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Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Khlong 1, Khlong Luang, Pathum Thani, 12120, Thailand
Ka-Lai Pang
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR, School of Biological Sciences, University of Portsmouth, King Henry Building, King Henry I Street, Portsmouth, PO1 2DY, UK
Souwalak Phongpaichit
Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand
E.B. Gareth Jones
National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Khlong 1, Khlong Luang, Pathum Thani, 12120, Thailand
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONKEY TO THE GENERA...LITERATURE CITED |
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The genus Haligena (Halosphaeriales, Ascomycota), with two accepted species, is encountered frequently in marine habitats, especially on wood in temperate regions. Phylogenetic analyses of Haligena elaterophora (type species) and H. salina were undertaken, with partial large subunit ribosomal DNA sequences, to determine their relationships with other closely related genera in the order. The genus was shown to be polyphyletic within the Halosphaeriales with the type species forming a basal clade to the order. Haligena salina constituted a sister clade with weak support of Neptunella longirostris in all analyses. Haligena elaterophora and H. salina differ significantly in the nature of their ascospore appendages: wider, more sticky and strap-like in H. elaterophora and spoon-shaped at the point of attachment; in H. salina they are longer and narrower, finely drawn out filaments. A new genus, Morakotiella, is introduced to accommodate H. salina.
Key words: Halosphaeriales, LSU rDNA, molecular systematics, Morakotiella
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONKEY TO THE GENERA...LITERATURE CITED |
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, with the type species H. elaterophora Kohlm. The unique characteristic of the species was the long bipolar strap-like appendages and multiseptate ascospores that characterize and clearly distinguish the genus from other members of the Halosphaeriaceae (Kohlmeyer 1961). A number of species later were assigned to the genus: H. amicta (Kohlm.) Kohlm. & E. Kohlm., H. spartinae E.B.G. Jones, H. unicaudata E.B.G. Jones & Le Camp.-Als. and H. viscidula Kohlm. & E. Kohlm. ( Jones 1962, Kohlmeyer and Kohlmeyer 1965, Jones and Le Campion-Alsumard 1970). Shearer and Crane (1980) transferred H. spartinae, H. unicaudata and H. viscidula to Halosarpheia because of their hamate polar appendages that uncoil to form long thread-like structures. Recent phylogenetic studies showed that they are not related to Halosarpheia and were transferred to Magnisphaera J. Campb. et al and Ascosalsum J. Campb. et al (Anderson et al 2001, Campbell et al 2003). Haligena amicta is distinct from Haligena in having appendages that arise from the episporium at various points in the spore wall ( Johnson et al 1987). In Haligena appendages are polar, arising as outgrowths of the ascospore wall. Thereforea new genus Appendichordella R.G. Johnson et al was introduced to accommodate H. amicta ( Johnson et al 1987).
Another species that has been accepted in Haligena is H. salina C.A. Farrant & E.B.G. Jones, which originally was identified as a Remispora-like species ( Jones 1985, Farrant and Jones 1986). Haligena salina differs from the type species in ascospore size, septation and especially appendage morphology; appendages spoon-shaped at the base, initially coiled and attached closely to the spore wall and separating to form a long thread-like filament (Farrant and Jones 1986). Thus only two species, H. elaterophora and H. salina, are retained in Haligena. Therefore in this study relationships of Haligena elaterophora and H. salina and their affinity to other genera in the Halosphaeriales are investigated with sequences of the large subunit ribosomal DNA (LSU rDNA).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONKEY TO THE GENERA...LITERATURE CITED |
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). Additional cultures were obtained by single-spore isolations from woody material collected at Marloes, South Wales, and Portsmouth, England. Isolates are maintained in the BIOTEC Culture Collection and coded as BCC and JS numbers (TABLE I). All cultures were grown in seawater glucose-yeast extract-peptone broth (GY P) (Abdel-Wahab et al 2001) on a rotary shaker at 200 rpm at 25 C.
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). DNA was extracted following the instruction of a Nucleospin® Plant DNA extraction kit (Macherey-Nagel, Germany).
The partial LSU rDNA was amplified with primers LROR-LR7 (Bunyard et al 1994) and JS1-JS8 (Landvik 1996). PCR reactions were carried out in total volume of 50 µL containing 50 ng DNA template, 1x PCR buffer, 1.5 mM MgCl2, 2 mM dNTPs, 0.2 µM each primer and 0.5 units of Taq Polymerase (DyNAzymeTM II DNA Polymerase Kit, FINNZYMES, Finland). Amplification cycles were performed following the procedure of Pang et al (2003). The PCR products were purified with NucleoSpin® Extract Kit (Macherey-Nagel, Germany), following the manufacturer’s instructions. PCR products were sequenced directly by the Bio Service Unit (BSU) laboratory with BigDye on an ABI 377 automated sequencer (Perkin Elmer). Forward and reverse primers: JS1, NL4, JS5, JS8, LROR, NL3 and NL4R, were used for the sequencing reactions (Bunyard et al 1994, Landvik 1996). Each sequence was checked for ambiguous bases and was assembled with Bioedit 5.0.6 (Hall 2001).
