Faecal Pellets of Lichenivorous Mites Contain Viable Cells of the Lichen-Formingascomycete Xanthoria Parietina and Its Green Algal Photobiont, lFebouxia Arboricola

Biological Journal of the Linnearz Society, 2002, 76, 259-268. With 14 figures

Faecal pellets of lichenivorous mites contain viable cells of the lichen-forming ascomycete Xanthoria parietina and its green algal photobiont, lFebouxia arboricola

FRANZ A. MEIER, SANDRA SCHERRER and ROSMARIE HONEGGER"

Insti tute of Plant Biology, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland

Received 9 August 2001; accepted for publication 1 March 2002

The bright yellow wall lichen, Xarzthoria parietina, is usually inhabited by oribatid mites (Acari) which do not only find shelter, but also graze on selected areas of the thallus. As X. parietina does not produce symbiotic vegetative propagules and its compatible photobiont, unicellular green algae of the genus Debouxia, are rare outside lichen thalli, we tested the hypothesis of dispersal of viable Trebouxia cells via acarine faeces. The lichenivorous mites, Trhypochtonius tectorum and Trichoribates trimaculatus, were isolated from thalli of X. parietina and cultured in the laboratory on a lichen diet. Light microscopic investigations of faecal pellets from mites that had been feeding on X . parietina indicated gut passage of intact ascospores and photobiont cells. In a series of experiments, viable algal and fungal cells contained in such faecal pellets were cultured. The taxonomic affiliation of these isolates was identified using molecular techniques, i.e. comparative investigations of nuclear ribosomal gene data (ITS 1 and 2, 5.8s rDNA) in the algal and fungal partners, and of the species-specific hydrophobin gene sequence in the fungal partner. Our culturing experiments demonstrated that the faecal pellets of both lichenivorous mites, upon feeding on X. parietina, contain viable ascospores and photobiont cells (Trebouxia arboricola) and thus might be a common and successful mode of vegetative short- and long-distance dispersal of this and numerous other lichen-forming ascomycetes and their photobionts. Future studies will have to elucidate the evolutionary significance of invertebrate interactions with lichens. 0 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 76, 259-268.

ADDITIONAL KEYWORDS: Acari - comparative rDNA sequence analysis culturing experiments - hydrophobin gene sequence - lichen-arthropod interactions - oribatid mites - Trhypochtonius tectorum - Trichoribates trimaculatus - vegetative dispersal of lichens.

INTRODUCTION

Lichens are the symbiotic phenotype of nutritionally specialized fungi (mostly ascomycetes) which derive fixed carbon from a population of either a green alga (mostly unicellular) or a cyanobacterium. These are referred to as the photobionts. The Latin binomial applied to a lichen refers to the fungal partner, the photoautotrophic inhabitants of lichen thalli having their own names and phylogenies. The photobiont has only been identified to species in less than 10% of lichens, and in only few species of lichen-forming

Torresponding author. E-mail: rohoneggQbotinst.unizh.ch

ascomycetes has the range of compatible photobiont taxa been investigated (reviews: Honegger, 199610, 1997). Today, photobiont-specific primers for PCR and comparative sequence analyses are powerful tools for studying the taxonomic diversity of lichen photobionts (Helms et al., 2001). Currently available data indicate that lichen symbiosis is moderately specific to specific, with one to few closely related algal or cyanobacterial taxa being compatible partners (reviews: Honegger, 1996b, 1997).

A large percentage of lichen-forming fungi disperse very efficiently by means of symbiotic vegetative propagules such as soredia, isidia and blastidia, and thallus fragmentation is probably a very widespread mode of dispersal of the symbiotic state (review:

0 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 76, 259-268 259

260 F. A. MEIER ET AL.

Honegger, 1997). However, the majority of lichen- fbrming fungi reproduce sexually by means of asco- or basidiospores and thus have to re-establish the symbiotic state a t each reproductive cycle. While some photobiont taxa are common and widespread outside lichen thalli, the most common unicellular green algal photobionts, representatives of the genus E-ebouxia de Puymaly 1 Trebouxiophyceae sensu Friedl, 19951, are not common elements of aerophilic algal commu- nities. The present study focuses on the question how germinating ascospores can re-lichenize with green algal photobiont taxa that are rare outside lichen thalli. Our experimental system was the golden-yellow wall lichen, Xanthoria parietina (L) Th.Fr., a common and widespread lichen-forming ascomycete with no symbiotic propagules except thallus fragmentation I Honegger. 1996a). but with very abundant ascospore formation in disk-shaped fruit-bodies (apothecia) which cover the subapical, central part of the foliose thallus.

