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Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803
Daniel A. Henk
Biology Department, Duke University, Box 90338, Durham, North Carolina 27708
Kevin G. Jones
Department of Biology, University of Virginia at Wise, Wise, Virginia 24293
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ABSTRACT |
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Species of Termitaria are lesion-forming ectoparasites occurring worldwide on a diverse group of termites. The reduced thallus consists of a basal cell layer from which haustorial cells penetrate the termite and a darkly pigmented sporodochium. One species, Termitaria snyderi, has been the subject of several morphological studies, but its phylogenetic position has remained enigmatic. Here we provide evidence of a close relationship between T. snyderi and the morphologically distinct ascomycetes, Kathistes analemmoides and K. calyculata, based on phylogenetic analysis of molecular characters derived from portions of the nuclear-encoded small-subunit ribosomal RNA gene (ssu rDNA) and supplemental evidence from the ß-tubulin gene. Trees were derived using parsimony and maximum-likelihood criteria. Bayesian analysis and parsimony bootstrap methods were used to assess support for the tree nodes.
Key words: asexual fungi, conidial fungi, insect-associated fungi, Isoptera, mitosporic fungi, ssu rDNA, Termitaria
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INTRODUCTION |
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). The life histories of the fungi are not well known, and hypotheses of phylogenetic relationships often have been based on scant morphological and life history data (Blackwell and Kimbrough 1976a, b, Blackwell et al 1980, 1986, Blackwell 1994). In addition to the more elaborate thalli of several Laboulbeniomycetes found on termites, the thalli of some termite ectoparasites, such as species of Laboulbeniopsis and Coreomycetopsis, have thalli composed of 3–15 linearly superposed cells; single-celled Amphoromorpha species reported from termites probably are the capilliconidia of species of Basidiobolus (Blackwell and Malloch 1989). None of these fungi has been cultured in agar medium.
A second group of termite fungi has more prominent, albeit reduced, thalli and includes species of Termitaria and Mattirolella. Termitaria snyderi conidia germinate and form thalli on the surface of the termite exoskeleton, and thalli can develop on any exposed body part, including the head, legs, antennae and abdomen. A thallus consists of a basal layer of cells, some or all of which may be haustorial mother cells. Each of the dark, thick-walled haustorial mother cells of the pseudoparenchymatous crust possesses a pale circular area at the base, actually an opening in the cell wall through which penetration pegs exit the cell to enter the host exoskeleton. From the penetration pegs the walled, nucleated haustoria penetrate through the exoskeleton to the basement membrane of the termite integument (Khan and Aldrich 1975). Above the basal layer, the cells of the subhymenium give rise to a compact palisade of conidiogenous cells that are enclosed by a border of dark-walled cells forming a sporodochium (Khan and Kimbrough 1974a). Khan and Aldrich (1973) reported basipetal production of conidia from a fixed locus within a closed conidiogenous cell; conidium release occurred upon synchronous rupture of the conidiogenous cell apices.
In addition to T. snyderi, four other species of Termitaria are known from termite hosts (Thaxter 1920, Kimbrough and Lenz 1982), and the genus is present in most regions where termites occur (Blackwell and Rossi 1986, Hojo et al 2001). Other species of termite parasites in Mattirolella (Kimbrough and Thorne 1982) and Termitariopsis cavernosa on legionary ants (Blackwell et al 1980) have a thallus morphology and ecology similar to those of T. snyderi (Blackwell and Rossi 1986), and we suspect that they are all related. It is, however, the relationships of the morphologically distinguished group to other fungi that is the question needing attention.
Termitaria and Mattirolella were suggested as relatives of several fungi based on morphological features: the flat stromatic growth of the primary thallus has been likened to that of the Asterinales (Thaxter 1920, Kimbrough and Thorne 1982); the darkly pigmented sporodochia with tightly held conidiogenous cells have been compared to Bloxamia; conidium ontogeny in Termitaria and Mattirolella was thought to be similar to the processes observed in Bloxamia truncata, Thielaviopsis, Chalara and Chalaropsis (Khan and Kimbrough 1974a, b). However, morphological and developmental studies merely have served to highlight the unique morphology of species of Termitaria and Mattirolella when compared to other fungi.
The distinctive morphology caused Khan and Kimbrough (1974b) to place Termitaria and Mattirolella in a new order of the Deuteromycotina, Termitariales. The classification of asexual fungi, however, has been de-emphasized since that time because it now is possible to use molecular characters to place asexual fungi among their closest sexual relatives. Here we report information on T. snyderi that provides strong support for a new hypothesis that this species is related to two species of arthropod-associated ascomycetes, Kathistes analemmoides and K. calyculata (Malloch and Blackwell 1990).
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METHODS |
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).
Culture –
Infected termite parts were removed aseptically and placed on 
strength cornmeal agar (7 g Difco cornmeal agar, 12 g DifcoBacto agar, 1 L tap water) or Molisch's agar (15 g agar, 20 g sucrose, 10 g peptone, 0.25 g Mg2SO4, 0.25 g K2HPO4, 1 L tap water), which is used specifically for entomopathogens (Harry Evans pers comm, 1994). A small amount of chlortetracycline hydrochloride (Sigma, St. Louis, Missouri) was sprinkled on the agar surface to retard bacterial growth. The fungal lesions on the agar were observed daily for 2 wk to monitor the slowly growing T. snyderi hyphae and to determine whether contaminants were present.
