Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society 1462 223229 Original Article SYSTEMATICS OF HALOSPHAERIA CUCULLATA K.-L. PANG ET AL.
Botanical Journal of the Linnean Society, 2004, 146, 223–229. With 5 figures
Okeanomyces, a new genus to accommodate Halosphaeria cucullata (Halosphaeriales, Ascomycota) KA-LAI PANG1*, E. B. GARETH JONES2, LILIAN L. P. VRIJMOED3 and SABARATNAM VIKINESWARY4 1
School of Biological Sciences, University of Portsmouth, King Henry Building, King Henry I Street, Portsmouth PO1 2DY, UK 2 National Centre for Genetic Engineering and Biotechnology, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand 3 Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR 4 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, 50603, Federal Territory, Malaysia Received December 2003; accepted for publication March 2004
The taxonomic affinity of Halosphaeria cucullata to Halosphaeria is reassessed based on a recent collection of this fungus. Halosphaeria cucullata is characterized by immersed, darkly coloured ascomata, clavate asci which deliquesce very early in development, and cylindrical ascospores with or without a polar cap-like appendage at one end. In a phylogenetic analysis of the LSU rDNA sequences from members of the Halosphaeriaceae, H. cucullata did not form a monophyletic clade with H. appendiculata, the type species of the genus. These results suggest that H. cucullata should not be included in Halosphaeria. Okeanomyces gen. nov. is proposed to accommodate this fungus. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 223–229.
ADDITIONAL KEYWORDS: Halosphaeria appendiculata – Halosphaeriaceae – marine fungi – Periconia prolifica – phylogeny.
INTRODUCTION Halosphaeria appendiculata Linder is the type species of the genus Halosphaeria Linder (Halosphaeriaceae, Halosphaeriales) and was one of the earliest lignicolous ascomycetes described from the marine environment (Barghoorn & Linder, 1944). This species is characterized by having polar and equatorial ascospore appendages that are deep spoon-shaped at the region of attachment. Appendages arise as an outgrowth of the episporium and are attached to the ascospore by an isthmus of electron-dense material (Jones, Johnson & Moss, 1984; Hyde, Moss & Jones, 1994). The appendages comprise a reticulate network of material. Twelve species were once assigned to the genus which included: H. appendiculata, H. cucullata *Corresponding author. E-mail:
[email protected]
(Kohlm.) Kohlm., H. hamata (Höhnk) Kohlm., H. maritima (Linder) Kohlm., H. mediosetigera Cribb & J. W. Cribb, H. pilleata (Kohlm.) Kohlm., H. quadricornuta Cribb & J. W. Cribb, H. quadriremis (Höhnk) Kohlm., H. salina (Meyers) Kohlm., H. stellata (Kohlm.) Kohlm., H. torquata Kohlm. and H. trullifera (Kohlm.) Kohlm. (Kohlmeyer, 1972). Based on ascospore appendage morphology and ontogeny, the genus was subsequently split into a number of different genera (Jones, Moss & Cuomo, 1983; Jones et al., 1984) and only Halosphaeria appendiculata and H. cucullata are retained in the genus (Jones, 1995; Hyde, Sarma & Jones, 2000). Halosphaeria cucullata is distinct from H. appendiculata in having bi-celled, hyaline and cylindrical ascospores with a cap-like appendage at one end (Kohlmeyer, 1964). However, this appendage is ephemeral and only readily observed in very young
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material. Often, a thin layer of material is formed around the ascospore when observed at the TEM level (E. B. G. Jones, pers. observ.). While H. cucullata has an anamorphic stage (Periconia prolifica Anastasiou), none has been reported for H. appendiculata (Shearer, 1986). This species was initially described by Kohlmeyer (1964) as Remispora cucullata Kohlm. but was later transferred to Halosphaeria in his revision of the family Halosphaeriaceae (Kohlmeyer, 1972). The placement of this species in Halosphaeria has been doubtful because of the significant differences in morphology between this species and H. appendiculata. Its position in Halosphaeria has long been questioned (Nakagiri & Tubaki, 1985). Recently, H. cucullata was collected on pneumatophores of Sonneratia sp. in Morib mangrove, Malaysia on several occasions. The morphology of this species was examined and compared with H. appendiculata. In addition, phylogenetic analysis of the partial large subunit ribosomal RNA gene was used to determine the phylogenetic relationship between H. appendiculata and H. cucullata, a species showing no morphological affinity to the former species, the type species of Halosphaeria.
