HQ111021 e HQ111010 e HQ111011 e HQ111017 AY227118 AF539475 AF539470
e AY227137 e e
DQ237877 DQ237876 AY150225 e HQ111026 e HQ111014 HQ111003 EU984304 EU984263 U17401 U32418 AF279395 AF164357
Bioresource Collection and Research Center, Hsinchu, Taiwan. Sequences generated in this study.
bootstrap, GTR, gamma þ invariant sites, number of gamma categories set at 5, heuristic search with the nearest-neighborinterchange, initial tree from neighbor-joining method.
HQ111032 e HQ111009 e AF539473 e AF491267 AF491268 KC692142b U46892 KC692144b KC692146b KC692148b KC692150b U46893 AY227120
e e KC692143b U46877 KC692145b KC692147b KC692149b KC692151b U46878 AF352077
3.
Results
The ascomatal wall of N. effusa is thin and composed of a few layers of cells, an outer 1e2 layers of angular cells and an inner 3e4 layers of elongated cells with large lumina (Fig. 1A, B). Nimbospora effusa has bi-celled ascospores enrobed by a sheath with a tuft of appendages protruding from one side of the ascospores at the mid-septum (Fig. 1C). In N. bipolaris, two tufts of appendages are present, one on either side of the midseptum (Fig. 1D). Nimbospora octonae also occurs commonly on wood in northern Taiwan and produces large ascomata with a long neck and a thick peridium composed of two strata (Fig. 2AeC).
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
4
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Fig. 1 e Nimbospora spp. A: Ascoma of N. effusa immersed in wood with asci already deliquesced. B: Thin peridium of N. effusa. C: Ascospore of N. effusa with a sheath and a tuft of appendages on one side of the septum. D: Ascospore of N. bipolaris with a sheath and two tufts of appendages, one on either side of the septum. Bars: A 20 mm; B 10 mm; C, D 5 mm.
Both strata are textura angularis; cells of the outer stratum are smaller while those of the inner stratum are larger and elongated with large lumina. Asci deliquesce early and can only be seen in early development (Fig. 2D). A gelatinous sheath wraps around the ascospores and swells in seawater (Fig. 2E, F). Our Bayesian phylogenetic analysis included partial sequences of the 18S and 28S rRNA genes from 69 representative taxa in 40 genera of the Halosphaeriaceae and Hypocreales and was designed to infer the phylogenetic relationships between the three Nimbospora species and other closely related
taxa in the Halosphaeriaceae (combined 18S þ 28S tree shown in Fig. 3). Hypocrea schweinitzii (Fr.) Sacc., Ceratocystis adiposa (E.J. Butler) C. Moreau and C. fimbriata Ellis & Halst. were used as outgroups to root the tree. The effective sample size (ESS) for all parameters is bigger than the estimate of the posterior distribution and over 200, suggesting that enough samples were taken to produce a reasonable estimate of the posterior distribution of each parameter. In the combined 18S þ 28S Bayesian analysis, the three isolates of N. effusa (type species) group with N. bipolaris in a
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
5
Fig. 2 e Ebullia octonae. A: Ascoma embedded deeply inside wood with a thick peridium at the basal region. B: Long neck. C: Thick, two-layered peridium. D: Clavate ascus. E, F: Ascospores with a sheath that extends in seawater. Bars: A 100 mm; B 25 mm; C 20 mm; D 10 mm; E, F 5 mm.
well-supported clade (PP ¼ 1, ML bootstrap ¼ 99), with Naufragella spinibarbata (Jørg. Koch) Kohlm. & Volkm.-Kohlm. forming a sister group (PP ¼ 0.98). The two isolates of N. octonae, however, are in a separate, well-supported clade (PP ¼ 1, ML bootstrap ¼ 100) with Haligena elaterophora Kohlm.
(PP ¼ 0.98), sister to the remaining Halosphaeriaceae. Similarly, N. effusa and N. bipolaris (PP ¼ 1) also cluster with Nauf. spinibarbata (PP ¼ 0.92) in the 28S tree, and so does N. octonae with H. elaterophora (PP ¼ 0.99). Many of the poorly supported groups in the Bayesian analyses of the 18S þ 28S and 28S rRNA
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
6
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Fig. 3 e A Bayesian phylogenetic tree based on a combined analysis of the 18S and 28S rRNA genes using BEASTv.1.7.2. Posterior probability (PP) and maximum likelihood bootstrap value are shown at the branches.
genes were not recovered in the ML bootstrap analysis, but the clade comprising N. effusa and N. bipolaris is strongly supported (ML bootstrap ¼ 99) and distantly related to the two isolates of N. octonae.
4.