Xanfhoria parietina and its green algal partners I Trehorrsia urhoricola Puymaly and related taxa) are physiologically facultative symbionts; both can be cultured independently. In nature, ascospore-derived germlings are likely to survive in a non-lichenized state but express the symbiotic phenotype exclusively in association with a compatible photobiont taxon. Ott 11987a,bJ assumed X. parietina germlings to acquire a compatible green algal partner by invading the thalli or soredia of adjacent, grey Ph.yscia species. In long-term observations such presumably parasitized Ph,vscia spp. turned out to be juvenile stages of Xunihoria polycarpa (Hoffm.) Rieber (Honegger et al., 1996), and rnolecular studies indicate that the photobionts of' Physcia spp. are not algal partners of X. parietinn (Beck et al., 1998). However, the full spectrum of compatible photobiont species of X. parietina remains unknown.

Wherever thalli of X . parietina are collected in the wild they are inhabited, like many other macrolichens, by mit.es. Less common inhabitants are springtails or other insects. All of them find shelter and some even graze on selected areas of the thallus (Figs 5,6,7,8). Consequently, the golden-yellow thallus surface is dotted with faecal pellets. The great diversity of lichen-invertebrate interactions was surveyed by Gerson & Seaward (1977). The association of mites with lichens. ranging from casual visits to high depen- dency, was investigated by Seyd & Seaward (1984) and Stubbs ( 1987, 1989, 1995). Detailed information on the biology and ecology of corticolous and saxicolous oribatid mites and their seasonal behaviour and population structure in south-western France was published by "rave (1963 ).

In the present study we focused on two species of oribatid mites ( Acariformes), Trhypochtonius tectorum

-

0 2002 The Linnean Society of London, BaologLcal Journal of the Lznnean Soaet,y, 2002, 76, 259-268

(Berlese 1896; Trhypochtonidae) and Trichoributes trimaculatus (C.L. Koch 1836; Ceratocetinae ), common inhabitants and consumers of X . parietina, and on their role as potential vectors of viable photohiont cells. Due to its distinct segmentation and its poorly sclerotinized, porous teguments, T tectorum is referred to as a primary species, whereas Tr. trim.ac- ulatus, with its compact body structure, its well sclerotinized tegument and extended tracheal system, is interpreted as a phylogenetically younger form (Woolley, 1961; Krantz, 1978).

MATERIAL AND METHODS

Xanthoria parietina was collected on the composite asbestos roof of the bicycle shelter of our institute. This roof is situated under a large beech tree (Fagus syluatica) whence nutrients are assumed to leach out; these facilitate luxuriant growth of X . parietina and grey Physcia spp. Both oribatid mites, 12.. tectorum and is trimaculatus, were part of the infauna of the thalli.

LABORATORY CULTURES OF MITES

Both mite species were kept in the laboratory a t about 20°C in glass containers that were covered with fine cotton gauze. They were fed thallus fragments of X . parietina with vegetative parts and apothecia. Lichens were subjected to regular wetting and drying cycles.

CULTURING VIABLE FUNGAL AND ALGAL CELLS FROM FAECAL PELLETS

Out of numerous trials, the following three methods gave the best results. In the first, suspensions derived from squeezed faecal pellets were spread over the surface of non-nutrient, agarized mineral medium (Bold's basal medium [BBM] according to Deason & Bold, 1960) with double the amount of nitrogen and with 0.005% Doxycyclin (SIGMA) as a broad-spectrum antibiotic. These (and all other) cultures were incubated a t 15 i 1°C in a 16/8h lightidark cycle. All cultures were checked a t regular intervals and fast-growing fungal contaminations were cut out. Developing slow-growing fungal colonies, which were assumed to be aposymbiotic X. parietina, were transferred to agarized BBM without antibiotics. Non- contaminated, dividing algal cells were transferred to BBM plates supplemented with 0.125% peptone and 0.25% glucose (1/4 strength Trebouria medium according to Ahmadjian, 1967) as routinely used in our laboratory. In the second method, faecal pellets were gently ground in a mortar and suspended in sterile distilled water. Small amounts of this suspension were