MATERIAL AND METHODS MORPHOLOGY Decaying pneumatophores of Sonneratia sp. were collected in Morib mangrove, Malaysia. Samples were examined immediately after returning to the laboratory. Material was mounted in seawater and examined under an interference contrast microscope for photography.
FUNGAL
CULTURES
Ceriosporopsis halima Linder LP45 (on driftwood from Denmark), Halosphaeria appendiculata CY3485 (on driftwood from Friday Harbour, U.S.A.), Halosphaeria cucullata LP67 (on pneumatophore of Sonneratia sp. from Morib, Malaysia) and Periconia prolifica CY2978 (on wood block of Sonneratia sp., Hong Kong) were grown in GYP broth (4 gL-1 glucose, 4 gL-1 yeast extract, 2 gL-1 peptone) in filtered seawater. Ceriosporopsis halima LP45 and H. cucullata LP67 were isolated by the senior author and deposited at the Portsmouth University Culture Collection, UK. Halosphaeria appendiculata CY3485 and P. prolifica CY2978 were obtained from the City University Culture Collection, Hong Kong.
DNA
EXTRACTION,
PCR
AND SEQUENCING
Genomic DNA was extracted using the DNeasy Plant DNA Extraction Kit (Qiagen) according to the manu-
facturer’s instructions. The nuclear large subunit rRNA gene was amplified from genomic DNA using JS1 (Landvik, 1996) and LR7 (Bunyard, Nicholson & Royse, 1994) primers. PCR reactions were performed in 50 mL containing c. 20 ng DNA, 0.2 mM of each primer, 0.2 mM of each dNTP, 1.5 mM MgCl2 and 1 U of Taq Polymerase (Gibco). The amplification cycle consisted of an initial denaturation step of 95∞C for 2 min followed by 35 cycles of (i) denaturation (95∞C for 1 min), (ii) annealing (55∞C for 1.5 min) and (iii) elongation (72∞C for 1.5 min), and a final 10-min elongation step at 72∞C. The PCR products were analysed by agarose gel electrophoresis and purified using QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. The purified PCR product was sent off to the Sequencing Service, University of Dundee, Scotland for direct sequencing using JS1 and JS5 primers of Landvik (1996) and LR5 and LR7 of Bunyard et al. (1994).
ALIGNMENT
AND PHYLOGENETIC ANALYSIS
Thirty-two sequences were aligned manually in Se-Al v. 1.0a1 (Rambaut, 1999) based on the alignment of our previous study (Pang et al., 2003b). GenBank accession numbers are listed in Table 1. All analyses were performed in PAUP* v.4.0b10 (Swofford, 2002). Two insertions in H. cucullata LP67 were excluded from the analysis. All gaps, the beginning and the end of the sequences were treated as missing data. Daldinia concentrica (Bolton) Ces. & De Not. was designated as the outgroup taxon in all analyses. A most parsimonious tree (MPT) was constructed through an heuristic search with stepwise addition of taxa, 100 replicas of random addition of taxa, treebisection-reconnection branch-swapping algorithm. Maximum likelihood analysis was performed with the MPT as the starting tree (tree-bisection-reconnection branch-swapping algorithm). One thousand parsimony bootstrap analyses (stepwise addition of sequence, ten replicas of random addition of taxa, tree–bisection–reconnection branching–swapping algorithm) were used to determine support of the clades.