Discussion
Nimbospora effusa and N. octonae are common marine ascomycetes on wood collected on the northern coast of Taiwan, while N. bipolaris is uncommon (Pang and Jheng 2012). All three species of Nimbospora have a thick mucilaginous sheath around the ascospores after release from the asci, a character uniting these species. This sheath and the sticky fibrillar appendages on ascospores of Nimbospora species are important for attaching to substrata to allow germination. Bayesian phylogenetic analysis revealed the monophyly of N. effusa and N. bipolaris in a clade (Fig. 3, arrow) comprising
several morphologically unrelated species. This wellsupported clade was also recovered by Sakayaroj et al. (2011), who studied the phylogeny of many oceanic taxa in the Halosphaeriaceae. Nimbospora effusa and N. bipolaris are similar in ascospore morphology; a thick, mucilaginous sheath wraps around the ascospores, which is penetrated by fibrillar appendages at the septum. One tuft of appendages is present on one side of the ascospore septum in N. effusa while in N. bipolaris, the appendages are on both sides of the septum. The sheath of N. bipolaris appears to be less eccentric than that of N. effusa (Read et al. 1993). At transmission electron microscope (TEM) magnifications, the appendage precursor material was observed in the cytoplasm and mesosporium at the equator at an early developmental stage in ascospores of N. bipolaris (Read et al. 1993). Fibrillar equatorial appendages are secreted through pores in the episporium and penetrate through the thick, mucilaginous sheath, when the mature ascospores are released from the asci.
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Although monophyletic with N. effusa and N. bipolaris, Naufragella spinibarbata differs significantly from them in terms of ascospore appendage morphology, an important characteristic for the delineation of genera in the Halosphaeriaceae (Sakayaroj et al. 2011). No sheath is present on ascospores of Nauf. spinibarbata, but two types of ascospore appendages are discernible; (1) a gelatinous strap-like appendage at the polar position that separates from the side of the ascospore to form a long wide band in seawater; and (2) hair-like appendages that arise from the sub-polar region of ascospores (Jones et al. 2009). Morphologically, Nohea Kohlm. & Volkm.-Kohlm., typified by Noh. umiumi Kohlm. & Volkm.-Kohlm., also resembles Nimbospora effusa and N. bipolaris by having fibrillar ascospore appendages, but two kinds of appendages are present in Noh. umiumi; (1) a subapical gelatinous pad wraps around the equator and unfurls in water to form long sticky filaments; and (2) two bundles of fibers attach to the spore wall at polar positions (Kohlmeyer and Volkmann-Kohlmeyer 1991). Nohea umiumi, however, is not monophyletic with N. effusa and N. bipolaris (Fig. 3). Nimbospora as presently defined is not a monophyletic genus. Phylogenetically, N. octonae is located in a lineage with Haligena elaterophora, sister to most other taxa of the Halosphaeriaceae (Jones et al. 2009; Sakayaroj et al. 2011), and is distantly related to the other two Nimbospora species. In addition to the sheath, N. octonae differs from N. effusa and N. bipolaris in many morphological characters, including the thick peridial structure of ascomata, the absence of equatorial tufts of appendages but the presence of subulate appendages. Ascomata of Nimbospora octonae are significantly larger (6001250 mm) than those of N. effusa (225240 mm) and N. bipolaris (204380 mm) (Koch 1982; Hyde and Jones 1985; Kohlmeyer 1985). Although forming a monophyletic group with Haligena elaterophora, N. octonae has little morphological resemblance with it. Ascomata of H. elaterophora are coriaceous or subcarbonaceous and black (Kohlmeyer 1961), while those of N. octonae are light-colored (Kohlmeyer 1985). In H. elaterophora, the ascospores are oblong with multiple septa with constrictions, lack a sheath, and the bipolar appendages are strap-like and polymorphic (Sakayaroj et al. 2005). No fibrillar or straplike appendages are present on ascospores of N. octonae. Instead, an exosporic sheath is present around the ascospores and subulate appendages occur at the polar and equatorial positions after the sheath deliquesces (Kohlmeyer 1985). Nimbospora octonae is morphologically most similar to Marinospora A.R. Caval. in the Halosphaeriaceae. In Marinospora spp., both polar and equatorial appendages are outgrowths of the mesosporium and episporium, with a fragmenting exosporic sheath forming tiny caps on the tips of the primary appendages (Johnson et al. 1984). The sheath of ascospores of Marinospora spp. is evanescent, while that of N. octonae is persistent, and no substructures are present on the subulate appendages (Kohlmeyer 1985). Another genus characterized by ascospores with a sheath is Tunicatispora K.D. Hyde, typified by T. australiensis K.D. Hyde (Hyde 1990). However, the sheath of T. australiensis is thin, persistent and skin-like, and a cap-like appendage arises through a hole at a polar position and later unfurls to form a long thread. Unlike
7
Marinospora, no DNA sequences are available for comparison. Because of these morphological and phylogenetic differences, a new genus Ebullia is established below for N. octonae.
5.
Taxonomy
Ebullia K.L. Pang, gen. nov. MycoBank no.: MB 803444. Ascomata subglobose to ampulliform, immersed, ostiolate, with long necks, coriaceous, dark brown to black above, hyaline to brown below. Peridium two-layered, much thicker at the base. Necks periphysate. Asci eight-spored, clavate, pedunculate, unitunicate, thin-walled, early deliquescing, developing at the base of the ascoma venter on a convex cushion of ascogenous cells. Ascospores ellipsoidal, oneseptate, not or slightly constricted at the septum, hyaline, with a gelatinous sheath enclosing 6e7 subulate equatorial appendages evenly distributed around the septum and one similar appendage at each end; swells in water. Type species: Ebullia octonae (Kohlm.) K.L. Pang, this paper. Etymology: ‘Ebullia’ meaning ‘bubble’ in Latin, in reference to the bubble-like sheath of the ascospores. Anamorph: Unknown. Ebullia octonae (Kohlm.) K.L. Pang, comb. nov. Fig. 2. h Nimbospora octonae Kohlm., Can. J. Bot. 63: 1122, 1985 (basionym). MycoBank no.: MB 805905. Distribution: Brunei, Hawaiian Islands, India, Taiwan. Holotype: J.K. 4474a (The New York Botanical Garden). Nimbospora Jørg. Koch, Nordic J. Bot. 2: 166, 1982. Nimbospora effusa Jørg. Koch, Nordic J. Bot. 2: 166, 1982. (type species) Fig. 1AeC. Nimbospora bipolaris K.D. Hyde & E.B.G. Jones, Can. J. Bot. 63: 611, 1985. Fig. 1D.
