Mycologia, 102(1), 2010, pp. 83–92. DOI: 10.3852/07-147 # 2010 by The Mycological Society of America, Lawrence, KS 66044-8897
Morphological and molecular characteristics of a poorly known marine ascomycete, Manglicola guatemalensis ( Jahnulales: Pezizomycotina; Dothideomycetes, Incertae sedis): new lineage of marine ascomycetes Satinee Suetrong1
Brachiosphaera tropicalis and Xylomyces chlamydosporus. In subclade B Manglicola strains form a sister group of the Aliquandostipite species Aliquandostipite crystallinus, A. khaoyaiensis, Jahnula siamensiae and Patescospora separans. Subclade C comprises Jahnula species, Jahnula aquatica, J. granulosa and J. rostrata, while Megalohypha aqua-dulces constitutes subclade D. Therefore Manglicola forms another lineage of marine fungi. Key words: Hypsostromataceae, Jahnulales, LSU rDNA, phylogeny, SSU rDNA
Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand, and Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Paholyothin Road, Khlong 1, Khlong Luang, Pathum Thani, 12120, Thailand
Jariya Sakayaroj Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Paholyothin Road, Khlong 1, Khlong Luang, Pathum Thani, 12120, Thailand
Souwalak Phongpaichit
INTRODUCTION
Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand
A number of publications document the collections of marine fungi in Thailand. Kohlmeyer (1984) was the first to report 15 marine fungi from Chonburi Province, and subsequent collections are listed in Jones et al. (2006). Senescent fronds of the brackish water palm Nypa fruticans have been examined for marine fungi, with Pilantanapak et al. (2005) listing 126 taxa. However none of these studies list M. guatemalensis, a rarely collected marine ascomycete (Kohlmeyer and Kohlmeyer 1971, Hyde 1988). The type species, Manglicola guatemalensis, was collected from dead roots of Rhizophora mangle in Guatemala (Kohlmeyer and Kohlmeyer 1971). Subsequent collections were made on intertidal prop roots of Rhizophora apiculata at Kpg. Danau, Brunei (Hyde 1988). Kohlmeyer and Kohlmeyer (1971) noted a close relationship of M. guatemalensis and the Pleosporaceae or Venturiaceae, while Huhndorf (1992, 1994) classified it in the Hypsostromataceae, order Incertae sedis. Thus its phylogenetic position is not clear. The objective of this study was (i) to document the morphological features of new collections of Manglicola guatemalensis and (ii) to determine higher order classification of the fungus based on combined SSU and LSU rDNA sequences.
E.B. Gareth Jones Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Paholyothin Road, Khlong 1, Khlong Luang, Pathum Thani, 12120, Thailand
Abstract: The poorly known marine ascomycete Manglicola guatemalensis from Trang and Trat provinces, Thailand, were collected in 2005 and 2006 on the brackish water palm Nypa fruticans. This fungus is known only from two previous collections. This paper reports on the morphological characteristics and molecular phylogeny of this unique marine bitunicate ascomycete. Manglicola guatemalensis has large clavate to obtusely fusiform ascomata, wide ostioles, bitunicate asci, cylindrical, thick-walled, unequally one-septate ascospores, constricted at the septum, apical cell larger, chestnut-brown and a smaller light brown basal cell. Ascospores germinate readily, always from the basal cell. Four isolates from different locations were selected for the phylogenetic study. Regions of the rDNA gene, including SSU and LSU, were sequenced and combined. Molecular data places M. guatemalensis in the Jahnulales with high bootstrap support; all strains are monophyletic. In the combined SSU and LSU analyses the Jahnulales comprises four subclades, A, B, C and D. Subclade A comprises Jahnula species and two anamorphic fungi,
MATERIALS AND METHODS
Collection of fungi.—Submerged basal parts of the Nypa fruticans fronds were collected in Mu Ko Chang National Park, in Trat Province (Oct 2005, 2006) and Trang Province (Nov 2005), placed in clean plastic bags and returned to the laboratory. Samples were examined after washing with freshwater to remove mud and sediments for fungi. Samples
Submitted 7 Aug 2007; accepted for publication 7 Jun 2009. 1 Corresponding author. E-mail:
[email protected]
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MYCOLOGIA
TABLE I.
