The evolution of spore size in Agarics: do big have big spores?

P. MEERTS Laboratoire de GeÂneÂtique et Ecologie veÂgeÂtales, Universite Libre de Bruxelles, ChausseÂe de Wavre 1850, B-1160 Bruxelles, Belgium (e-mail: [email protected])

Keywords: Abstract ; As a ®rst attempt to investigate evolutionary patterns of spore size in Agarics, I Basidiomycotina; tested whether this trait was correlated to the size of the fruit-body fungal development; (basidiocarp). Based on phylogenetically independent contrasts, it was shown fungi; that big species had on average 9% longer, 9% wider and 33% offspring size; more voluminous spores (all with P < 0.05, one-tailed tests) than small PIC; congeneric species (a three-fold difference in cap diameter was used to reproductive strategy; discriminate big and small mushrooms). It is argued that larger spore size does scaling. not consistently confer higher ®tness in fungi, owing to aerodynamic constraints. Surprisingly, the cap±spore correlation was strongly lineage- speci®c. Thus, spore volume correlated signi®cantly with cap diameter in ®ve of 16 large genera (four positive and one negative correlation). Positive cap± spore correlations are interpreted in terms of developmental constraints, mediated by hyphal swelling during cap expansion. The possible mechanisms which can account for the breakdown of this constraint in the majority of genera investigated are discussed.

produce and liberate spores as ef®ciently as possible Introduction (Webster, 1980; PoÈ der, 1983), there is little doubt that Offspring size is subject to optimization by natural fruit-body (basidiocarp) morphology has been subjected selection because larger offspring tend to have a higher to optimizing selection, particularly in Homobasidiomy- ®tness but are more costly to produce (Lloyd, 1987; cetes (McNight & Roundy, 1991; PoÈ der, 1992; PoÈ der & Morris, 1987; Silvertown, 1989). Trade-offs between Kirchmair, 1995). offspring size and number are often masked by variation Basidiospores are unicellular, haploid propagules dis- in parental size because larger organisms, having higher persed by air. This mode of dispersion, in addition to absolute allocation to reproduction, can increase both narrow habitat requirements, must result in high rates of offspring size and offspring number at the same time density-independent mortality, a selective regime known (Stearns, 1992; Begon et al., 1996). Thus, interspeci®c to favour small offspring size (Stearns, 1992; Begon et al., scaling is a pervasive source of variation in offspring size 1996). In Agarics, i.e. Homobasidiomycetes with a stipe, a in plants and animals (Stearns, 1992). In ¯owering pileus (cap) and a hymenophore consisting of lamellae plants, for instance, seed size is often correlated positively (gills), spore volume varies by about three orders of with fruit size and vegetative size (Primack, 1987; magnitude among species (Pegler & Young, 1971; Singer, Thompson & Rabinowitz, 1989; Niklas, 1994). 1975; Webster, 1980). In this paper, I test the simple Compared to animals and plants, the evolutionary hypothesis that spore size is correlated with basidiocarp ecology of offspring size in the third kingdom of multi- size. I anticipate a positive correlation, on the grounds that cellular eucaryotes, i.e. fungi, has received much less basidiocarp growth occurs mostly through cellular swell- attention (but see Ingold, 1971; Kreisel, 1984; Parmasto ing and spores are produced by unicellular sporocysts & Parmasto, 1987). As a structure whose function is to (basidia) (Webster, 1980; Oberwinkler, 1982) (Fig. 1).

Correspondence: P. Meerts, Laboratoire de GeÂneÂtique et Ecologie veÂgeÂtales, Materials and methods Universite Libre de Bruxelles, ChausseÂe de Wavre 1850, B-1160 Bruxelles, Belgium. The taxonomic groups considered are the and E-mail: [email protected] the Russulales. Spore dimensions [length (L) and width

J. EVOL. BIOL. 12 (1999) 161±165 Ó 1999 BLACKWELL SCIENCE LTD 161 162 P. MEERTS

Fig. 1 Schematic diagram of a typical Agaric fruit-body. PM: primary mycelium (haploid hyphae), SM: secondary mycelium (dicaryotic hyphae); S: stipe; C: cap; TS: transverse section of a gill; H: ; T: trama; B: basidium; B1: caryogamy; B2: meiosis; B3: spore forma- tion.