VIABLE ASCOSPORES AND ALGAE IN FAECAL, PELLETS OF LICHENIVOROUS MITES 261

kept in hanging drop cultures and microscopically investigated a t regular intervals. The third method involved transferring faecal pellets to Parafilm strips that were fixed to microscopy slides and incubated in moist chambers, i.e. Petri dishes with slightly wetted filter paper, a t 15 f 1°C. From time to time, the drying filter paper was remoistened. Approximately 200 faecal pellets were investigated in this type of culturing experiment.

LIGHT MICROSCOPY (LM) Lichen thalli and apothecia with grazing marks were photographed with a ZEISS Tessovar dissecting microscope and KODAK film EPJ 320 T. Faecal pellets, upon incubation on Parafilm, were photographed with a NIKON CoolPix mounted on a WILD M5A dissecting microscope. Faecal pellet preparations and hanging drop cultures were examined and photo- graphically documented in a ZEISS Photomikroskop I1 equipped with epifluorescence.

SCANNING ELECTRON MICROSCOPY (SEM) Mites or thallus fragments with grazing marks were chemically fixed by exposure to the vapour of a 4% aqueous solution of osmium tetroxide. Mites were air dried and mounted on aluminium specimen stubs. Lichens were mounted following dehydration in acetone and critical point drying. Specimens were sputter coated with gold and examined in either a STEREOSCAN S 4100 (Cambridge Instruments, UK) or a HITACHI S 4000 scanning electron microscope at 20kV and micrographs were made on KODAK Professional Film TMX 120.

NUCLEAR RIBOSOMAL DNA (RDNA) ANALYSIS

Genomic DNA was isolated from either fresh thalli or aposymbiotically cultured fungal mycelia or green algal colonies, both of which had been isolated from faecal pellets of lichenivorous mites that had been kept on thalli of X. parietina. Samples were ground with a micropestle in a 1.5-mL reaction tube which was kept in liquid nitrogen. Genomic DNA was isolated using the GFX PCR, DNA and Gel Band Purification Kit (AMERSHAM PHARMACIA Biotech Ltd). 100 pL of capture buffer was added to the ground material and the samples were incubated for 10min at 60°C. After centrifugation the supernatant was loaded on the columns which were preloaded with 1OOpL of capture buffer. The polymerase chain reaction (PCR) was performed using the specific gene primers ITS 4: 5'-TCCTCCGCTTATTGATATGC-3' (forward) and ITS 5: 5'-GGAAGTAAAGTCGTAA CAAGG-3' (reverse), as characterized by White et al.

(1990), on a PERKIN ELMER Gene Amp. PCR-System 9600 thermal cycler.

PCR amplification products were loaded on agarose gels where single bands were obtained. These were cut out and purified with the GFX kit (see above) for sequencing. Sequencing reactions were performed with ABI PRISM Big Dye TM Terminator Cycle Sequencing Ready Reaction kit (APPLIED BIOSYS- TEMS) following the instructions of the manufacturer. The sequences were analysed using the Genetics Computer Group (GCG) version 10 software of the Wisconsin Sequence Analysis Package.

HYDROPHOBIN H1 GENE SEQUENCE ANALYSIS

The species-specific hydrophobin gene sequence (GenBank Accession number A52872271 was analysed as described by Scherrer et al. (2002). The following primers were used for PCR: 5'-CCGAGAACCTGGTC GAGC-3' (forward) and 5'AATTCGCTCCGAGGC GAATG-3' (reverse). Thirty cycles with the following profile were programmed: denaturation 1 min a t 94"C, annealing 30s at 5PC, extension lmin at 72"C, fol- lowed by final extension 6min at 72°C.

