This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Henk, D. A. Articles by Blackwell, M. Search for Related Content PubMed Articles by Henk, D. A. Articles by Blackwell, M. Agricola Articles by Henk, D. A. Articles by Blackwell, M.

Laboulbeniopsis termitarius, an ectoparasite of termites newly recognized as a member of the Laboulbeniomycetes

     Biology Department, Duke University, Box 90338, Durham, North Carolina 27708

Alex Weir

     Faculty of Environmental and Forest Biology, SUNY College of Environmental Science and Forestry, 350 Illick Hall, 1 Forestry Drive, Syracuse, New York 13210

Meredith Blackwell

     Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Minute fungi associated with termites have caused taxonomic problems in the past due to their autapomorphic and highly reduced morphologies. DNA sequence data from one such enigmatic fungus, Laboulbeniopsis termitarius, supports its phylogenetic position as member of a laboulbeniomycete clade within the Ascomycota. This clade is composed entirely of fungi associated with arthropods, often as parasites, and the inclusion of L. termitarius supports the single origin of thallus development by means of enlargement and division of the spore.

Key words: ascomycetes, Laboulbeniales, phylogeny, Reticulitermes flavipes, taxonomy

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Laboulbeniopsis termitarius has posed a taxonomic question because of its autapomorphic traits combined with microscopic size and inability to grow in culture. When Thaxter (1920) relegated several minute ectoparasites of termites to "the limbo of genera incertae sedis," he anticipated that, once a greater diversity of similar fungi were discovered, additional information on their morphological traits might be revealed to place these fungi among their relatives. However, he could not have anticipated the development of molecular methods that promise to link these and all fungi to their near relatives regardless of morphology.

Here we discuss the phylogenetic placement of Laboulbeniopsis termitarius, a minute ectoparasite described by Thaxter (1920) from the ectoskeleton of the termite Eutermes morio collected at Grand Etang, Bahamas. Although L. termitarius and other termite ectoparasites often have been considered rare, a trained biologist usually is able to find them in almost any termite population. Since 1920, L. termitarius has been mentioned in the literature only a few times. However, information on its geographical distribution and termite host range has increased significantly (Kimbrough and Gouger 1970, Blackwell 1980, Blackwell and Rossi 1986), and the phylogenetic placement of L. termitarius has figured prominently in hypotheses concerning the evolution of the Laboulbeniales, a group of approximately 2000 species of ascomycete fungi associated with arthropods (Blackwell 1994).

The thallus of L. termitarius consists of a basal attachment cell and three linearly superposed cells with the most distal cell cleaving internally (Fig. 1), a process described as ascospore delimitation (Blackwell and Kimbrough 1976). Thaxter (1920) provisionally grouped L. termitarius with the genus Thaxteriola, now recognized as an anamorph of Pyxidiophora species, a relatively recently recognized genus of the Laboulbeniales clade (Blackwell and Malloch 1989, Blackwell 1994, Weir and Blackwell 2001). It is interesting to note that species of Thaxteriola and several other taxa with minute thalli comprising superposed cells were included in an informal "Laboulbeniales Imperfecti" (Gäumann and Dodge 1928). Laboulbeniopsis termitarius was rediscovered in 1970 by Kimbrough and Gouger, and Blackwell and Kimbrough (1976) added morphological data from an ultrastructural study to suggest the fungus was an ascomycete. They noted that, while there were similarities to the members of the order Laboulbeniales, including linear thallus construction and insect-associated habit, other morphological traits, such as the presumed uniascal thallus with one-celled spores and apparent absence of haustoria, excluded L. termitarius from the order as it was defined at the time. In this study we address the phylogenetic position of L. termitarius using DNA sequence data.

View larger version (114K): [in this window] [in a new window]   FIG. 1. Phase contrast photograph of Laboulbeniopsis termitarius thallus on the cuticle of Reticulitermes flavipes. The darkened basal attachment cell (bc) superficially resembling those found in the Laboulbeniales is affixed to a part of a termite antenna (ta) adjacent to a prominent seta (ts). The two stalk cells (sc) connect the basal attachment cell to the ascogenous cell (ac). One-celled spores (s) are visible within the ascus as is the apical ring (ar) through which spores emerge. Scale bar = 10 µm

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA extraction – Genomic DNA extraction was carried out using modified protocols of Lee and Taylor (1990). Thirty to 40 thalli were removed from infected individuals of a single collection and placed in a micro-centrifuge tube. The thalli were disrupted in SDS buffer that then was incubated at 65 C for 10 minutes. SDS and protein were precipitated in 7.5 M ammonium acetate for 10 minutes at -20 C. The precipitate was pelleted by centrifugation, and the supernatant was removed to a clean tube. Nucleic acids were precipitated in isopropanol at 4 C for 1 h, then pelleted by centrifugation. The supernatant was removed and DNA was eluted in water. NS17 (Gargas and Taylor 1992) and NS4 primers (White et al 1990) were used to amplify SSU rDNA. Products were purified with a Prep-A-Gene DNA Purification Kit (BioRad Laboratories, Hercules, California) and then used as template for sequencing with an ABI Cycle Sequencing Kit using primers NS2 and NS3 (White et al 1990). Sequence data were read with an ABI Prism 310 Genetic Analyzer.