RESULTS MORPHOLOGY A recent collection of Halosphaeria cucullata allowed a detailed examination of its morphology to reassess its taxonomic affinity to Halosphaeria. Identity of this isolate was once uncertain because of the lack of ascospore appendages from all the ascomata examined. The production of its anamorph, Periconia prolifica, from 20 single spore isolates (including isolate
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Table 1. Sequences included in the phylogenetic analysis of partial LSU rRNA gene and their GenBank accession number Taxa
GenBank accession number
Aniptodera chesapeakensis Shearer & M. Miller Arenariomyces trifurcatus Höhnk Ascosalsum viscidulum (Kohlm. & E. Kohlm.) J. Campb., J. L. Anderson & Shearer Ceriosporopsis halima Linder LP45 Ceriosporopsis halima Linder Corollospora maritima Werderm. Cucullosporella mangrovei K. D. Hyde & E. B. G. Jones Daldinia concentrica (Bolton) Ces. & De Not. Halosarpheia fibrosa Kohlm. & E. Kohlm. Halosphaeria appendiculata Linder Halosphaeria appendiculata Linder CY3485 Halosphaeria cucullata (Kohlm.) Kohlm. LP67 Halosphaeriopsis mediosetigera (Cribb & J. W. Cribb) T. W. Johnson Lignincola laevis Höhnk Magnisphaera spartinae (E. B. G. Jones) J. Campb., J. L. Anderson & Shearer Marinospora longissima (Kohlm.) Cavaliere Microascus trigonosporus C. W. Emmons & B. O. Dodge Nais inornata Kohlm. Natantispora retorquens (Shearer & J. L. Crane) J. Campb., J. L. Anderson & Shearer Neptunella longirostris (Cribb & J. W. Cribb) K. L. Pang & E. B. G. Jones Nereiospora comata (Kohlm.) E. B. G. Jones, R. G. Johnson & S. T. Moss Nimbospora effusa J. Koch Nohea umiumi Kohlm. & Volkm.-Kohlm. Ophiodeira monosemeia Kohlm. & Volkm.-Kohlm. Periconia prolifica Anastasiou CY2978 Petriella setifera (J. C. Schmidt) Curzi Phaeonectriella lignicola R. A. Eaton & E. B. G. Jones Saagaromyces ratnagiriensis (S. D. Patil & Borse) K. L. Pang & E. B. G. Jones Tirispora unicaudata E. B. G. Jones & Vrijmoed Varicosporina ramulosa S. P. Meyers & Kohlm. Xylaria curta Fr. Xylaria hypoxylon (L.) Grev.
U46882 U46883 AY150223 AY490787 U47844 U46884 AY150219 U47828 AF396872 U46885 AY090892 AY490788 U46887 AF539471 AY150221 AF491266 U47835 AF539476 AF396874 AF539473 AF491267 U46892 U46893 U46894 AY090891 AF043596 AY150224 AF539470 AY150225 U44092 U47840 U47841
LP67 sequenced in this study) confirmed its identity as H. cucullata (Kohlmeyer, 1969). Ascomata of H. cucullata are either immersed or erumpent and dark in colour (Figs 1, 2). Asci are arisen from a hymenium (Fig. 3) but deliquesce early in development. Ascospores are cylindrical in shape and uniseptate but no cap-like appendage was observed in our specimens (Fig. 4).
SEQUENCE
ANALYSIS
In the maximum parsimony analysis, two MPTs resulted from 260 parsimony-informative characters, each with a tree length of 1192 steps; the consensus tree is shown in Figure 5. The bootstrap supports on most of the nodes within the Halosphaeriales were poor except the well-supported groups in the dotted-line boxes, which include H. appendiculata, H. cucullata and P. prolifica. Halosphaeria cucullata
is not monophyletic with H. appendiculata and phylogenetically supported by the nodes separating these two taxa (61% and 100%). Halosphaeria cucullata forms a well-supported clade with Periconia prolifica, its anamorph (100%). Similar topology was obtained from maximum likelihood analysis for the relationship between H. appendiculata and H. cucullata, with the difference in the group comprising H. appendiculata, Neptunella longirostris (Cribb & J. W. Cribb) Pang & E. B. G. Jones and Nohea umiumi Kohlm. & Volkm.-Kohlm. The two H. appendiculata isolates are not monophyletic; one groups with Neptunella longirostris and the other with Nohea umiumi, but all with low bootstrap support.
DISCUSSION Data from both morphological observation and phylogenetic analysis of the LSU rRNA gene suggest that
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1
2
3
4
Figures 1–4. Okeanomyces cucullatus. Fig. 1. Immersed, dark-coloured ascoma in pneumatophore of Sonneratia sp. Scale bar = 20 mm. Fig. 2. Neck. Scale bar = 10 mm. Fig. 3. Immature, clavate asci in very young material. Scale bar = 10 mm. Fig. 4. Two-celled, fusiform ascospore without any appendage. Scale bar = 5 mm.