GenBank accession numbers of Manglicola guatemalensis and Jahnula appendiculata sequences used GenBank numbers
Genera
Classification
Isolate number
BCC number
Manglicola guatemalensis Kohlm. and Kohlm.
Dothideomycetes, Incertae sedis
SAT00180
BCC 20079
SAT00179 GR00249
BCC 24217 BCC 20156
GR00250
BCC 20157
PF00009
BCC 11400
PF0008
BCC 11445
Jahnula Dothideomycetes, appendiculata Incertae sedis Pinruan, K.D. Hyde and E.B.G. Jones
were kept moist by spraying with sterilized distilled water. Sporulating fungi were examined, identified, illustrated and single-spore isolations made. Fungal isolates.—Single ascospore isolations of M. guatemalensis were made on cornmeal seawater agar (CMA/SW) with antibiotics (streptomycin sulfate 0.5 g/L, penicillin G 0.5 g/L) and germinated overnight. Germinating spores were transferred to a fresh agar plate and incubated 2 wk at 25 C and deposited in the BIOTEC Culture Collection (BCC). Dried specimens were deposited in the BIOTEC Bangkok Herbarium (BBH). DNA extraction, amplification and sequencing.— Fungal biomass was harvested through cheesecloth, washed several times with sterile distilled water, frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. Fifty to one hundred milligrams ground fungal mycelia was placed in 400 mL lysis buffer (O’Donnell et al 1997). The rDNA was amplified with Taq DNA polymerase from FERMENTAS Taq DNA polymerase (recombinant) using PCR Model MJ Research DYAD ALD. Primers used for amplification include the small subunit (SSU) and large subunit (LSU) of rDNA (White et al 1990, Bunyard et al 1994, Landvik 1996). The PCR products were purified with Nucleo SpinH Extract Kit (Macherey-Nagel, Germany), following the manufacturer’s instructions. PCR products were directly sequenced by Macrogen Inc., Korea. Each sequence was checked for ambiguous bases and assembled with BioEdit 6.0.7 (Hall 2004). Sequence alignment and phylogenetic analyses.—The consensus sequences for each DNA region were multiple aligned by Clustal W 1.6 (Thompson et al 1994) along with other sequences obtained from GenBank. Accession numbers for selected taxa obtained from GenBank were provided (TABLE I, FIG. 16). The dataset was refined visually in
Locality Trang Province, Thailand Trang Province Mu Ko Chang National Park, Trat Province Mu Ko Chang National Park, Trat Province Chaloem Phrakiat Somdet Phra Thep Wildlife, Narathiwat Province Chaloem Phrakiat Somdet Phra Thep Wildlife, Narathiwat Province
SSU
LSU
FJ743443
FJ743449
FJ743441 FJ743442
FJ743447 FJ743448
FJ743444
FJ743450
FJ743440
FJ743446
FJ743439
FJ743445
BioEdit 6.0.7 (Hall 2004). Saccharomyces cerevisiae and Candida valdiviana were chosen as outgroup for all analyses. The phylogenetic analyses of the combined SSU and LSU rDNA sequence data were performed with PAUP* 4.0b 10 (Swofford 2002). Gaps were treated as missing data. Maximum parsimony analyses on settings were performed. (a) Unweighted parsimony: 100 replicates of random stepwise addition of sequence and tree-bisection reconnection (TBR) branch-swapping algorithm. All characters were given equal weight. The consistency indices (CI), retention indices (RI) and rescaled consistency indices (RC) were calculated for each tree. Nucleotide transformation based on the transition : transversion (ti : tv) ratio was estimated with maximum likelihood score in PAUP* 4.0b 10 (Swofford 2002). The rate ratio of transition : transversion was 1.7. (b) Weighted (step matrix) parsimony was performed to weight transversion 1.7 times over transition; 100 replicates of random stepwise addition of sequence. (c) Reweighted parsimony: characters were reweighted according to their RC: 100 replicates of random stepwise addition of sequence. Tree topologies from parsimony analyses were tested with the KashinoHasegawa (K-H) maximum likelihood test (Kishino and Hasegawa 1989) to find the most likely tree. Bootstrap supports (Felsenstein 1985) were calculated for all parsimony analyses by 1000 bootstrap replicates (full heuristic searches, 10 replicates of random stepwise addition of sequence). (d) Bayesian phylogenetic analysis was performed in MrBayes 3.0.b4 (Huelsenbeck and Ronquist 2001) with a uniform GTR + I + G model, 5 000 000
SUETRONG ET AL: MANGLICOLA GUATEMALENSIS generations in four chains with sampling every 100 generations. The first 5000 generations were discarded as burn-in. A 50% majority rule consensus tree of all remaining trees, as well as the posterior probabilities (PP), was calculated.