Sequence alignment and phylogenetic analyses.— –
The consensus sequences for each species were multiple aligned by Clustal W 1.6 (Thompson et al 1994) along with other sequences obtained from the GenBank database. The dataset was refined visually in Se-Al v1.0a1 (Rambaut 1999) and Bioedit 5.0.6, 6.0.7 (Hall 2001, 2004). Daldinia concentrica (Bolton) Ces. and Xylaria hypoxylon (L) Grev. were chosen as outgroup for all analyses. Two insertion regions were observed, one at a position 835-1035 of Halosarpheia trullifera (AF396875
[GenBank]
), H. unicellularis (AF396876
[GenBank]
), H. salina (AY094182
[GenBank]
), H. salina (AY864843
[GenBank]
) and H. salina (AY864844
[GenBank]
) and the other at a position 1148-1243 of Halosarpheia unicellularis (AF396876
[GenBank]
), H. fibrosa (AY094183
[GenBank]
) and Saagaromyces ratnagiriensis (AF539470
[GenBank]
). Inclusion and exclusion of all insertion regions had no effect on the tree topology in all analyses. Therefore the insertion regions were included in all analyses.
The phylogenetic analyses were performed with PAUP 4.0b10 (Swofford 2002) with maximum parsimony analysis applying heuristic searches with this setting: 100 replicates of random stepwise addition of sequence and tree-bisection-reconnection (TBR) branch-swapping algorithm. Gaps were treated as missing data; all characters were given equal weight. The consistency indices (CI), retention indices (RI) and rescaled consistency indices (RC) were calculated for each tree generated. Weighted parsimony analysis was performed with a step matrix to weight nucleotide transformations based on the reciprocal of the observed transition to transversion (ti : tv) ratio, which was estimated by maximum likelihood score setting in PAUP* (Swofford 2002). Moreover characters were reweighted according to their RC with PAUP* default setting for reweighting character. Bootstrap analysis (Felsenstein 1985) was performed for un-weighted and weighted parsimony with full heuristic search on 1000 replicates (10 replicates of random stepwise addition of sequence and TBR branch-swapping algorithm).
The proportion of invariable sites, gamma distribution shape parameter and base frequency were estimated from maximum likelihood score setting in PAUP*. Maximum likelihood analysis was employed with a heuristic search (as is stepwise addition sequence and TBR branch-swapping algorithm) according to these estimated values.
Bayesian phylogenetic inference was calculated with MrBayes 3.0b4 with general time reversible (GTR) model of DNA substitution and a gamma distribution rate variation across sites (Huelsenbeck and Ronquist 2001). Four Markov chains were run from random starting trees for 2 000 000 generations and sampled every 100 generations. The first 100 000 generations were discarded as burn-in of the chain. A majority rule consensus tree of all remaining trees, as well as the posterior probabilities, was calculated. The alignments were deposited in TreeBase: study accession number = S1228, matrix accession numbers = M2135, M2136.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONKEY TO THE GENERA...LITERATURE CITED |
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, 2). The weighted parsimony (step matrix of 1.38) resulted in two MPTs, which gave the same topology as unweighted maximum parsimony, 1641.06 steps long, CI = 0.607, RI = 0.612 and RC = 0.372. The weighted parsimony (characters reweighted) resulted in a single MPT with a tree length of 623.77 steps, CI = 0.863, RI = 0.804 and RC = 0.693 (FIG. 1).
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Bayesian inference provided a topology similar to other analyses. Although a minor difference in the position of Nohea umiumi was noted, this difference does not affect the overall topology of the tree and the conclusions drawn.
The position of Haligena within the Halosphaeriales clearly was supported by all analyses (FIGS. 1
, 2) and shown to be polyphyletic. Two isolates of H. elaterophora and three isolates of H. salina were shown as separate but monophyletic clades with high bootstrap values. Haligena elaterophora always was shown on a basal branch to the rest of the Halosphaeriales with 82% bootstrap values and 100% posterior probabilities support in weighted parsimony and Bayesian inference, respectively (FIGS. 1, 2). All three H. salina sequences grouped together with high bootstrap support as a sister clade of Neptunella longirostris with 75% bootstrap obtained from parsimony analysis and below 95% posterior probabilities from Bayesian analysis.