RESULTS

hl3ORATORY CULTURES AND FEEDING HABITS OF THE MITES

Cultivation of Dhypochtonius tectorum (Figs 1 ,2) and Dichoribates trimaculatus (Figs 3, 4) on moulds such as Penicillium and Dichoderma spp. or on nutrient media containing bakers yeast, cereals and vita- mins, as recommended for the culture of oribatid mites (Woodring, 1962; Travk, 1963; Evison, 1981; Reutimann, 1985), resulted in low fertility and com- paratively high mortality (c. 70%). In contrast, the provisioning of apothecia-bearing thallus fragments of X. parietina, subjected to regular wetting and drying cycles, as the sole nutrition gave distinctly better results. On this diet, the number of D. tectorum indi- viduals increased from 30 to c. 300, and of I: trimaculatus from 30 to c. 800 within 12 months of culturing. Neither in nature nor in laboratory experiments were the thalli of adjacent Phaeopyscia orbicularis grazed (Fig. 7). Both mite species differed in their juvenile development. Dichoribates trimaculatus was strictly oviparous, whilst Dhypochtonius tectorum appeared to be ovoviviparous, its embryonal development taking place within the eggshell that is embedded in the uterus of the moribund female. Pronuptiae of this species, upon hatching in spring, first feed on the viscera of the dead female prior to switching, as deu- tonymphae and adults, to a diet consisting of putrifiy- ing plant material and lichens.

0 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 76, 259-268


Figures 1-6. Scanning electron microscopy of lichenivorous oribatid mites and their grazing marks on thalli of the lichen- forming ascomycete Xanthorin parietina and its green algal photobiont, Debouria arboricola. Figs 1, 2. 37-hypochtonius twtorum: lateral view of the adult (Fig. 1) with its segmented body structure (arrow) and rather weak, porous integument, and fiontal view of the prosoma (Fig. 2 ) with its strong, dented chelicerae (CH). Figs 3, 4. Dichoribates trimnculatus, dorsal view of'the adult (Fig. 3) with unsegmented dorsal shield (arrow) and solid, compact integument, and frontal view of the prosoma (Fig. 4) with spatula-like labium (L). Figs 5 , 6. Grazing marks, caused by 7: trintaculatus i n laboratory culture, on X . paric~tinn. The upper cortex (UC) and algal layer (PH refers to individual cells of the photobiont, 7: arbori- c d n J were grazed off'; consequently, the conglutinate strands of the medullary hyphae (M) are exposed. Faecal pellets ( F P ) are sticking to the thallus surface and the grazed area.

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VIABLE ASCOSPORES AND ALGAE IN FAECAL PELLETS OF LICHENIVOROUS MITES 263

In laboratory cultures, the Xanthoria-dwelling mite Tr. tectorum was mainly night-active. With their strong, dented chelicerae (Fig. 2) the deutonymphs and adults were found to graze upon the upper cortex and algal layer of any part of the Xanthoria, whereas larvae preferred the hymenial, subhymenial and algal layers of young apothecia (Fig. 8). Gut passage of the ingested material was rather fast. Faecal pellets of adults and deutonymphs were dark brown to black, those of larvae light brown to greenish (Figs 9-11). Under laboratory conditions at c. 20°C the development from the larval to the adult stage lasted 3-6 months, depending on the season in which the animals were collected in the wild.

Trichoribates trimaculatus was mainly day-active during approximately 6 hours. It grazed on all parts of the thalli (Figs 5-7) but seemed to prefer the lobe margins and the hymenial, subhymenial and algal layers of apothecia. As in Trhyphochtonius tectorum, the lower cortex and rhizinae were never ingested. Upon accumulation in the gut, salivated food por- tions were condensed to boluses and enveloped by a peritrophic membrane. Such ovoid pellets were subse- quently transported to the expanding caeca (Fig. 9) where they stayed for another resting period prior to excretion during the following period of activity. Adhe- sion of faecal pellets to the substratum seems to be mediated by the slimy peritrophic membrane (Fig. lo), which is not a biomembrane, but a proteinaceous secretion product of the digestive tract. The coloration of the pellets largely depended on the parts of the lichen that had been ingested: brown pellets contained mainly ascospores and remains of paraphyses, green ones were rich in algal cells (Fig. lo), and yellow to orange ones (Fig. l l a ) contained mainly remains of the upper cortical layer in which the mycobiont-derived secondary metabolites (bright yellow anthraquinones such as parietin and parietinic acid; Fig. l l c ) accumulate. Obviously these anthraquinones passed the digestive tract unaltered and did not harm the con- sumer. The chlorophylls in pellets rich in either Trebouxia cells or algal cell debris revealed a strong autofluorescence in green light (Fig. llb), but chlorophyll fluorescence does not indicate algal cell viability. Our LM observations of the contents of faecal pellets suggest that mainly young Trebouxia cells, which were still enclosed in the degrading mother cell wall, remained intact.