H. cucullata is not related to Halosphaeria (H. appendiculata), and a new genus is proposed to accommodate it. Differences in ascospore and ascospore appendage morphology suggest that H. cucullata has no affinity to Halosphaeria. There is no, or only a single polar cap-like deciduous appendage to ascospores in H. cucullata, whereas polar and equatorial spoonshaped appendages are observed in H. appendiculata. Moreover, the ascospore of H. cucullata is more cylin-
drical (length/width = 5.1) as compared to those of H. appendiculata (length/width = 2.5). From these data alone it is apparent that H. cucullata should be removed from Halosphaeria based solely on morphological grounds, as has previously been argued by Nakagiri & Tubaki (1985). Ascospore and ascospore appendage morphology and ontogeny are still the most important delimiting characters in the Halosphaeriales because: (a) most ascomatal characters (colour, texture, etc.) are very susceptible to
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Nohea umiumi
HALOSPHAERIALES
Neptunella longirostris Halosphaeria appendiculata Halosphaeria appendiculata CY3485 Nimbospora effusa Tirispora unicaudata
61
Natantispora retorquens Lignincola laevis
96
Phaeonectriella lignicola
71 100
Aniptodera chesapeakensis Nais inornata
100
Okeanomyces cucullatus Periconia prolifica
87
Corollospora maritima Varicosporina ramulosa
52
Arenariomyces trifurcatus Nereiospora comata Halosphaeriopsis mediosetigera Saagaromyces ratnagiriensis Cucullosporella mangrovei Halosarpheia fibrosa Magnisphaera spartinae
100
83
Ophiodeira monosemeia Ascosalsum viscidulum
100
100
Ceriosporopsis halima Ceriosporopsis halima LP45 Marinospora longissima
96
Microascus trigonosporus Petriella setifera
96
Xylaria hypoxylon Xylaria curta Daldinia concentrica
Figure 5. Consensus tree of two MPTs from parsimony analysis of the large subunit ribosomal RNA gene sequence of 32 taxa from the Halosphaeriales. Consistency Index = 0.5092, Retention Index = 0.5673, Tree length = 1192.
change by environmental conditions and substrata; (b) there is a general lack of information about ascoma morphology, e.g. number of peridial cell type and cell layers; (c) asci deliquesce in most taxa of the order, by that limiting their use as a systematic character; and (d) ascospore and ascospore appendage morphology and ontogeny are the most consistent traits. However, parallel evolution or adaptation to environmental conditions has been noted in genera with polar unfurling appendages (Pang et al., 2003b).
This separation of H. appendiculata and H. cucullata is supported by the molecular data. The bootstrap supports of the clades within the Halosphaeriales are weak, as demonstrated by other studies (Pang et al., 2003a, b) especially when many taxa are included in the analysis to resolve the relationship between H. cucullata and other genera in the order. However, the group comprising H. appendiculata and H. cucullata is robust and their separation is well-supported. In the maximum likeli-
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hood analysis, the two H. appendiculata isolates are not monophyletic, and this may be accounted for by the shorter length of the H. appendiculata sequence retrieved from the GenBank. Periconia prolifica is the anamorph of H. cucullata, a connection that was established by Kohlmeyer (1969). In our study, isolation of H. cucullata always results in the anamorph. Thus, based on these results, H. cucullata is removed from Halosphaeria and a new genus, Okeanomyces, is proposed as it has no morphological affinity with other genera in the Halosphaeriales.
Substrata: Intertidal and drifting wood, test panels, mangrove roots and pneumatophores, and seedlings of Rhizophora mangle, submerged branches of Pluchea ¥ fosbergii, endocarp of Cocos nucifera; sometimes in the calcareous lining of teredinid tubes.
TAXONOMY
REFERENCES
OKEANOMYCES K. L. PANG & E. B. G. JONES GEN. NOV.