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TAXONOMY |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONKEY TO THE GENERA...LITERATURE CITED |
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Typus generis: M. salina (C.A. Farrant & E.B.G. Jones) Sakayaroj.
Ascomata globosa, subglobosa, immersa vel partim immersa, ostiolata, papillata, coriacea, nigra, collo hyaline. Catenophyses praesentes. Asci clavati vel fusiformis, pedicellati, unitunicati, leptodermi, pristine deliquescentes. Ascosporae 1-septatae, ellipsoidalis, hyalina, ad septa constrictae, verrucosus pagina, appendices bipolares. Appendages filum, denique, polares, as basim cochleariformes, attenuatae, canaliculatae, ad extensionem apicis, sporae affixae.
Ultrastructurally spore wall composed of two layers: electron-dense episporium and a wide electron transparent mesosporium. Appendage fibrillar, bounded by a delimiting membrane.
Ascomata immersed or partly immersed or superficial, globose, subglobose, ostiolate, black, perithecial wall coriaceous. Neck short, cylindrical and periphysate. Asci thin-walled, unitunicate, pedunculate, fusiform to clavate, deliquescing early. Catenophyses present or absent. Ascospores 1-septate, ellipsoidal, slightly constricted, hyaline, appendaged. Appendages polar, initially wrapped around the ascospore wall, later separating to form long filaments that are spoon-shaped at the place of attachment to the spore wall, attenuate, channeled, more than 50 µm long. Ascospore wall under TEM two-layered, outer episporium electron-dense, inner wall layer mesosporium less electron-dense, at each pole wall bulging outward with electron material within the mesosporium and beneath the episporium. Appendage origin not determined but bounded by a thin electron-dense delimitating membrane attached to ascospore apices by fine threads. Appendage substructure fibrillar to amorphous, electron-dense. Under SEM appendage comprising fine fibrillar material running the length of the appendages becoming amorphous and sometimes deliquescing.
Typus. – Morakotiella salina (C.A. Farrant & E.B.G. Jones) Sakayaroj
Etymology. – "Morakot" refers to Professor Morakot Tanticharoen, Director BIOTEC Thailand, for her continued support of fungal taxonomy in Thailand and "ella" = diminutive
Morakotiella salina (C.A. Farrant & E.B.G. Jones) Sakayaroj comb. nov. FIGS. 8–12.
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Holotype: IMI 297765.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONKEY TO THE GENERA...LITERATURE CITED |
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, in their treatment of Halosarpheia species with bipolar unfurling appendages, stated that "transmission electron microscope and scanning electron microscope studies on a limited number of species to date do not indicate any heterogeneity in structure or ontogeny" and that "all the appendages are reported to develop the same way, by extrusion through pores in the episporium wall." These statements do not consider the diversity in structure of the polar appendages. For example, the development of ascospore appendages from a pore in Magnisphaera spartinae (E.B.G. Jones) J. Campb. et al differs significantly from that of the pore fields in Saagaromyces ratnagiriensis (S.D. Patil & Borse) K.L. Pang & E.B.G. Jones ( Jones and Moss 1980, Baker et al 2001). In Cucullosporella mangrovei (K.D. Hyde & E.B.G. Jones) K.D. Hyde & E.B.G. Jones, the substructure of the appendage comprises two distinct elements: folded fibrogranular electron-dense material and fine fibrils (Alias et al 2001). However we have much to learn about how appendages in species with bipolar unfurling appendages are formed, as appreciated by Campbell et al (2003). While distinct fibers are apparent in the appendages within the delimiting membrane in asci in some species, in others the appendage may be extruded as a gel-like material, only later to aggregate to form fibers (Nakagiri and Ito 1994, E.B.G. Jones, unpubl). Another consideration is the condition under which appendages are formed as it was shown for the appendages of Aniptodera salsuginosa Nakagiri & Tad. Ito (Nakagiri and Ito 1994). In this case, the salinity of the water exerts a profound influence on the morphology of the appendages.
Likewise the appendage structure of Haligena species differ. In H. elaterophora and H. salina the appendages initially are wrapped around the ascospores and then uncoil to form long polar appendages (FIGS. 3–12). These characteristics are relevant when comparing the appendages of H. salina and Panorbis viscosus. However H. salina and P. viscosus differ at the ultrastructural level and molecular results (FIGS. 1, 2). Haligena salina originally was identified as a Remispora-like species (Farrant and Jones 1986), however, it cannot be referred to Remispora because they are distantly related morphologically and phylogenetically (tree not shown). Appendages of H. salina also resemble the appendages of Halosphaeria appendiculata but they differ at the ultrastructural level: in H. salina equatorial appendages are lacking and appear amorphous, while in H. appendiculata they are present and are reticulate, with a substructure composed of both electron-dense and less electron-dense material (Hyde et al 1994). Our recent molecular results confirm that they are distantly related (FIGS. 1, 2).