Taxa analyzed – The taxa included in the analyses, along with their Genbank accession numbers, were Ambrosiozyma platypodis L36984, Ascobolus denudatus AF121076, Balansia sclerotica U32399, Candida albicans M60302, Capnodium dermatum AF006724, Ceramothyrium linnaeae AF02271, Cladonia subcervicornis AF0854, Elaphomyces maculatus U45440, Endomyces scopularum AF267229, Graphium calicioides AB007655, Hesperomyces coccinelloides AF407575, Kathistes analemnoides AF313767, Kathistes clyculata AF313768, Leucostoma persoonii M83259, Laboulbeniopsis termitarius AY21810, Melanospora zamiae U78356, Melanospora fallax U47842, Microascus trigonosporus L36987, Monascus purpureus M83260, Morchella esculenta U42642, Neurospora crassa X04971, Ophiostoma piliferum U20377, Ophiostoma ulmi M83261, Petriella setifera U32421, Protomyces inouyei D11377, Pyxidiophora sp.1 AF313769, Pyxidiophora SPO3 AY21811, Scorias spongiosa AF006726, Sphaerostilbella aureonitens U32415, Stigmatomyces limnophorae AF407576, Talaromyces flavus M83262, Taphrina deformans U20376, Termitaria snyderi AY21812, Xylaria hypoxylon U20378, Zodiomyces vorticellarius AF407577, Capniomyces stellatus AF007531, Smittium culisetae D29950, Mucor mucedo X89434, Entomophthora muscae D29948, Chytridium confervae M59758 and Neocallimastix frontalis X80341.

Sequences – Sequences were aligned manually with data from GenBank, and the alignment is available in TreeBASE. Ambiguous and missing regions were excluded from the analyses. Maximum-parsimony analyses were made using PAUP 4.0b8 (Swofford 2001). Heuristic tree searches were executed using the tree bisection-reconnection branch swapping algorithm, starting from 100 random addition sequence replicates. The trees derived from maximum-parsimony analysis were compared using likelihood scores and the Shimodaira-Hasegawa test (Shimodaira and Hasegawa 1999). Each tree was allowed to estimate substitution and rate heterogeneity parameters independently of the other trees. Support for the internal branches in the resulting trees was obtained by bootstrap analysis (Felsenstein 1985) with 1000 replications.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Nearly 20% of the termites collected were infected with L. termitarius. About 1100 bp were amplified with primers NS17 and NS4, and two independent sequences of about 1000 base pairs of the nuclear encoded small subunit ribosomal DNA region were obtained for use in the analyses. The sequences aligned easily with the other species included in the analyses.

Phylogenetic analyses including 41 species, with Neocallomastix frontalis and Chytridium confervae as outgroup taxa produced four most-parsimonious trees 970 steps in length with CI values of 0.5247. The Laboulbeniales clade, including two species of Pyxidiophora and L. termitatius, was grouped with 100% bootstrap support (Fig. 2). The only topological difference among the most-parsimonious trees was in the placement of taxa in the Laboulbeniales clade. Specifically, rearrangements placing L. termitarius as sister to i) the Pyxidiophora species, ii) the non-Pyxidiophora species, or iii) the entire clade were equally parsimonious. Also, the placement of Zodiomyces vorticellarius differed in one of the trees, placing it as sister to all other taxa in the Laboulbeniomycetes clade. The most-likely tree was the one showing a sister relationship between L. termitarius and the Pyxidiophora species, and although not significantly worse, the tree showing Z. vorticellarius sister to the rest of the clade was the least likely of the trees.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Phylogram of the most likely of the four most-parsimonious trees. Numbers above the nodes represent bootstrap support. Laboulbeniopsis termitarius is shown in bold. Branches not present in a strict consensus of the four most-parsimonious trees are highlighted

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In the introduction, we alluded to the existence of other minute arthropod-associated fungi with linearly superposed cells. Fewer than a dozen such species were described more than 80 years ago in the genera Amphoromorpha Thaxter, Endosporella Thaxter, Laboulbeniopsis Thaxter, Coreomycetopsis Thaxter, Amphoropsis Speg., Myriopodophila Speg. and Entomocosma Speg. (Spegazzini 1918, Thaxter 1914, 1920). We believe most of these are related to the fungi of the Laboulbeniales clade, either as dispersal anamorphs of Pyxidiophora (identical or similar to Thaxteriolla) or other relatives of L. termitarius (Blackwell et al 1986, Blackwell 1994). One candidate for a close relationship to L. termitarium is Coreomycetopsis oedipus. The taxa share several common traits, including an attachment cell that is identical at the ultrastructural level (Blackwell 1994). Our attempts to extract and amplify DNA from C. oedipus thus far have been futile, primarily because the fungus is rarer than L. termitarius, requiring greater material from the field.