DNA extraction –
DNA extraction from fungal lesions on termites was carried out by adaptation of the protocols of Cenis (1992) and Ferreira and Glass (1996). Three thalli were dissected from a single infected termite and placed in a 1.5 mL microfuge tube. The dry fungal material was irradiated at 700 W for 5 min in a domestic microwave oven and then dispersed in 100 µL extraction buffer (0.2 M Tris-HCl, pH 8.0; 0.25 M NaCl; 25 mM EDTA; 0.5% SDS) by using a vortex. Further homogenization of the material was performed with a disposable micropestle (Kontes, Vineland, New Jersey), and the extract was incubated at 65 C for 5 min. One-half volume of 7.5 M ammonium acetate was added to the homogenate, followed by incubation at -20 C for 10 min. Cellular debris and precipitated proteins were pelleted by centrifugation (15 min, 16 000x g), and the supernatant, transferred to a clean tube. Nucleic acids were precipitated by addition of an equal volume of propan-2-ol, and recovered by centrifugation, as described above. DNA was rinsed twice with 500 µL 70% ethanol, air dried, and dissolved in 100 µL sterile water. Aliquots of the DNA solution were used undiluted in PCR reactions. To extract DNA from cultured material, the sparse aerial hyphae were collected under a dissecting microscope by contact with a toothpick dipped in sterile water. Adherent hyphae were dispersed in 100 µL of extraction buffer in a 1.5 mL microfuge tube. Subsequent processing of the sample was performed as described above with the omission of the microwave treatment. DNA samples were diluted 1/10 for use in PCR reactions.
DNA amplification, polymerase chain reaction and sequencing –
Sequence data were obtained from the 5' end of the ssu rRNA gene and from a portion of the gene encoding ß-tubulin. PCR amplifications were performed with 100 µL PCR Supermix (Gibco BRL, Bethesda, MD) containing 10 µM each of the appropriate primers (see below). For ssu rDNA, primers NS17 (Gargas and Taylor 1992) and NS4 (White et al 1990) were used in a cycling protocol consisting of 94 C for 5 min, followed by 35 cycles of 95 C for 1 min; 54 C for 1 min; and 72 C for 1.5 min. Tubulin amplifications were performed using primers ßt2a and ßt2b (Glass and Donaldson 1995, Donaldson et al 1995) in a cycling procedure consisting of 94 C for 5 min, followed by 35 cycles of 94 C for 1 min; 65 C for 1 min; 72 C for 1 min with a 5 s increase in extension time per cycle. Aliquots of the reactions were subjected to electrophoresis through 1% agarose gels, and PCR products were recovered with a Prep-A-Gene DNA Purification Kit (Bio-Rad Laboratories, Hercules, California). DNA sequencing reactions were performed with primers NS17, NS2, NS3 and NS4 (18S rDNA), and ßt2a and ßt2b for tubulin products, with the Taq dideoxy Terminator Cycle Sequencing Kit (Perkin-Elmer GeneAmp PCR System 2400, PE Applied Biosystems, Foster City, California). Reactions were analyzed on an ABI Prism Model 377 automated sequencer (PE Applied Biosystems, Foster City, California).
Phylogenetic analysis –
Sequences of 43 taxa accessioned in GenBank are: Alternaria alternata (U05194), Ambrosiozyma platypodis (L36984), Aspergillus fumigatus (M60300), Balansia sclerotica (U32399), Blastomyces dermititidis (M55624), Capronia mansonii (X79318), Candida albicans (M60302), Candida tropicalis (M55527), Ceratocystis fimbriata (U32418), Ceratocystis virescens (U32419), Chaetomium globosum (U20379), Claviceps paspali (U32401), Cryphonectria parasitica (L42441), Daldinia concentrica (U32402), Diaporthe phaseolorum (L36985), Haematonectria haematococca (U32413), Halosphaeriopsis mediosetigera (U32420), Hypomyces polyporinus (U32410), Hypoxylon atroroseum (U32411), Kathistes analemmoides (AF313767), Kathistes calyculata (AF313768), Leptosphaeria maculans (U04238), Leucostoma persoonii (M83259), Microascus trigonosporus (L36987), Morchella esculenta (L36998), Neurospora crassa (X04971), Ophiostoma ulmi (M83261), Ophiostoma piliferum (U20377), Protomyces inouyei (D11377), Petriella setifera (U32421), Peziza badia (L37539), Pleospora herbarum (U05201), Pyxidiophora sp. 1 (IMI-1989) (AF313769), Pyxidiophora sp. 2 (Baton Rouge, Chalara anamorph) (AY212811*), Saccharomyces cerevisiae (J01353), Sphaerostilbella aureonitens (U32416), Subbaromyces splendens (U63552), Talaromyces flavus (M83262), Taphrina deformans (U00971), Termitaria snyderi (culture) (AY280724* and AY280725, AY278207*), Termitaria snyderi (lesion) (AY212812*, AY278206*), Xylaria curta (U32417) and Xylaria hypoxylon (U20378). Sequences acquired in this study that were aligned with the multialignment program Clustal W (Thompson et al 1994) are marked with an asterisk. The alignment (TreeBASE matrix SN1427–4151) was optimized visually, and ambiguous regions were excluded from the analyses. Ambrosiozyma platypodis, Candida albicans, Candida tropicalis, Protomyces inouyei, Saccharomyces cereviseae and Taphrina deformans were designated outgroup taxa based on the results of many previous studies showing that these groups lie outside the clade of ascocarpic ascomycetes.