Acknowledgments K.L. Pang would like to thank National Science Council Taiwan for research grants (NSC101-2621-B-019-001-MY3) and Mr. Ren-Sheng Jheng for help.
references
Bunyard BA, Nicholson MS, Royse DJ, 1994. A systematic assessment of Morchella using RFLP analysis of the 28S ribosomal RNA gene. Mycologia 86: 762e772. Drummond AJ, Rambaut A, 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 214. Hyde KD, 1990. Intertidal fungi from warm temperate mangroves of Australia, including Tunicatispora australiensis, gen. et sp. nov. Australian Systematic Botany 3: 711e718.
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
8
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Hyde KD, Jones EBG, 1985. Marine fungi from Seychelles. I. Nimbospora effusa and Nimbospora bipolaris sp. nov. from driftwood. Canadian Journal of Botany 63: 611e615. Johnson RG, Jones EBG, Moss ST, 1984. Taxonomic studies of the Halosphaeriaceae: Remispora Linder, Marinospora Cavaliere and Carbosphaerella Schmidt. Botanica Marina 27: 557e566. Jones EBG, Sakayaroj J, Suetrong S, Somrithipol S, Pang KL, 2009. Classification of marine Ascomycota, anamorphic taxa and Basidiomycota. Fungal Diversity 35: 1e187. Koch J, 1982. Some lignicolous marine fungi from Sri Lanka. Nordic Journal of Botany 2: 163e169. Kohlmeyer J, 1961. Pilze von der no¨rdlichen Pazifik-ku¨ste der U.S.A. Nova Hedwigia 3: 80e86. Kohlmeyer J, 1985. Nimbospora octonae, a new marine ascomycete from Hawaii. Canadian Journal of Botany 63: 1122e1125. Kohlmeyer J, Volkmann-Kohlmeyer B, 1991. Illustrated key to the filamentous marine fungi. Botanica Marina 34: 1e61. Pang KL, Jheng JS, 2012. A checklist of marine fungi of Taiwan with description of Kitesporella keelungensis gen. et sp. nov. Botanica Marina 55: 459e466. Rambaut A, 1999. Se-Al. Department of Zoology, University of Oxford, Oxford. http://evolve.zoo.ox.ac.uk/Se-Al/Se-Al.html. Accessed 8 Aug 2002.
Rambaut A, 2009. FigTree v1.3.1. Department of Zoology, University of Oxford, Oxford. http://tree.bio.ed.ac.uk/ software/figtree/. Accessed 21 Dec 2009. Read SJ, Jones EBG, Moss ST, 1993. Ultrastructural observations on Nimbospora bipolaris (Halosphaeriaceae, Ascomycetes). Philosophical Transactions of the Royal Society of London Series B: Biological Sciences 339: 483e489. Sakayaroj J, Pang KL, Jones EBG, 2011. Multi-gene phylogeny of the Halosphaeriaceae: its ordinal status, relationships between genera and morphological character evolution. Fungal Diversity 46: 87e109. Sakayaroj J, Pang KL, Phongpaichit S, Jones EBG, 2005. A phylogenetic study of the genus Haligena (Halosphaeriales, Ascomycota). Mycologia 97: 804e811. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731e2739. White TJ, Bruns T, Lee S, Taylor JW, 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 application. Academic Press, San Diego, pp 315e322.
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/myc
Full paper
A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae Ying Chu a, Siti Aisyah Alias b, Mohammed Rizman-Idid b, Sheng-Yu Guo b, Ka-Lai Pang c,* a
Department of Life Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC Institute of Ocean and Earth Sciences, University Malaya, 50603 Kuala Lumpur, Malaysia c Institute of Marine Biology and Center of Excellence for the Oceans, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan, ROC b
article info
abstract
Article history:
Nimbospora is a genus in the Halosphaeriaceae with three species: N. effusa (the type species), N.
Received 2 March 2013
bipolaris and N. octonae. All species have two-celled ascospores with a prominent sheath. A
Received in revised form
second type of appendage is present in two species, a single tuft of fibrillar appendages is
25 February 2014
present in N. effusa and two occur in N. bipolaris. Nimbospora effusa and N. bipolaris are
Accepted 25 February 2014
morphologically similar, but there are major morphological differences in ascomatal
Available online xxx
morphology between N. effusa/N. bipolaris and N. octonae. In this study, we investigated the morphology of N. effusa and N. octonae and the phylogenetic relationships of the three Nimbo-
Keywords:
spora species based on partial sequences of 18S and 28S rRNA genes. Bayesian analysis sug-
Ascomycota
gested that Nimbospora is not monophyletic. Nimbospora effusa groups with N. bipolaris in a well-
Marine fungi
supported clade, with Naufragella spinibarbata forming a sister group. Nimbospora octonae,
Sordariomycetes
however, clusters with Haligena elaterophora in a separate, well-supported clade. The ascomata
Taxonomy
of N. octonae differ from those of N. effusa and N. bipolaris by their thick peridium, and ascospores that lack equatorial tufts of appendages but possess polar and equatorial subulate appendages after the sheath is dissolved. Based on these characters and the correlating phylogenetic distance, the new genus Ebullia is established to accommodate N. octonae. ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
1.