(w)] and cap diameter are from the standard ¯ora of above) were selected randomly from each of 54 genera Moser (1983). Therein, spore and cap dimensions are showing suf®ciently large variation in cap size among reported as the two extremes of the species' normal species, from a total of about 95 genera comprising two variation range; typically, the range of values is about 10± species or more. The genera included were (total 25% of the average value for spore dimensions (e.g. spore number of species in parentheses, round ®gures): length: 10±12 lm) and 50±100% for cap diameter (e.g. 2± (67), Agrocybe (18), (36), Bolbitius 4 cm). Cap size indeed shows large phenotypic plasticity (6), Calocybe (13), Camarophyllus (12), Clitocybe (94), depending on age and growth conditions (Webster, Clitopilus (9), Collybia (33), Conocybe (30), Coprinus (92), 1980). The average value of the two extremes of the (530), Crepidotus (25), Cystoderma (13), normal variation range was used for subsequent analysis. Cystolepiota (8), Entoloma (150), Flammulaster (19), Species for which only a single value was available (e.g. Galerina (57), Gerronema (8), Gymnopilus (13), Hebeloma cap diameter `up to 3 cm') were not considered. Cap (53), Hohenbuehelia (12), (57), Hygrophorus diameter was preferred over other basidiocarp size mea- (46), Hypholoma (16), Inocybe (170), Lactarius (89), surements because it is positively correlated with total Lentinellus (9), (52), Leucoagaricus (13), Le- hymenophore area and thus, with total spore number ucocoprinus (15), Leucopaxillus (14), Lyophyllum (19), (PoÈ der, 1983). Spore volume was calculated as that of a Macrolepiota (11), Marasmius (32), Melanoleuca (31), revolution ellipsoid: 4p/3 (L/2)*(w/2)2 (Gross, 1972). Two Micromphale (6), Mycena (130), (31), Panellus, different approaches were then conducted. Pholiota (34), Pluteus (47), Psathyrella (100), Pseudo- baeospora (4), Psilocybe (17), Rhodocybe (11), Ripartites (6), Russula (160), Squamanita (7), Stropharia (16), Phylogenetically independent contrasts (PICs) Tephrocybe (24), Tricholoma (67), Tubaria (11), Volvariella Phylogenetically independent contrasts (PICs) (Harvey (16). Spore length, width and volume were then & Pagel, 1991) were constructed by the following compared between big and small mushrooms by means procedure. A three-fold difference in average cap of two-sample paired t-tests. diameter was chosen as the size difference criterion for discriminating between big and small mushrooms. Spore±cap correlations within genera This criterion was a compromise between maximizing the number of PICs included in the analysis and Those 16 genera of Agaricales and Russulales comprising minimizing overlaps in the size range of small and big more than 50 species were used to investigate correla- species. One big and one small species (as de®ned tions between cap size and spore size at the within-genus

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level. For the largest three genera (Cortinarius, Entoloma and Inocybe), a subsample of about one-third of all species were randomly selected; for the other 13 genera (Agaric- us, Clitocybe, Coprinus, Galerina, Hygrocybe, Hygrophorus, Lactarius, Lepiota, Mycena, Pluteus, Psathyrella, Russula, Tricholoma), all species for which the relevant data were available were considered. For Coprinus, in which some species have dorsi-ventrally compressed spores, two measures of spore width were included where appropri- ate (w1 and w2) and spore volume was calculated accordingly. All data were transformed to natural loga- rithms before analysis. Associations between cap diam- eter, spore length, spore width and spore volume were computed as Pearson correlation coef®cients.

Results

Phylogenetically independent contrasts On average, big mushrooms had 9% longer, 9% wider and 33% more voluminous spores than small mush- Fig. 2 Phylogenetically independent contrasts of spore volume between big and small mushroom species (i.e. at least a three- rooms; these differences were, however, signi®cant only fold difference in cap diameter) in each of 54 genera of Agarics. at a one-tailed error rate (mean values ‹ standard errors Each point represents one pair of species. The diagonal line over 54 species, length: big 8.37 ‹ 0.41 lm, small represents equal spore size for big and small mushrooms. 7.71 ‹ 0.35 lm, t ˆ 1.73 P ˆ 0.045; width: big 5.19 ‹ 0.28 lm, small 4.75 ‹ 0.21 lm, t ˆ 1.93 P ˆ 0.029; volume: big 169 ‹ 31 lm3, small 120 ‹ 19 lm3, and ®tness might therefore be quite different than in t ˆ 1.85 P ˆ 0.035) (Fig. 2). Compared to their 54 animals and plants and the optimal spore size might small counterparts, the spores of the large species were conceivably vary depending on way of life. For instance, longer in 29 cases, wider in 35 cases and more volumi- mushrooms which colonize twigs and living leaves tend nous in 34 cases. to have relatively larger spores because these are more easily captured by the host (Ingold, 1971); conversely, low wind speed and high vegetation density might favour Correlations within individual genera smaller spores in forest-¯oor species, but this does not The correlation patterns with cap diameter were very similar for all spore size measurements so that detailed Table 1 Pearson correlation coef®cients (r) between cap diameter results are presented only for spore volume (Table 1). At and spore volume in the 16 largest genera of Agarics in Europe. a tablewide error rate, spore volume was signi®cantly n = number of species considered. Coef®cients in bold type remain correlated with cap diameter in four of 16 genera signi®cant when a tablewide error rate is applied. ***P < 0.001, (positive correlation in Agaricus, Coprinus and Cortinarius, **P < 0.01, *P < 0.05, ns not signi®cant. negative correlation in Psathyrella). n r