Equally weighted maximum-parsimony (MP) analysis was carried out with PAUP 4.0b10 (Swofford 2001) to perform a heuristic search starting from 100 random-addition sequence replicates and swapping with the TBR algorithm. Support using MP was assessed with 1000 bootstrap replicates (Felsenstein 1985). Equally parsimonious trees were compared with maximum-likelihood scores using the Shimodaira-Hasegawa test. Analyses were done with a variety of samplings among diverse ascocarpic ascomycetes, but additional information on the taxa of interest was not obtained. Bayesian Markov Chain Monte Carlo (B-MCMC) analysis was carried out with the program MrBAYES 2.01 (Huelsenbeck and Ronquist 2001). The general time reversible model with gamma shape estimate of rate heterogeneity and estimated base frequencies and invariant sites was implemented in the B-MCMC analysis. The analysis consisted of 200 000 generations of four chains sampling every 100 generations. The first 24 000 generations were discarded as burn in time, leaving 1760 trees as the sample representing the posterior probability distribution.
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RESULTS |
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Parsimony analysis resulted in two most-parsimonious trees that were incongruent only in the placement of Morchella esculenta and Peziza badia. The most-likely tree (-ln L = 6129.43615) (Fig. 1) showed a sister relationship between M. esculenta and P. badia, while the other tree (-ln L = 6129.58604; P = 0.445) showed these taxa as a grade leading to the remaining Euascomycetes. B-MCMC analysis and parsimony bootstrap provided similar levels of support for nodes throughout the tree. Both of the trees placed T. snyderi and the two species of Kathistes within a single clade excluded from the perithecial ascomycetes with 100% Bayesian analysis and bootstrap support.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Kathistes is a relatively recently described and little known ascomycete genus that contains three species (Malloch and Blackwell 1990). The species are known only from dung and are characterized by hyaline perithecial ascomata with light brown necks, basally clustered evanescent asci, several-septate hyaline ascospores and unique multicellular structures of undetermined function (sporidiomata) associated with the base of mature perithecia. The sporidiomata contain minute conidium-like cells that presumably escape through a "nozzle" formed by a single elongated wall cell, but there is no other information on the function or fate of these cells. Two species, K. analemmoides and K. calyculata, have been studied in culture, and they are characterized by ascospore germination by budding to produce yeast-like growth that may darken in color to tan; only scant mycelial growth occurs in culture with age. Species of Kathistes are associated with arthropods for dispersal in dung habitats, but they are not known to be arthropod parasites or associates of termites.
By contrast T. snyderi has a darkly pigmented sporodochium enclosing tightly packed conidiogenous cells as its only known state and apparently is restricted to termite hosts. Thus, based on our current knowledge, species of Kathistes and Termitaria do not have comparable states in their life cycles. Because the morphological traits could not be compared among the taxa to increase confidence in the grouping of these fungi, B-MCMC was used as a model-based method by which we specifically addressed the potential spurious results of long-branch attraction that are possible under some circumstances in parsimony analysis (Felsenstein 1978). B-MCMC, in fact, did support the hypothesis.
Our strategy to find close relatives of T. snyderi was dependent on the inclusion of a diverse group of insect-associated ascomycetes in our phylogenetic database, and, although we have discovered that K. analemmoides and K. calyculata are its closest relatives, we have not been able to resolve the clade among other ascomycetes, a recurring problem in attempts to discover the relationships of some major ascomycete clades. For example, such a problem occurred in the placement of the Laboulbeniomycetes (represented in Fig. 1 by Pyxidiophora), a group that includes another previously problematic termite parasite, Laboulbeniopsis (Henk et al 2003). Lack of resolution has been particularly frustrating in our attempts to track evolutionary transformations in morphology and life histories in insect-associated lineages with highly divergent morphologies. Our inability to determine the near relatives of the Termitaria-Kathistes clade severely reduces the information we can infer from the tree on morphological traits and life history strategies.
At this time we are unable to suggest additional ascomycetes for sampling but hope for the accidental discovery of other members of the Termitaria-Kathistes clade from among the remaining, largely unsampled, diversity of ascocarpic ascomycetes. However, the common problem associated with reconstructing rapid ancient radiations, probably represented here by the deeper, poorly supported branches in both maximum-parsimony and maximum-likelihood analyses, might require that additional genes be applied to achieve a solution (Lanyon 1993).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication May 8, 2003.
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LITERATURE CITED |
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