Introduction
Nimbospora Jørg. Koch is a genus in the Halosphaeriaceae, Microascales (Sordariomycetes, Ascomycota) with N. effusa Jørg. Koch as the type species (Koch 1982). Currently, there are three lignicolous species in Nimbospora, namely N. effusa, N.
bipolaris K.D. Hyde & E.B.G. Jones (Hyde and Jones 1985) and N. octonae Kohlm. (Kohlmeyer 1985). Originally described from driftwood collected from a coastal area of Sri Lanka in the Indian Ocean, N. effusa is a typical member of the Halosphaeriaceae, with globose ascomata, catenophyses, thinwalled, deliquescing asci and hyaline ascospores with
* Corresponding author. Tel.: þ886 2 24622192x5319; fax: þ886 2 24633152. E-mail address: [email protected] (K.-L. Pang). http://dx.doi.org/10.1016/j.myc.2014.02.003 1340-3540/ª 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
2
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
appendages. Ascospores of N. effusa are surrounded by a thick sheath, and a tuft of fibrillar appendages is attached to one side of the ascospore septum, which extends after the asci are dissolved (Koch 1982). Nimbospora bipolaris is similar to N. effusa in morphology. Instead of having one tuft of fibrillar appendages at the spore equator, N. bipolaris has two tufts of appendages, one on each side of the central septum, and the sheath is constricted at the equator and less eccentric than in N. effusa (Read et al. 1993). Although an exosporic sheath is present around the ascospores of N. octonae, no tuft-like equatorial appendages occur. Instead, subulate appendages are present at the equatorial and polar positions of the ascospores after the sheath has deliquesced (Kohlmeyer 1985). During our ongoing study of the diversity of marine fungi in Taiwan, all three known Nimbospora species were collected. A morphological study was initiated to re-examine the features of these species, especially the peridial wall structure based on paraffin sections. Because partial 18S and 28S rRNA genes were available for most species of the Halosphaeriaceae, these genes were consequently sequenced and analyzed by Bayesian inference or maximum likelihood to determine (1) if Nimbospora as currently delimited is monophyletic; and (2) the phylogenetic relationships between Nimbospora and other genera in the family.
2.
Materials and methods
2.1.
Collection, identification and isolation
Driftwood/trapped wood was collected in northeastern Taiwan. Wood samples were placed in large Zip-lock plastic bags and incubated at room temperature in the laboratory. Ascomata of Nimbospora species on wood were cut open with a razor blade under a SZ61 stereomicroscope (Olympus, Tokyo). Centrum material was transferred to a drop of sterile, natural seawater on a glass slide. The morphology of asci and ascospores was observed under a BX51 microscope (Olympus) and photographs made on a DP20 Microscope Camera (Olympus). For isolation, a spore suspension of Nimbospora species was made by transferring centrum material to a drop of sterile, natural seawater on a sterilized glass slide. The spore mass was dispersed evenly in the drop of seawater with sterilized tweezers and identifications were confirmed by observation under a compound microscope. More sterile, natural seawater was added and dispensed onto the surface of a cornmeal seawater agar (CMAS) plate (Difco, Sparks, MD, USA) supplemented with 0.5 g/L each of penicillin G and streptomycin sulfate (BioShop, Burlington, ON, Canada). The plate was incubated at 25 C for 1e3 days. Germinated single spores were picked up and transferred to fresh CMAS plates. The plates were incubated at 25 C and cultures are deposited at the Bioresource Collection and Research Centre (BCRC), Hsinchu, Taiwan (ROC).
2.2.
Section of ascomata
For sections of ascomata, pieces of wood with ascomata were cut out and fixed in FAA solution (5% formaldehyde and 5%
glacial acetic acid in 50% ethanol) overnight at 4 C. The fixed samples were washed three times in 50% ethanol. Samples were then dehydrated in a graduated t-butanol/ethanol/ water series (10/40/50, 20/50/30, 35/50/15, 55/45/0, 75/25/0, 100/0/0, 100/0/0, in percentage by volume), and infiltrated gradually and embedded in paraffin. Paraffin sections (7 mm) were cut on a FRM-200P rotary microtome (Physitemp Instruments, Taipei), floated on water at 42 C to relax compression and mounted on microscope slides. Dried sections were de-paraffinised and rehydrated through a graded series of ethanol. The sections were stained with 1% safranin O in 50% ethanol (10 s) and 0.5% Orange G in 95% ethanol (30 s). After washing and dehydration, each stained section was permanently mounted with a cover slip and Histokitt (Assistent, Sonheim/Rho¨n, Germany). Specimens were observed on the Olympus BX51 microscope and light micrographs were taken.
2.3.