RESULTS MORPHOLOGY OF MANGLICOLA GUATEMALENSIS
Ascomata 1100–1750 mm high, 290–640 mm diam at center, 82.5–280 mm diam at base, 100–200 mm diam at apex; obtusely clavate to obtusely fusiform; stipitate, epapillate, coriaceous, olive-brown, aggregated (FIG. 1) or solitary (FIGS. 2, 4), ascoma wall differentiated into several layers of polygonal, thick-walled cells; ascoma superficial seated on the substratum with a hypostroma, composed of pseudoparenchymatous cells and brown hyphae 5–20 mm diam; ostiolate; stipe composed of a cortex of polygonal, brown cells (FIG. 2). Ascoma attached by a hypostroma immersed in the host tissue of N. fruticans. Peridium 30–55 mm thick, composed of 3–5 layers of cells (FIGS. 3–5). Immature ascomata are differentiated into a wall composed of several layers of polygonal, thick-walled cells and smaller, thin-walled cells that appear dark. Pseudoparaphyses 1.23–2.5 mm diam, narrow, numerous, septate, simple, trabeculate (arrowed PR) between asci arising from the base of the centrum (FIG. 6), but reticulate and anastomosing in the upper part of the centrum and arising from the venter wall (FIG. 4 arrowed RPR). Asci 440–640 3 30– 50 mm, 8-spored, cylindrical, bitunicate, thick-walled, developing at the base of the ascoma venter between the pseudoparaphyses. In mature ascomata branching and reticulate pseudoparaphyses stand between the asci (FIGS. 2, 6, 7). Ascomata contain 3–5 mature asci and are visible through the thin ascoma wall (FIG. 2). Asci with apical apparatus, comprising a lens-shape disk and more clearly defined with ascus maturity (FIG. 9). Immature ascospores are hyaline, 1-septate, and not constricted at the septum. Ascospores from the Mu Ko Chang collection: 87.5–137.5 3 20– 47.5 mm (FIGS. 11, 12); and Trang collection: 92.5– 125 3 22.5–40 mm (FIG. 13), uniseriate, fusiform, apiculate, unequally 1-septate, constricted at the septum; apical cell larger, orange-brown, light brown, dark brown to chestnut-brown; basal cell, turbinate, light brown; gelatinous appendages (FIG. 10) cover both apices; apical and basal appendages are cylindrical. Ascospores germinate readily, always from the basal smaller cell (FIG. 8). Anamorph. Not known. Colonies on potato dextrose seawater agar (PDA/ SW) effuse and immersed; cream to light brown, producing a brown pigment. Colonies grow slowly on
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CMA/SW and PDA/SW reaching ca. 2–3 cm diam (FIG. 14) in 30 d at room temperature (21–25 C), mycelium 10–18.7 mm wide, hypha smooth-walled, septate and constricted at the septa (FIG. 15). Habitat. Superficial on frond bases of Nypa fruticans and Rhizophora mangle. Distribution. Brunei, Guatemala and Thailand. Material examined. Mu Ko Chang National Park, Trat Province BBH 17801; Nypa forest, Trang Province. Isolates of M. guatemalensis BCC 20156–20157, 25032–25053, Mu Ko Chang National Park, Trat Province; BCC 20079, 20167–20168, 24217, Nypa forest, Trang Province. PHYLOGENY