CULTURING EXPERIMENTS WITH FAECAL PELLETS

In hanging drop cultures of squeezed faecal pellets, some intact photobiont cells and ascospores were dis- covered (not shown), the latter germinating within 3-5 days incubation. Ascospores contained in faecal pellets were also germinating on agarized mineral medium

(BBM) where they grew out during several months into small colonies of 1-2mm in diameter (Fig. 12). In our laboratory, X. parzetina is routinely cultured from single ascospores. Thus, samples were available for morphotypic comparisons of growth rate, colony structure and coloration, hyphal dimensions, etc. Promis- ing fungal colonies were subcultured and used for DNA extraction and analysis. As hanging drop cultures could be kept for only limited periods of time before they were completely overgrown by fungal and bacterial contaminants, the viability of the green algal cells was investigated in Parafilm strip cultures (see Methods). When kept on Parafilm stripes that were fixed on microscopy slides and incubated in humid growth chambers, i.e. Petri dishes with moist filter paper a t 15 f 1"C, the faecal pellets and their contents could be kept for several months. Even here the majority of pellets (c. 150 out of 200) had to be sooner or later discarded due to the outgrowth of faster-growing non-lichenized fungi. Within the first 30 days of incubation of squeezed pellet contents in hanging drop cultures, an increase in size was noted among the majority of intact photobiont cells (not shown). Aplanospore formation, visible as outbursts of green algal cells on the pellet surface, occurred within several months in c. 2% of all pellets that were kept on Parafilm stripes (Figs 13), i.e. in four out of 200 pellets which had been transferred to Parafilm. Within the next months, the increasing cell number in the photobiont cell population surpassed the volume of the faecal pellet, with cauliflower-like algal outgrowths bursting out of the surface of the pellet (Fig. 14). Such outgrowths were subcultured, examined in the light microscope and their rDNA was extracted and analysed (see below).

RECORD OF IDENTITY OF THE AF'OSYMBIOTIC FUNGAL AND ALGAL ISOLATES FROM FAECAL PELLETS

Light microscopic examination allowed a first identifi- cation of the algal isolates, the large, lobate central chloroplast with central pyrenoid and the laterally positioned nucleus being distinctive features of the green algal genus Trebouxia. The ellipsoid, bipolar ascospores, as observed in hanging drop cultures of faecal pellets, were also easily identified mainly due to the massive thickenings of their walls. Comparative rDNA analyses of the aposymbiotic fungal (Fig. 12) and algal cultures derived from faecal pellets with thallus fragments collected in nature and with referenced data unequivocally confirmed the taxonomic identity of pellet-derived fungal and algal subcultures. For X. parietina, the rDNA data (alignment: 539bp) revealed 100% homology of the pellet subcultures and the thalli, and 97% conformity with the X. parietina thallus referenced by Lohtander et al. (2000).



a

10

PM

200 pm

14

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V M L E ASCOSPORES AND ALGAE IN FAECAL PELLETS OF LICHENIVOROUS MITES 265

The hydrophobin gene data revealed 100% homology of the pellet subcultures and published data on thalli of this collecting site (Scherrer et al., 2000; Scherrer & Honegger, unpubl.). Two independent DNA analyses of pellet-derived algal subcultures revealed 100% identity in 650bp overlap, and 99.9% identity with Debouxia arboricola (Z 68703) as submitted by Fried1 (Bhattacharya et al., 1996).

DISCUSSION The present study is the first report on gut passage of viable ascospores and algal cells derived from a lichen species that had been grazed by oribatid mites. The recovery in our highly artificial laboratory cultures on Parafilm stripes was rather low (2% of pellets with outgrowing Debouxia arboricola). It could be that the recovery in nature is higher, provided that the faecal pellets are deposited on a suitable substratum as regards to structure, composition and topology.