Molecular analysis
The isolates were grown on potato dextrose seawater agar plates (Difco) for at least 2 weeks at 25 C. Mycelium was scraped off the agar surface and ground into powder in a mortar and pestle in liquid nitrogen. The DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used for genomic DNA extraction following the manufacturer’s instructions. Extracted DNA was used directly for PCR reactions with the following ingredients: 0.2 mM of each primer (NS1/NS4, White et al. 1990; LROR/LR6, Bunyard et al. 1994), 0.2 mM of each dNTP, 2.5 mM MgCl2 and 1 U of Taq Polymerase (Invitrogen, Sa˜o Paulo, Brazil). The amplification cycle consisted of an initial denaturation step of 94 C for 5 min followed by 35 cycles of (i) denaturation (94 C for 0.5 min), (ii) annealing (55 C for 0.5 min) and (iii) elongation (72 C for 0.5 min) and a final 11 min elongation step at 72 C. The PCR products were analyzed by agarose gel electrophoresis and shipped to Tri-I Biotech, Inc., Taiwan, for purification and direct sequencing with the same primers. Returned sequences were checked for ambiguity, assembled and deposited in GenBank (Table 1). These sequences and those from the GenBank were manually aligned in Se-Al v1.0a1 (Rambaut 1999). The alignments (TreeBASE accession no. 14728) were entered into BEAUti v1.7.2 for generation of XML files for Bayesian analysis in BEASTv.1.7.2 (Drummond and Rambaut 2007). Two data sets were analyzed (28S, 18S þ 28S) with the following analytical settings: GTR, estimated base frequency, gamma þ invariant sites, number of gamma categories set at 4, a strict clock with estimated evolutionary rate and normal rate distribution, the Yule process as the speciation model, with 20 million for 28S and 50 million generations for the combined 18S þ 28S data sets, with parameters and trees sampled every 1000 generations. Convergence of the analyses was checked in Tracer v1.5 (Drummond and Rambaut 2007) and the effective sample size (ESS) of the parameter statistics >200 was ensured. The first 10% of the trees were treated as burn-in and discarded. A summary tree was produced using TreeAnnotator v1.7.2 (Drummond and Rambaut 2007) and viewed and edited in FigTree v1.3.1 (Rambaut 2009). For the combined 18S þ 28S data set, a maximum likelihood bootstrap analysis was performed in Mega 5.2.2 (Tamura et al. 2011) with the following settings: 1000
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
3
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Table 1 e Sequences used in the phylogenetic analysis.
Table 1 (continued)
Taxa
Taxa
Culture no.
GenBank accession no. 28S
Halosphaeriaceae, Microascales Alisea longicolla CP2464b BF Aniptodera ATCC 32818 chesapeakensis Antennospora GR89/e quadricornuta Arenariomyces JK5409A/e trifurcatus Ascosacculus A444-1D/e aquaticus Ascosacculus A108-7D heteroguttulatus Ceriosporopsis halima JK 5473F Corollospora luteola NBRC 31315 Corollospora maritima JK4834 Corollospora MD832/e portsaidica Haiyanga salina GR85/e Haligena elaterophora PP4705/e Haligena elaterophora JS147/e Halosarpheia fibrosa PP5159/e Halosarpheia japonica IMI 397961 Halosarpheia trullifera PP4268/e Halosarpheia CY2980/e unicellularis Halosphaeriopsis ATCC 16934 mediosetigera Havispora CY5278/e longyearbyenensis Humicola siamensis IT110 Kochiella crispa BCC 33507/e Magnisphaera e/A221-1A spartinae Magnisphaera A409-1B stevemossago Marinospora BBH 28307/e calyptrata Marinospora PP0868/e longissima Morakotiella salina CY3437/e Nais inornata ATCC 200453/J115-5C Natantispora lotica A333-1A Natantispora A231-1D retorquens Naufragella BCC 33482/e spinibarbata Naufragella PP6886/e spinibarbata Nautosphaeria BBH 28308/e cristaminuta Neptunella PP4648/e longirostris Nereiospora comata PP2520/e Nereiospora cristata PP5988/e Nimbospora bipolaris BCRC 34920a Nimbospora effusa JK5104A Nimbospora effusa BCRC 34922a Nimbospora effusa BCRC 34866a Nimbospora octonae BCRC 34925a Nimbospora octonae BCRC 34926a Nohea umiumi JK5103F Oceanitis cincinnatula A318-1C
18S
EU118365 EU118370 U46882 U46870 EF383130
e
U46883
e
AY227136 e AY227121 AF352085 U47844 JN941490 U46884 AB361016
U47843 JN941656 U46871 e
EF383131 AY864845 AY864846 AF396872 HQ009884 AF396875 AF396876
e e e e HQ009885 e e
U46887
U32420
HQ111023 e DQ237875 DQ237874 HQ111019 e AY150221 AF352076 AY227134 AY227140 HQ111035 e AF491266 e
Oceanitis scuticella CP2421/CP2464a Oceanitis unicaudata CY1333/e Oceanitis viscidula ATCC 24310/e Ocostaspora LP53/e apilongissima Okeanomyces LP67/e cucullatus Ondiniella torquata BCC 34303/e Ophiodeira JK5164A monosemeia Panorbis viscosus JK5380A/JK5433C Periconia prolifica e/e Phaeonectriella PP7008/A157-1D lignicola Pseudolignincola IT41 siamensis Remispora maritima BBH 28309 Remispora pilleata BBH 28305/e Remispora BCC 15555/e quadriremis Remispora CY5279/e spitsbergenensis Remispora stellata JS150/e Sagaaromyces abonnis 5304A/K5304A Sagaaromyces glitra PP4672/e Sagaaromyces CY0732/e ratnagiriensis Thalespora IT200 appendiculata Tirispora unicaudata CY2370/e Toriella tubulifera BCC 33511/e Tubakiella galerita BCC 33500 Ceratocystidaceae, Microascales Ceratocystis adiposa CBS 600.74 Ceratocystis fimbriata C89 Hypocreaceae, Hypocreales Hypocrea schweinitzii ATCC 90178/e a b
AY864843 AF539476 AY227124 AY227128
Culture no.