The most-likely tree grouping Pyxidiophora and L. termitarius in a subclade of the Laboulbeniales is somewhat surprising. This relationship would require one of two equally parsimonious life history changes. In one scenario, germ tube germination of both conidia and ascospores would have been suppressed in the ancestor to the clade (Fig. 2) and partially regained in Pyxidiophora, which has conidium germination by germ tubes. The second case would require independent losses from an ancestor with ascospore germ tube germination in each subclade (L. termitarius and the other Laboulbeniales) and development of the thallus from an ascospore. This second scenario could be convergent evolution associated with a life cycle restricted to arthropod hosts, as in L. termitarius and Laboulbeniales, unlike the complex life history of Pyxidiophora, in which several life-cycle states are maintained on different hosts. Discovery of a more closely related sister taxon is desirable to help discern the morphological and life history changes that occurred in the evolution of the Laboulbeniomycetes.

	ACKNOWLEDGMENTS

 
This research was supported in part by the National Science Foundation (Phylogeny of Laboulbeniales, DEB-9615520, and REU supplements) and a Howard Hughes Medical Institute grant through the Undergraduates Biological Sciences Education Program to Louisiana State University. Dr. Kevin G. Jones provided valuable assistance with techniques throughout the study.

	FOOTNOTES

 
¹ Corresponding author. E-Mail: dah{at}duke.edu

Accepted for publication January 28, 2003.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Alexopoulos CJ, Mims CW, Blackwell M., 1996 Introductory mycology. 4th ed. New York: John Wiley & Sons. 868 p

Blackwell M., 1980 New records of termite-associated fungi from Georgia. J Invert Pathol 35:101-104

Blackwell M., 1994 Minute mycological mysteries: the influence of arthropods on the lives of fungi. Mycologia 86:1-17

———, Kimbrough JW., 1976 Ultrastructure of the termite-associated fungus Laboulbeniopsis termitarius. Mycologia 68:541-550

———, Malloch D., 1989 Similarity of Amphoromorpha and secondary capilliconidia of Basidiobolus. Mycologia 81:735-741

———, Rossi W., 1986 Biogeography of fungal ectoparasites of termites. Mycotaxon 25:581-601

———, Bridges JR, Moser JC, Perry TJ., 1986 Hyperphoretic dispersal of a Pyxidiophora anamorph. Science 232:993-995[Abstract/Free Full Text]

Felsenstein J., 1985 Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791

Gargas A, Taylor JW., 1992 Polymerase chain reaction (PCR) primers for amplifying and sequencing 18S rDNA from lichenized fungi. Mycologia 84:589-592

Gäumann EA, Dodge CW., 1928 Comparative morphology of the fungi. New York: McGraw-Hill. 701 p

Kimbrough JW, Gouger R., 1970 Structure and development of the fungus Laboulbeniopsis termitarius. J Invert Pathol 6:205-213

Lee SB, Taylor JW., 1990 Isolation of DNA from fungal mycelia and single spores. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR protocols. A guide to methods and applications. San Diego, California: Academic Press. p 282–287

Shimodaira H, Hasegawa M., 1999 Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol 16:1114-1116

Spegazzini C., 1918 Observaciones microbiológicas. Anales Soc Cient Ci Argent 85:311-323

Swofford DL., 2001 PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4d65. Sinauer Associates. Sunderland, Massachusetts:

Thaxter R., 1914 On certain peculiar fungus-parasites of living insects. Bot Gaz (Crawfordsville) 58:235-253

———. 1920 Second note on certain peculiar fungus-parasites of living insects. Bot Gaz (Crawfordsville) 69:1-27

Weir A., Blackwell M., 2001 Molecular data support the Laboulbeniales as a separate class of Ascomycota, Laboulbeniomycetes. Mycol Res 105:1182-1190

White TJ, Bruns T, Lee SB, Taylor JW., 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols. A guide to methods and applications. San Diego, California: Academic Press. p 315–352

This article has been cited by other articles:

				M. Blackwell, D. A. Henk, and K. G. Jones Extreme morphological divergence: phylogenetic position of a termite ectoparasite Mycologia, November 1, 2003; 95(6): 987 - 992. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Henk, D. A. Articles by Blackwell, M. Search for Related Content PubMed Articles by Henk, D. A. Articles by Blackwell, M. Agricola Articles by Henk, D. A. Articles by Blackwell, M.