Analyses of the combined SSU and LSU rDNA sequence data were performed along with various orders of the Dothideomycetes (Jahnulales, Pleosporales, Patellariales, Capnodiales, Myriangiales and Dothideales) from GenBank. The dataset consisted of 64 taxa, with Saccharomyces cerevisiae and Candida valdiviana as outgroup. Maximum parsimony analyses on different settings (unweighted, weighted [step matrix] and reweighted) gave the same topology for all analyses. The unweighted parsimony dataset consists of 2377 total characters; 1344 characters are constant, 464 characters are parsimony informative and 579 variable characters are parsimony uninformative. This analysis resulted in 14 MPT 2359 steps long (CI 5 0.618, RI 5 0.780, RC 5 0.482). The difference between these MPT is in the swapping branching pattern in Jahnula (trees not shown). The reweighted parsimony resulted in 18 MPT with a tree length of 1456.86549 steps, CI 5 0.755, RI 5 0.840, RC 5 0.634, which had the same topology as unweighted parsimony (trees not shown). The weighted step matrix parsimony yielded 91 MPT with 3052.20 steps long, CI 5 0.619, RI 5 0.782 and RC 5 0.484. Its topology was identical to one of the MPT in unweighted parsimony analysis. Tree length from weighted step matrix parsimony was longer than unweighted parsimony and had lower consistency and rescaled consistency indices. Moreover Bayesian inference also provided identical topology to other analyses. The result of the K-H test for all trees generated from unweighted, reweighted and weighted parsimony is provided (TABLE III). The trees produced by weighted analysis gave the best phylogenetic hypothesis for the dataset (FIG. 16). In all analyses the Dothideomycetidae can be divided into five major groups (Capnodiales/Myriangiales, Dothideales, Jahnulales, Patellariales and Pleosporales), in which they form a monophyletic
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MYCOLOGIA
FIG. 1. Mature ascomata of M. guatemalensis on surface of N. fruticans, partially immersed in mud (MU). Bar 5 500 mm. FIG. 2. Ascoma superficial seated on the substratum with a hypostroma, composed of pseudoparenchymatous cells (PC) and dark ascospores (AS) in the mature asci (AC), visible through thin wall; spores exuded at ostiole (OS). Bar 5 500 mm. FIG. 3. Peridium (PE) composed of 3–5 layers of cells. Bar 5 10 mm. FIGS. 4, 5. Longitudinal section of ascoma.
SUETRONG ET AL: MANGLICOLA GUATEMALENSIS
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FIG. 8. Germinating ascospore, with germ tube developing laterally from the small basal cell. Bar 5 50 mm. FIG. 9. Ascus tip with an apical apparatus (AP) comprising a lens-shape disk. Bar 5 20 mm. FIG. 10. Ascospore in ascus with apical (AA) and basal (BA) appendages. Bar 5 20 mm. FIG. 11. Ascospores of M. guatemalensis collected from Mu Ko Chang National Park, Trat Province in 2006. Bar 5 50 mm. FIG. 12. Ascospores of M. guatemalensis collected from Mu Ko Chang National Park, Trat Province in 2005. Bar 5 50 mm. FIG. 13. Ascospores of M. guatemalensis collected from Nypa forest, Trang Province in 2005. Bar 5 50 mm. FIG. 14. Culture on seawater potato dextrose agar at room temperature for 30 d. FIG. 15. Hyphal fusion. Bar 5 50 mm. r Ascoma covered with mud (arrowed MU, Fig. 5). Periphyses (PP) are simple; reticulate (arrowed RPR) and anastomosing pseudoparaphyses in the upper part of the centrum arising from the venter wall; ostiolar canal (OC), trabeculate pseudoparaphyses (PR) arise between the asci from the base of the centrum. Bars 5 50 mm. FIG. 6. Trabeculate pseudoparaphyses numerous, septate, simple. Bar 5 10 mm. FIG. 7. Cylindrical asci. Bar 5 100 mm.