e AF050482 AF352080 AF352086
HQ111034 e
GenBank accession no. 28S
18S
EU118367 AY150222 AY150223 HQ111005
EU118369 e e e
AY490787 e HQ111038 e U46894 U46879 AY227133 AF352083 AY090891 e AY150224 AF050484 DQ237873 DQ237872 HQ111012 HQ111002 HQ111021 e HQ111010 e HQ111011 e HQ111017 AY227118 AF539475 AF539470
e AY227137 e e
DQ237877 DQ237876 AY150225 e HQ111026 e HQ111014 HQ111003 EU984304 EU984263 U17401 U32418 AF279395 AF164357
Bioresource Collection and Research Center, Hsinchu, Taiwan. Sequences generated in this study.
bootstrap, GTR, gamma þ invariant sites, number of gamma categories set at 5, heuristic search with the nearest-neighborinterchange, initial tree from neighbor-joining method.
HQ111032 e HQ111009 e AF539473 e AF491267 AF491268 KC692142b U46892 KC692144b KC692146b KC692148b KC692150b U46893 AY227120
e e KC692143b U46877 KC692145b KC692147b KC692149b KC692151b U46878 AF352077
3.
Results
The ascomatal wall of N. effusa is thin and composed of a few layers of cells, an outer 1e2 layers of angular cells and an inner 3e4 layers of elongated cells with large lumina (Fig. 1A, B). Nimbospora effusa has bi-celled ascospores enrobed by a sheath with a tuft of appendages protruding from one side of the ascospores at the mid-septum (Fig. 1C). In N. bipolaris, two tufts of appendages are present, one on either side of the midseptum (Fig. 1D). Nimbospora octonae also occurs commonly on wood in northern Taiwan and produces large ascomata with a long neck and a thick peridium composed of two strata (Fig. 2AeC).
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
4
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Fig. 1 e Nimbospora spp. A: Ascoma of N. effusa immersed in wood with asci already deliquesced. B: Thin peridium of N. effusa. C: Ascospore of N. effusa with a sheath and a tuft of appendages on one side of the septum. D: Ascospore of N. bipolaris with a sheath and two tufts of appendages, one on either side of the septum. Bars: A 20 mm; B 10 mm; C, D 5 mm.
Both strata are textura angularis; cells of the outer stratum are smaller while those of the inner stratum are larger and elongated with large lumina. Asci deliquesce early and can only be seen in early development (Fig. 2D). A gelatinous sheath wraps around the ascospores and swells in seawater (Fig. 2E, F). Our Bayesian phylogenetic analysis included partial sequences of the 18S and 28S rRNA genes from 69 representative taxa in 40 genera of the Halosphaeriaceae and Hypocreales and was designed to infer the phylogenetic relationships between the three Nimbospora species and other closely related
taxa in the Halosphaeriaceae (combined 18S þ 28S tree shown in Fig. 3). Hypocrea schweinitzii (Fr.) Sacc., Ceratocystis adiposa (E.J. Butler) C. Moreau and C. fimbriata Ellis & Halst. were used as outgroups to root the tree. The effective sample size (ESS) for all parameters is bigger than the estimate of the posterior distribution and over 200, suggesting that enough samples were taken to produce a reasonable estimate of the posterior distribution of each parameter. In the combined 18S þ 28S Bayesian analysis, the three isolates of N. effusa (type species) group with N. bipolaris in a
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
5
Fig. 2 e Ebullia octonae. A: Ascoma embedded deeply inside wood with a thick peridium at the basal region. B: Long neck. C: Thick, two-layered peridium. D: Clavate ascus. E, F: Ascospores with a sheath that extends in seawater. Bars: A 100 mm; B 25 mm; C 20 mm; D 10 mm; E, F 5 mm.
well-supported clade (PP ¼ 1, ML bootstrap ¼ 99), with Naufragella spinibarbata (Jørg. Koch) Kohlm. & Volkm.-Kohlm. forming a sister group (PP ¼ 0.98). The two isolates of N. octonae, however, are in a separate, well-supported clade (PP ¼ 1, ML bootstrap ¼ 100) with Haligena elaterophora Kohlm.
(PP ¼ 0.98), sister to the remaining Halosphaeriaceae. Similarly, N. effusa and N. bipolaris (PP ¼ 1) also cluster with Nauf. spinibarbata (PP ¼ 0.92) in the 28S tree, and so does N. octonae with H. elaterophora (PP ¼ 0.99). Many of the poorly supported groups in the Bayesian analyses of the 18S þ 28S and 28S rRNA
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
6
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Fig. 3 e A Bayesian phylogenetic tree based on a combined analysis of the 18S and 28S rRNA genes using BEASTv.1.7.2. Posterior probability (PP) and maximum likelihood bootstrap value are shown at the branches.
genes were not recovered in the ML bootstrap analysis, but the clade comprising N. effusa and N. bipolaris is strongly supported (ML bootstrap ¼ 99) and distantly related to the two isolates of N. octonae.