As lichen-forming fungi do not normally produce sexual or asexual reproductive structures in aposymbiotic culture, they can only be identified with molecular markers. Comparative rDNA sequence analyses are routinely performed in molecular sys- tematic studies. The hydrophobin gene sequence structure encodes for a species-specific, hydrophobic cell wall surface protein which plays an important role for the functioning of the symbiosis (Scherrer et al., 2000, 2002; Honegger, 2001). Peculiarities of hydrophobins, a class of fungal cell wall proteins with very little amino acid sequence homology with the exception of 8 cysteine residues in a conserved pattern, and their functions in diverse fungus-host interac-

tions are summarized by Wessels (1999, 2000). Hydrophobin gene sequences would be excellent taxonomic markers but, due to their very high rate of sequence divergence, primers can be used only within a limited range of closely related taxa (Scherrer et al., 2000; Scherrer & Honegger, unpubl. data). Lichen photobionts of the genus Trebouxia can be identified a t the genus level by light microscopy (experts identify them even a t the species level with microscopy techniques), the large, lobate central chloroplast with central pyrenoid and the laterally located nucleus being the most prominent features, but molecular markers are preferably used for determining the species. Thus, our experimental approach unequivocally shows that the cultured fungal and algal isolates derived from faecal pellets are the lichen-forming ascomycete X. parietina and its green algal photobiont, Trebouxia arboricola. Besides numerous viable spores of non-lichenized fungi, faecal pellets also contained large numbers of viable bacterial cells of unknown origin and function.

Although we were not yet able to demonstrate in culturing experiments that lichen thalli grow out of such faecal pellets, we hypothesize dispersal of X. parietina via oribatid mites to be a common and widespread feature. This hypothesis is based on the following observations. Firstly, oribatid mites are extremely common inhabitants and grazers of X. parietina. Specimens of this very widespread lichen origi- nating from all over Europe, from North America, South Africa, Australia and New Zealand all contained mites (partly the same species as in our study site) and revealed grazing marks (Honegger, unpubl. data). Sec- ondly, large numbers of juvenile thalli of X. parietina of about the same size as faecal pellets were observed

Y

Figures 7-14. Light microscopy of mite-derived grazing marks on the thallus ofX. parietina, faecal pellets and their contents and pellet-derived cultures of the fungal and algal partner of this lichen. Fig. 7. Marginal thallus lobes of X . parietina ( X P ) , with extensive grazing marks caused by Tk, trimaculatus in laboratory culture. Adjacent Physcia orbicularis (PHY) was not affected. Fig. 8. Grazing marks on apothecia (AP) of X. parietina, caused by T tectorum in laboratory cultures. In many places, the hymenium was eaten down to the subhymenial layer. Groups of photobiont cells in the algal layer underneath the subhymenium are recognized as green spots. The whole surface of the lichen, grazing marks included, is dotted with dark faecal pellets. Fig. 9. Lateral view of the praenymphal stage of I: tectorum in transmitted light, with prospective faecal pellets (FP) in the gut and extended caeca. Fig. 10. Bright field microscopy of a faecal pellet from a laboratory culture of 72: trimaculatus, with surrounding peritrophic membrane (PM), fragments of the hymenial layer ( H Y ) of the fungal fruiting body, and intact, juvenile cells of the photobiont, I: arboricola (PH). Fig. 11. Two faecal pellets from a laboratory culture of I: tectorum in bright field (a) and fluorescence light microscopy (b-c) using green light (510-560nm) to visualize chlorophyll autofluorescence in red (b), and violet light (405 nm) to induce anthraquinone autofluorescence in bright yellow. Fig. 12. Three colonies of the lichen-forming ascomycete X. parietina, each growing out from a faecal pellet- derived ascospore, after 3 months incubation at 15 ? 1°C on agarized mineral medium. Fig. 13. Faecal pellet (FP) overlying a Parafilm stripe, after 7 months incubation in a humid growth chamber (Petri dish) at 15 f 1"C, showing cells of the green algal photobiont, I: arboricola (PH), which grow out of the pellet surface. Fig. 14. Faecal pellet (FP) overlying a Parafilm stripe, after 15 months incubation, with cauliflower-like outgrowth of I: arboricola cells, the unicellular green algal photobiont of the lichen-forming ascomycete X. parietina. Please note: the poor resolution in Figs 13 and 14 is due to insufficient focal depth at this high magnification.