Barghoorn ES, Linder DH. 1944. Marine fungi: their taxonomy and biology. Farlowia 1: 395–467. Bunyard BA, Nicholson MS, Royse DJ. 1994. A systematic assessment of Morchella using RFLP analysis of the 28S ribosomal RNA gene. Mycologia 86: 762–772. Hyde KD, Moss ST, Jones EBG. 1994. Ascospore ultrastructure of Halosphaeria appendiculata (Halosphaeriaceae). Botanica Marina 37: 51–56. Hyde KD, Sarma VV, Jones EBG. 2000. Morphology and taxonomy of higher marine fungi. In: Hyde KD, Pointing SB, eds. Marine mycology – a practical approach. Hong Kong: Fungal Diversity Press, 172–204. Jones EBG. 1995. Ultrastructure and taxonomy of the aquatic ascomycetous order Halosphaeriales. Canadian Journal of Botany 73: S790–S801. Jones EBG, Johnson RG, Moss ST. 1984. Taxonomic studies of the Halosphaeriaceae: Halosphaeria Linder. Botanica Marina 27: 129–143. Jones EBG, Moss ST, Cuomo V. 1983. Spore appendage development in the lignicolous marine pyrenomycetes Chaetosphaeria chaetosa and Halosphaeria trullifera. Transactions of the British Mycological Society 80: 193– 200. Kohlmeyer J. 1964. A new marine ascomycete from wood. Mycologia 56: 770–774. Kohlmeyer J. 1969. Marine fungi of Hawaii including the new genus Helicascus. Canadian Journal of Botany 47: 1469–1487. Kohlmeyer J. 1972. A revision of Halosphaeriaceae. Canadian Journal of Botany 50: 1951–1963. Landvik S. 1996. Neolecta, a fruit-body-producing genus of the basal ascomycetes, as shown by SSU & LSU rDNA sequences. Mycological Research 100: 199–202. Nakagiri A, Tubaki K. 1985. Teleomorph and anamorph relationships in marine ascomycetes (Halosphaeriaceae). Botanica Marina 28: 485–500. Pang KL, Vrijmoed LLP, Kong RYC, Jones EBG. 2003a. Lignincola and Nais, polyphyletic genera of the Halosphaeriales (Ascomycota). Mycological Progress 2: 29–36. Pang KL, Vrijmoed LLP, Kong RYC, Jones EBG. 2003b. Polyphyly of Halosarpheia (Halosphaeriales, Ascomycota): implications on the use of unfurling ascospore appendages as a systematic character. Nova Hedwigia 77: 1–18.
Type species: Okeanomyces cucullatus (Kohlm.) K. L. Pang & E. B. G. Jones Diagnosis: Ascomata subglobosa vel ellipsoidea, immersa vel subimmersa, fulva vel nigra, coriacea vel subcarbonacea. Periphyses absentes. Asci clavati, cum pedunculis brevibus, leptodermi, pristine deliquescentes. Catenophyses praesentes. Ascosporae cylindricae, parietibus tenuis, cum vel sine appendicibus. Description: Ascomata subglobose or ellipsoidal, immersed or semi-immersed, brown or black, coriaceous or subcarbonaceous. Periphyses absent. Asci clavate, with short peduncles, thin-walled, early deliquescing. Catenophyses present. Ascospores cylindrical, thin-walled, with or without a single cap-like appendage. Etymology: From the Greek ‘okeanos’ meaning ocean in reference to the habitat of the fungus.
OKEANOMYCES CUCULLATUS (KOHLM.) K. L. PANG & E. B. G. JONES COMB. NOV. (FIGS 1–4) Basionym: Remispora cucullata Kohlm. Mycologia 56: 770. 1964. Synonym: Halosphaeria cucullata (Kohlm.) Kohlm. Can. J. Bot. 50: 1956. 1972. Anamorph: Periconia prolifica Anastasiou. Holotype: J. K. no. 1686 in Herb. Mus. Bot. Berol. (B) Distribution: Australia, Bahamas, Belize, Brazil, Brunei, Great Abaco, Guatemala, Hawaii, India, Japan, Malaysia, Mauritius, Mexico, South Africa, Thailand, United States.
ACKNOWLEDGEMENTS K. L. Pang thanks the City University of Hong Kong for a postgraduate studentship and the Croucher Foundation, Hong Kong, for a postdoctoral fellowship.
© 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 223–229
SYSTEMATICS OF HALOSPHAERIA CUCULLATA Rambaut A. 1999. Se-Al. Department of Zoology, University of Oxford, Oxford OX1 4JD, UK. http://evolve.zoo.ox.ac.uk/ Se-Al/Se-Al.html Shearer CA. 1986. The significance of teleomorph/anamorph connections in the classification of marine Ascomycotina. In:
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Moss ST, ed. The biology of marine fungi. Cambridge: Cambridge University Press, 253–262. Swofford DL. 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods), v.4.0b10 Sunderland, MA: Sinauer Associates.
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