4.
Discussion
Nimbospora effusa and N. octonae are common marine ascomycetes on wood collected on the northern coast of Taiwan, while N. bipolaris is uncommon (Pang and Jheng 2012). All three species of Nimbospora have a thick mucilaginous sheath around the ascospores after release from the asci, a character uniting these species. This sheath and the sticky fibrillar appendages on ascospores of Nimbospora species are important for attaching to substrata to allow germination. Bayesian phylogenetic analysis revealed the monophyly of N. effusa and N. bipolaris in a clade (Fig. 3, arrow) comprising
several morphologically unrelated species. This wellsupported clade was also recovered by Sakayaroj et al. (2011), who studied the phylogeny of many oceanic taxa in the Halosphaeriaceae. Nimbospora effusa and N. bipolaris are similar in ascospore morphology; a thick, mucilaginous sheath wraps around the ascospores, which is penetrated by fibrillar appendages at the septum. One tuft of appendages is present on one side of the ascospore septum in N. effusa while in N. bipolaris, the appendages are on both sides of the septum. The sheath of N. bipolaris appears to be less eccentric than that of N. effusa (Read et al. 1993). At transmission electron microscope (TEM) magnifications, the appendage precursor material was observed in the cytoplasm and mesosporium at the equator at an early developmental stage in ascospores of N. bipolaris (Read et al. 1993). Fibrillar equatorial appendages are secreted through pores in the episporium and penetrate through the thick, mucilaginous sheath, when the mature ascospores are released from the asci.
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Although monophyletic with N. effusa and N. bipolaris, Naufragella spinibarbata differs significantly from them in terms of ascospore appendage morphology, an important characteristic for the delineation of genera in the Halosphaeriaceae (Sakayaroj et al. 2011). No sheath is present on ascospores of Nauf. spinibarbata, but two types of ascospore appendages are discernible; (1) a gelatinous strap-like appendage at the polar position that separates from the side of the ascospore to form a long wide band in seawater; and (2) hair-like appendages that arise from the sub-polar region of ascospores (Jones et al. 2009). Morphologically, Nohea Kohlm. & Volkm.-Kohlm., typified by Noh. umiumi Kohlm. & Volkm.-Kohlm., also resembles Nimbospora effusa and N. bipolaris by having fibrillar ascospore appendages, but two kinds of appendages are present in Noh. umiumi; (1) a subapical gelatinous pad wraps around the equator and unfurls in water to form long sticky filaments; and (2) two bundles of fibers attach to the spore wall at polar positions (Kohlmeyer and Volkmann-Kohlmeyer 1991). Nohea umiumi, however, is not monophyletic with N. effusa and N. bipolaris (Fig. 3). Nimbospora as presently defined is not a monophyletic genus. Phylogenetically, N. octonae is located in a lineage with Haligena elaterophora, sister to most other taxa of the Halosphaeriaceae (Jones et al. 2009; Sakayaroj et al. 2011), and is distantly related to the other two Nimbospora species. In addition to the sheath, N. octonae differs from N. effusa and N. bipolaris in many morphological characters, including the thick peridial structure of ascomata, the absence of equatorial tufts of appendages but the presence of subulate appendages. Ascomata of Nimbospora octonae are significantly larger (6001250 mm) than those of N. effusa (225240 mm) and N. bipolaris (204380 mm) (Koch 1982; Hyde and Jones 1985; Kohlmeyer 1985). Although forming a monophyletic group with Haligena elaterophora, N. octonae has little morphological resemblance with it. Ascomata of H. elaterophora are coriaceous or subcarbonaceous and black (Kohlmeyer 1961), while those of N. octonae are light-colored (Kohlmeyer 1985). In H. elaterophora, the ascospores are oblong with multiple septa with constrictions, lack a sheath, and the bipolar appendages are strap-like and polymorphic (Sakayaroj et al. 2005). No fibrillar or straplike appendages are present on ascospores of N. octonae. Instead, an exosporic sheath is present around the ascospores and subulate appendages occur at the polar and equatorial positions after the sheath deliquesces (Kohlmeyer 1985). Nimbospora octonae is morphologically most similar to Marinospora A.R. Caval. in the Halosphaeriaceae. In Marinospora spp., both polar and equatorial appendages are outgrowths of the mesosporium and episporium, with a fragmenting exosporic sheath forming tiny caps on the tips of the primary appendages (Johnson et al. 1984). The sheath of ascospores of Marinospora spp. is evanescent, while that of N. octonae is persistent, and no substructures are present on the subulate appendages (Kohlmeyer 1985). Another genus characterized by ascospores with a sheath is Tunicatispora K.D. Hyde, typified by T. australiensis K.D. Hyde (Hyde 1990). However, the sheath of T. australiensis is thin, persistent and skin-like, and a cap-like appendage arises through a hole at a polar position and later unfurls to form a long thread. Unlike
7
Marinospora, no DNA sequences are available for comparison. Because of these morphological and phylogenetic differences, a new genus Ebullia is established below for N. octonae.