88
MYCOLOGIA
TABLE II. Comparison of the morphological features of Manglicola guatemalensis collected in this study with those of Kohlmeyer and Kohlmeyer 1971 M. guatemalensis (Trang and Trat provinces) Submerged Nypa fronds (data in TABLE I)
M. guatemalensis (Kohlmeyer and Kohlmeyer 1971) Dead roots of Rhizophora mangle
1100–1750 mm diam 290–640 mm diam 100–200 mm diam 82.5–280 mm diam 30–55 mm wide 0.75–1.25 mm diam Trabeculate 1.25–2.5 mm diam Cylindrical, bitunicate 440–640 3 30–50 mm, 8-spored Fusiform, uniseriate, apiculate, unequally 1-septate, constricted at the septum; apical appendage cylindrical and basal appendages are cylindrical, prominent at the apical end of spore Mu Ko Chang: 87–137.5 3 20–47.5 mm Trang: 92.5–125 3 22.5–40 mm
835–1275 mm diam 95–165 mm diam 100–185 mm diam 95–165 mm diam 36–50 mm wide 2–3 mm diam Simple or reticulate 2–4 mm diam Cylindrical, bitunicate 275–326 3 24–28 mm, 8-spored Fusiform, uniseriate, apiculate, unequally 1-septate, constricted at the septum; deliquescing appendage cover both apices; apical appendage cylindrical, basal appendage subglobose 80–109 3 18–34 mm
Character Ascomata: High At center At apex At base Peridium Periphyses Pseudoparaphyses Asci Ascospores
group. The interordinal relationships within the Dothideomycetidae showed a stable branching pattern but with weak bootstrap support in some major clades, namely Pleosporales/Patellariales and Dothideales/Capnodiales/Myriangiales (FIG. 16). Four isolates of Manglicola guatemalensis strains form a monophyletic group and are well placed in the Jahnulales with high bootstrap support and posterior probabilities. The Jahnulales form a well supported clade within the Dothideomycetes, along with orders Pleosporales, Patellariales, Dothideales and Capnodiales/Myriangiales. Four subclades (A, B, C and D) are apparent within the Jahnulales (FIG. 16): Subclade (A) comprised Jahnula species Jahnula appendiculata, J. australiensis, J. bipileata, J. bipolaris, J. sangamonensis, J. seychellensis and J. sunyatsenii and two anamorphic
TABLE III.
Result of Kishino-Hasegawa maximum likelihood tests of the combined SSU and LSU
Analysis I. Unweighted parsimony II. Weighted parsimony (Reweighted) III. Weighted parsimony (Step matrix to weight transversion 1.7 times over transition) *
fungi, Brachiosphaera tropicalis and Xylomyces chlamydosporus, and form a monophyletic clade with strong statistical support (96% bootstrap support and 1.00 PP). The three Jahnula appendiculata strains form a close relationship with J. bipolaris, J. sangamonensis, J. seychellensis, J. sunyatsenii, while Aliquandostipite species formed a separated clade with weak support. Subclade B comprised Aliquandostipite species Aliquandostipite crystallinus, A. khaoyaiensis, A. siamensiae and Patescospora separans with strong statistical support (99% bootstrap support and 1.00 posterior probabilities), while Manglicola guatemalensis strains formed a sister clade but with weak support. Subclade C comprises Jahnula aquatica, J. granulosa and J. rostrata, which formed a well supported group (63% bootstrap support and 1.00 posterior probabilities), while subclade D consisted of two strains of Mega-
Number of MPT
Tree length (steps)
Consistency indices (CI)
Retention Rescaled indices indices (RI) (RC)
21n Likelihood
DifferenceP-value 1n L
14
2359
0.618
0.780
0.482
15195.07402
10.11296
0.000*
18
1458.86549
0.755
0.840
0.634
15194.87119
9.91012
0.000*
91
3052.20
0.619
0.782
0.484
15184.96107
(best)
—
A significant difference at P , 0.05.