266 F. A. MEIER E T AL.

developing on bare bark with no trace of free-living Debouxia cells in the vicinity of adult thalli with abundant grazing marks (Honegger, unpubl. data). With their migratory behaviour, mites deposit faecal pellets within a few centimetres distance of the thalli. Stubbs (1987) reported an average action radius of adult oribatids of about 18 cm. However, mites can be passively transported over large distances by wind currents. Passive transport of mites over intermediate or possibly even long distances (top-to-bottom transport in trees or on other substrata) may occur via water flows as mites of the Ceratozidae survive sub- mersion in water for several weeks (Meier, unpubl. data) due to their ability to accumulate large amounts of air in their tracheal system. Air bubbles captured on the dorsal part of the body prevent the mite from drowning and likely provide it with oxygen for a con- siderable period of time.

With their slimy peritrophic membrane, acarine faecal pellets seem to be nearly ideal propagules of X. parzetina and its green algal photobiont and also of a wide range of other lichens which are grazed by oribatid mites. Faecal pellets adhere not only to diverse substrata, but probably also to the extremities of larger invertebrates such as bugs or spiders, and of birds, all of which might serve as significant vectors for short- and long-distance dispersal of lichen- forming fungi and their photobionts. The evolutionary significance of such multiple interactions on the population genetics and dispersal of lichen-forming fungi and their phobotionts will be explored.

It remains to be seen whether gut passage of viable fungal and algal cells also occurs in other consumers of X . parietina such as springtails and other insects. Slugs and snails are less regular consumers of this particular species, but some gastropods were shown to ingest this and other lichen-forming fungi and their photobionts (Baur & Baur, 1997), e.g. in studies on the potential role of mycobiont-derived secondary metabolites as feeding deterrents (Stahl, 1904). From light microscopy studies on cell shape and measurements of chlorophyll fluorescence of Debouxia cells contained in faecal pellets of the land snail Helicogona lapicida L., which had been feeding on X. parietina on the Baltic island of Oland, Froberg et al. (2001) concluded that some ingested cells of the green algal photobiont were likely to remain viable after passing the digestive tract. As such, lichenivorous gastropods might be potent vectors of a wide range of lichen-forming fungi and their photobionts. A report on a peculiar type of cylindrical vegetative propagule, observed in SEM preparations of the terrestrial lichen Solorina crocea (Jahns, 1987), is likely to depict faecal pellets, possibly of molluscs as these release long, thread-like faeces which tend to fragment into cylindrical units.

The oribatid mite Humerobates arborea was shown to be a vector of soredia, a type of symbiotic propagules formed by large numbers of species of lichenized ascomycetes (Stubbs, 1995). It is possible that lichen- dwelling arthropodes fulfil additional roles, e.g. by carrying spermatia a t their extremities and thus facilitating fertilization. Insect-mediated fertilization was demonstrated in fungus-plant symbioses such as Epichloe endophytes of grasses (Bultman et al., 1995), where spermatia were shown to survive gut passage in Phorbia flies (Bultman et al., 1998), and in rust fungi which attract insects by means of floral mimicry, i.e. yellow to orange, flower-like coloration of the sys- temically infected leaves and production of attrac- tively scented spermatial stages (Roy, 1993; Raguso & Roy, 1998; Pfunder & Roy, 2000; Naef et al., 2002). Only few species of lichen-forming ascomycetes have so far been investigated with regards to their mating system (Murtagh, Dyer & Crittenden, 2000; Murtagh et al., 1999; Kroken & Taylor, 2001). It is not known whether X. parietina is heterothallic or homothallic (cross-fertilized or self-fertilized) and whether its microconidia serve as male gametes. Indeed, nothing is known about the population genetics of X. parietina and its photobiont.

We conclude that lichen-invertebrate interactions are likely to comprise many other yet unknown options and thus might be a fascinating area for future interdisciplinary studies.

ACKNOWLEDGEMENTS

Our sincere thanks are due to Dr Joseph Trave for identifying the mites, to Constance S. Stubbs for kindly sending us a copy of her Masters thesis, to Undine Zippler for excellent technical assistance, to Jean-Jacques Pittet for his competent help with the artwork, to Chris Hardy for improving the English of our manuscript, and to the Swiss National Science Foundation (grant Nr. 31-52981.97 to R.H) for gener- ous financial support.

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Faecal Pellets of Lichenivorous Mites Contain Viable Cells of the Lichen-Formingascomycete Xanthoria Parietina and Its Green Algal Photobiont, lFebouxia Arboricola