5.
Taxonomy
Ebullia K.L. Pang, gen. nov. MycoBank no.: MB 803444. Ascomata subglobose to ampulliform, immersed, ostiolate, with long necks, coriaceous, dark brown to black above, hyaline to brown below. Peridium two-layered, much thicker at the base. Necks periphysate. Asci eight-spored, clavate, pedunculate, unitunicate, thin-walled, early deliquescing, developing at the base of the ascoma venter on a convex cushion of ascogenous cells. Ascospores ellipsoidal, oneseptate, not or slightly constricted at the septum, hyaline, with a gelatinous sheath enclosing 6e7 subulate equatorial appendages evenly distributed around the septum and one similar appendage at each end; swells in water. Type species: Ebullia octonae (Kohlm.) K.L. Pang, this paper. Etymology: ‘Ebullia’ meaning ‘bubble’ in Latin, in reference to the bubble-like sheath of the ascospores. Anamorph: Unknown. Ebullia octonae (Kohlm.) K.L. Pang, comb. nov. Fig. 2. h Nimbospora octonae Kohlm., Can. J. Bot. 63: 1122, 1985 (basionym). MycoBank no.: MB 805905. Distribution: Brunei, Hawaiian Islands, India, Taiwan. Holotype: J.K. 4474a (The New York Botanical Garden). Nimbospora Jørg. Koch, Nordic J. Bot. 2: 166, 1982. Nimbospora effusa Jørg. Koch, Nordic J. Bot. 2: 166, 1982. (type species) Fig. 1AeC. Nimbospora bipolaris K.D. Hyde & E.B.G. Jones, Can. J. Bot. 63: 611, 1985. Fig. 1D.
Acknowledgments K.L. Pang would like to thank National Science Council Taiwan for research grants (NSC101-2621-B-019-001-MY3) and Mr. Ren-Sheng Jheng for help.
references
Bunyard BA, Nicholson MS, Royse DJ, 1994. A systematic assessment of Morchella using RFLP analysis of the 28S ribosomal RNA gene. Mycologia 86: 762e772. Drummond AJ, Rambaut A, 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology 7: 214. Hyde KD, 1990. Intertidal fungi from warm temperate mangroves of Australia, including Tunicatispora australiensis, gen. et sp. nov. Australian Systematic Botany 3: 711e718.
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003
8
m y c o s c i e n c e x x x ( 2 0 1 4 ) 1 e8
Hyde KD, Jones EBG, 1985. Marine fungi from Seychelles. I. Nimbospora effusa and Nimbospora bipolaris sp. nov. from driftwood. Canadian Journal of Botany 63: 611e615. Johnson RG, Jones EBG, Moss ST, 1984. Taxonomic studies of the Halosphaeriaceae: Remispora Linder, Marinospora Cavaliere and Carbosphaerella Schmidt. Botanica Marina 27: 557e566. Jones EBG, Sakayaroj J, Suetrong S, Somrithipol S, Pang KL, 2009. Classification of marine Ascomycota, anamorphic taxa and Basidiomycota. Fungal Diversity 35: 1e187. Koch J, 1982. Some lignicolous marine fungi from Sri Lanka. Nordic Journal of Botany 2: 163e169. Kohlmeyer J, 1961. Pilze von der no¨rdlichen Pazifik-ku¨ste der U.S.A. Nova Hedwigia 3: 80e86. Kohlmeyer J, 1985. Nimbospora octonae, a new marine ascomycete from Hawaii. Canadian Journal of Botany 63: 1122e1125. Kohlmeyer J, Volkmann-Kohlmeyer B, 1991. Illustrated key to the filamentous marine fungi. Botanica Marina 34: 1e61. Pang KL, Jheng JS, 2012. A checklist of marine fungi of Taiwan with description of Kitesporella keelungensis gen. et sp. nov. Botanica Marina 55: 459e466. Rambaut A, 1999. Se-Al. Department of Zoology, University of Oxford, Oxford. http://evolve.zoo.ox.ac.uk/Se-Al/Se-Al.html. Accessed 8 Aug 2002.
Rambaut A, 2009. FigTree v1.3.1. Department of Zoology, University of Oxford, Oxford. http://tree.bio.ed.ac.uk/ software/figtree/. Accessed 21 Dec 2009. Read SJ, Jones EBG, Moss ST, 1993. Ultrastructural observations on Nimbospora bipolaris (Halosphaeriaceae, Ascomycetes). Philosophical Transactions of the Royal Society of London Series B: Biological Sciences 339: 483e489. Sakayaroj J, Pang KL, Jones EBG, 2011. Multi-gene phylogeny of the Halosphaeriaceae: its ordinal status, relationships between genera and morphological character evolution. Fungal Diversity 46: 87e109. Sakayaroj J, Pang KL, Phongpaichit S, Jones EBG, 2005. A phylogenetic study of the genus Haligena (Halosphaeriales, Ascomycota). Mycologia 97: 804e811. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731e2739. White TJ, Bruns T, Lee S, Taylor JW, 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 application. Academic Press, San Diego, pp 315e322.
Please cite this article in press as: Chu Y, et al., A molecular reappraisal of Nimbospora (Halosphaeriaceae, Microascales) and a new genus Ebullia for N. octonae, Mycoscience (2014), http://dx.doi.org/10.1016/j.myc.2014.02.003