Marine ascomycetes have adapted to life in the marine environment in a number of ways: as indicated by their great diversity in morphology, such as early deliquescing asci, lack of apical apparatus and variously appendaged ascospores ( Jones 1995). Polar and equatorial ascospore appendages aid in the entrapment and attachment to suitable substrata by their sticky nature and in increasing the surface area for attachment ( Jones 1994).
The deliquescent nature of the asci of many marine ascomycetes was considered as a unifying character of the Halosphaeriales by many authors (Cain 1972, Berbee and Taylor 1992, Blackwell 1994, Spatafora and Blackwell 1994). With the transfer of Lulworthia and Lindra to a new order, the Lulworthiales, this character no longer can be considered as a unifying feature. Spatafora et al (1998) argued that evanescent asci are a continuation of the lineage arising from entomopathogenic ascomycetes. However deliquescent asci are common in a number of unrelated taxa that have made the transition from terrestrial to the marine environment: e.g. Halonectria E.B.G. Jones (Bionectriaceae); Amylocarpus Curr. (Leotiomycetidae) and Dryosphaera Jørgen Koch & E.B.G. Jones (Sordariomycetes).
Delineation of genera within the Halosphaeriales has relied heavily on the ascospore and appendage ontogeny, due to their great variation in morphology ( Jones 1994). This similarity is often the result of environmental adaptation to life in the marine milieu ( Jones 1995, Shearer 1993). This has led to many marine genera being circumscribed incorrectly (e.g. the inclusion of such species as Arenariomyces trifurcatus and Nereiospora comata in Corollospora). Ultra-structural studies clearly demonstrated distinct differences in ascospore appendage ontogeny, observations later supported by molecular sequences of the SSU rDNA (Campbell et al 2002).
Haligena was shown to be clearly delineated within the Halosphaeriales, but it is polyphyletic, with H. salina distantly related to the type species, H. elaterophora. Haligena elaterophora constitutes the basal clade to the order with high support for all analyses. The molecular study by Campbell et al (2003) and Pang et al (unpubl) support the exclusion of Magnisphaera spartinae and Ascosalsum unicaudatum from Haligena, as does our recent molecular observations.
Haligena elaterophora can be differentiated from H. salina by both morphological and molecular evidence. Haligena elaterophora possess smooth, multiseptate ascospores with constriction and the appendages are wider, strap-like, and polymorphic (FIGS. 4–7). Ascospores of H. salina are smaller than those of the former species; they have a warty ascospore wall surface (in the original collection) composed of an electron-transparent mesosporium and an electron-opaque episporium, continuous beneath the appendages (FIGS. 6, 10 in Farrant and Jones 1986). Its appendages are long, narrower, drawn out and attenuated at their tips and distinctly spoon-shaped at their point of attachment. Pseudoparenchymatous cells in the ascoma centrum of H. elaterophora break up to form catenophyses while these cells might be absent or present in some collections of H. salina. However we did not observe catenophyses in material from which our isolates were derived.
The three isolates of H. salina formed a monophyletic clade and showed a high number of base substitutions that caused a long branch length in isolate AY094182
[GenBank]
. The closest sister taxon of H. salina is Neptunella longirostris, but they are not congeneric and have weak phylogenetic support (FIGS. 1, 2). They significantly differ in the morphology of the ascomata and ascus structure: in H. salina the ascus deliquesce early, while in N. longirostris the ascus is persistent and with an apical pore.
Haligena salina differs from other genera with uncoiling appendages by the mode of attachment of the appendage to the ascospore wall; appendages are coiled around the spore, spoon-shaped at the point of attachment, channeled along its length, amorphous with distinct striations running the length of the appendage (visible under SEM) and arising as an outgrowth of the spore wall. Based on these morphological features and molecular data, a new genus Morakotiella, is proposed for H. salina.
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KEY TO THE GENERA IN THE HALOSPHAERIALES WITH POLAR UNFURLING APPENDAGES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSTAXONOMYDISCUSSIONKEY TO THE GENERA...LITERATURE CITED |
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Keys to the halosphaeriaceous taxa with unfurling polar appendages ( Jones 1995, Campbell et al 2003) are unsatisfactory because of the overlapping characters of many of the genera. In addition ultrastructural studies of spore appendage ontogeny are available only for a few of these genera (Alias et al 2001, Baker et al 2001).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Email: jariyask{at}biotec.or.th
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LITERATURE CITED |
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