SUETRONG ET AL: MANGLICOLA GUATEMALENSIS
89
FIG. 16. Phylogram generated from weighted parsimony analysis (step matrix) from combined SSU and LSU rDNA sequences. Parsimony bootstrap value greater than 50% and Bayesian posterior probabilities greater than 0.95 are given respectively above and below each clade.
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MYCOLOGIA
lohypha aqua-dulces, which is a basal clade to the Jahnulales. DISCUSSION
Morphological observation.—All Thai collections were made on the submerged basal parts of the N. fruticans fronds, with water fluctuating between saline and brackish. In all cases collections were from highly organically polluted waters and substrata covered by a heavy layer of enriched mud. This was often so thick it obscured the ascomatal stalk. The Jahnulales are characterized by broad, pale brown to brown; septate wide hyphae; ascomata often on a stalk; ascospores 1 to multiple septate, brown with sheaths or prominent polar appendages; or both; occurring on submerged wood or other lignocellulosic substrata, from freshwater habitats (up to the present), primary in tropical or subtropical locations (Hyde 1993, Pang et al 2002, Pinruan et al 2002, Raja et al 2005, Raja and Shearer 2006). The type species is Jahnula aquatica, collected on Salix wood in water from Germany. Common features of M. guatemalensis with the Jahnulales include stipitate ascomata, bitunicate asci, reticulate pseudoparaphyses and 1-septate brown ascospores. Manglicola guatemalensis differs from other bitunicate ascomycetes by its large ascomata, wide ostiole, large unequally 1-septate ascospores and mangrove habitat on R. mangle and the frond bases of N. fruticans. The structure of the ascomatal stalk warrants comment because differences can be noted with those of other Jahnulales species. Inderbitzin et al. (2001) illustrated the stalk as a row of single cells up to 50 mm wide, considerably wider than those normally found in ascomycetes, and 1.6 mm long. Ascomatal stalks reported by Hyde and Wong (1999) may be composed of single, wide cells of J. aquatica, J. bipolartis and Patescospora separans (Pang et al 2002). However finer fungal hyphae can also be observed within the large stalk cells of J. poonythii (Hyde and Wong 1999) and in J. siamensiae (Pang et al 2002). Huhndorf (1994) described a second Manglicola species, M. samuelsii from dead culms of bamboo collected in Guyana. Both species have large, elongate ascomata that extend conspicuously above the substratum. They also share the characteristics of a softtextured, pseudoparenchymatous ascomatal wall, trabeculate pseudoparaphyses, cylindrical asci attached to a central pad of interwoven hyphae at the base of the centrum and septate ascospores that are brown with paler end cells. Manglicola guatemalensis differs from M. samuelsii in a number of respects: host and habitat, number of asci per ascomata and degree of ascospore septation. Manglicola guatemalensis is
marine, asci are few and ascospores are 1-septate, while M. samuelsii is terrestrial, found on bamboo on tepui surrounded by lowland rainforest and has numerous asci and 3-septate ascospores. Huhndorf (1994) referred Manglicola to the Hypsostromataceae, a family with no known relationship to any group in the Dothideomycetes (Loculoascomycetes) but ‘‘probably with affinities to the Melanommatales.’’ Characteristics that unite Manglicola and the Hypsostromataceae include superficial, large, elongate ascomata (stalked) with a soft-texture, trabeculate pseudoparaphyses, stipitate asci attached in a basal arrangement in the centrum and fusiform, septate ascospores (Huhndorf 1994). However phylogenetic analysis of LSU rDNA sequence of Hypsostroma saxicola shows that it is well placed in the Pleosporales (Huhndorf pers comm). Phylogenetic evaluation.