Agaricus 57 0.688*** Discussion Clitocybe 80 0.169 ns Why are evolutionary changes in cap size only weakly Coprinus 74 0.292** Cortinarius 142 0.310*** correlated with changes in spore size in Agarics? A ®rst Entoloma 73 )0.063 ns likely explanation might lie in the fact that larger spores Galerina 50 )0.172 ns do not consistently have a higher ®tness in fungi, owing Hygrocybe 47 0.363* to aerodynamic constraints. Theoretical aspects of parti- Hygrophorus 43 )0.220 ns cle transfer by air indicate that spore size is negatively Inocybe 40 0.269 ns correlated with dispersability and positively correlated Lactarius 46 )0.254 ns with impaction ef®ciency (Whitehead, 1969; Dix & Lepiota 43 0.269 ns Webster, 1995). Thus, small spores are less prone to Mycena 119 0.090 ns being trapped by obstacles before reaching a suitable Pluteus 44 0.042 ns substrate because they tend to follow the airstream Psathyrella 89 )0.324** Russula 96 0.141 ns around a potential collecting object (Whitehead, 1969; Tricholoma 57 0.081 ns Ingold, 1971). The relationship between offspring size

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seem to have ever been tested. The positive correlation (Agaricus, Coprinus). In Russula, basidium size is probably between spore size of small and large species across uncoupled from cap size because specialized cells genera (Fig. 2) does support the idea that spore size tends (`sphaerocysts') account for most of the expansion process to be canalized within lineages. of the basidiocarp (Reijnders, 1963). The timing of the Secondly, unlike plant fruits and animal body cavities, hyphal in¯ation process, relative to basidium formation are open, not closed, structures in which (see Hammad et al., 1993), might also be of importance in total spore mass represents only a limited proportion of the evolution of cap±spore correlations. Speci®cally, if the the energy devoted to reproduction (Webster, 1980). charging of the basidium with protoplast, which sets the These peculiarities might loosen the constraint imposed upper limit of total spore volume (Corner, 1948), is by basidiocarp size on spore size. Clearly, fungal spores achieved before the onset of the expansion process, there are not homologous either structurally or functionally of would be limited opportunity for a correlation to evolve. plant seeds and animal eggs. The morphogenetic basis of the negative cap±spore corre- Surprisingly, however, the pattern of correlation of lation in Psathyrella is, however, less clear and would spore size with basidiocarp size is strongly lineage- deserve further investigation. speci®c. What might be the mechanism underlying the In conclusion, the results reveal unsuspected differen- positive correlation found in four genera? At the intra- ces between genera of Agarics in the pattern of covari- speci®c level, spore±cap correlations have been circum- ation of spore and basidiocarp size, the ecological and stantially reported on several occasions (e.g. Hanna, evolutionary signi®cance of which remains to be eluci- 1926; Watling, 1975; CleÂmencËon, 1979). Buller (1922, dated. On the whole, the factors which govern spore size 1924) already noted that hairs on the pileus and spores of evolution in fungi need further investigation. abnormally small fruit-bodies of Coprinus lagopus were smaller than in normal fruit-bodies. Recent progress in the study of morphogenesis of the Agaric fruit-body Acknowledgments (reviewed in Wells & Wells, 1982; Chiu & Moore, 1996) J. Rammeloo and A. De Kesel commented on an earlier might offer a mechanistic explanation to these observa- draft of the manuscript. This paper is dedicated to the tions. It is now generally accepted that Agarics primarily memory of P. Heinemann. depend on cell in¯ation (hyphal swelling) for fruit-body expansion (Moore et al., 1979; Reijnders & Moore, 1985; Moore, 1996). On the other hand, basidiospores are References produced by unicellular meiosporocysts (basidia). A tight correlation between basidium volume and total spore Begon, M., Harper, J.L. & Townsend, C.R. 1996. Ecology. Blackwell Science, Oxford. volume per basidium does exist in Basidiomycetes (Cor- Buller, A.H.R. 1922. Researches on Fungi. 3. Longman, Green and ner, 1948; PoÈder, 1986), indicating that spore size is Co., London. strongly constrained by basidium size. The size of hyphal Buller, A.H.R. 1924. Researches on Fungi. 4. Longman, Green and articles therefore appears as a possible mediator of the Co., London. spore±cap correlation. Chiu, S.W. & Moore, D. (eds). 1996. Patterns in Fungal Develop- Two conditions must be ful®lled for cell size to generate a ment. 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