—The primary objective of the study was to determine the higher order classification of Manglicola guatemalensis. The data presented here conclusively shows that Manglicola guatemalensis groups within the Jahnulales with high bootstrap and posterior probabilities (Dothideomycetes, Incertae sedis Hibbett et al 2007), a monophyletic order (Pang et al 2002) and most closely related to the loculoascomycete order Pleosporales. Campbell et al (2007) studied phylogenetic relationships among taxa in the Jahnulales inferred from SSU and LSU rDNA sequences. Kohlmeyer and Kohlmeyer (1971) said that M. guatemalensis should be referred to the Pleosporaceae or Venturiaceae, but our phylogenetic data does not support this hypothesis. However the Jahnulales and Pleosporales both possess pseudoparaphyses that Lumbsch and Lindemuth (2001) regarded as an important phylogenetic discriminator to differentiate the Pleosporales from Dothideales. The taxonomic status of the Jahnulales remains unresolved with Hibbett et al (2007) referring it to the Dothideomycetes Incertae sedis, although Eriksson (2006) in the outline of the Ascomycota lists it in the class. Schoch et al. (2006) also excluded it from their analyses, referring it to the Dothideomycetes Incertae sedis. However they indicated that analyses of nuclear SSU sequences in GenBank showed that it was separated from the other orders (Botryosphaeriales, Capnodiales, Dothideales, Myrangiales, Pleosporales) considered in their treatise. Sequencing a wider range of genes and wider taxa sampling might help resolve the taxonomic position of this unique group of ascomycetes. The ordinal description of the Jahnulales requires broadening with the inclusion of Manglicola and the description of further taxa (Pinruan et al 2002, Raja et al 2005, Raja and Shearer 2006).
SUETRONG ET AL: MANGLICOLA GUATEMALENSIS A new lineage of marine ascomycetes.—The Jahnulales hitherto have been an ascomycete order known only from freshwater habitats (Pang et al 2002, Campbell and Shearer 2003, Raja et al 2005, Raja and Shearer 2006), and the inclusion of Manglicola represents another marine fungal lineage. While a number of marine lineages have been reported for the Basidiomycota (Hibbett and Binder 2001, Hibbett and Thorn 2001, Maekawa et al 2005, Binder and Hibbett 2006, Binder et al 2006, Jones et al 2009) and the Sordariomycetes (Spatafora et al 1998, Sakayaroj et al 2005, Schoch et al 2006), few studies have focused on the origin of marine Dothideomycetes. Many Dothideomycetes are known from the intertidal zone (especially in mangrove habitats) or brackish areas of an estuary. However few have been reported from submerged substrata. Thus the Dothideomycetes might be a transitional group from terrestrial to totally marine ecosystems (Shearer 1993, Jones 2000). Therefore the Jahnulales are an interesting assemblage of genera because they are primarily freshwater dwellers with the exception of Manglicola. TAXONOMY
Measurement of ascomata, asci and ascospores are consistently larger than those of the type material (TABLE II). Although there is some overlap in these measurements they might be sufficiently different to warrant the description of a new species. This cannot be tested phylogenetically because cultures of the Guatemala and Brunei collections were not available. A variation in the morphology and dimension of the ascospores in the Thai collections has been noted (FIGS. 11–13). Ascospores vary from orange brown to chestnut-brown (FIG. 11), brown to dark brown (FIG. 12) and light brown (FIG. 13). A collection from Mu Ko Chang had much narrower ascospores than those of others.
ACKNOWLEDGMENTS
We thank Dr Sayanh Somrithipol for discussions on morphological characters of Manglicola in culture, Kazuaki Tanaka and Yukio Harada for exchange sequences and discussion, Aom Pinnoi and Umpava Pinruan for providing photographic assistance, Rattaket Choeyklin for collecting samples, Anupong Klaysuban for lab assistance and Dr Sabine Huhndorf for sharing her unpublished results. This work was supported by the TRF/BIOTEC program for Biodiversity Research and Training Grant BRT R_249001, R_251006. For their continued interest and support we also thank BIOTEC; Graduate School, Prince of Songkla University; Prof Morakot Tanticharoen; Dr Kanyawim Kirtikara; and Dr Lily Eurwilaichitr.
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LITERATURE CITED
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