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

The evolution of spore size in Agarics: do big mushrooms have big spores? P. MEERTS Laboratoire de GeÂneÂtique et Ecologie veÂgeÂtales, Universite Li...
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The evolution of spore size in Agarics: do big mushrooms 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

Basidiocarp; Basidiomycotina; fungal development; fungi; offspring size; PIC; reproductive strategy; scaling.

As a ®rst attempt to investigate evolutionary patterns of spore size in Agarics, I tested whether this trait was correlated to the size of the fruit-body (basidiocarp). Based on phylogenetically independent contrasts, it was shown that big mushroom species had on average 9% longer, 9% wider and 33% more voluminous spores (all with P < 0.05, one-tailed tests) than small congeneric species (a three-fold difference in cap diameter was used to discriminate big and small mushrooms). It is argued that larger spore size does not consistently confer higher ®tness in fungi, owing to aerodynamic constraints. Surprisingly, the cap±spore correlation was strongly lineagespeci®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.

Introduction Offspring size is subject to optimization by natural selection because larger offspring tend to have a higher ®tness but are more costly to produce (Lloyd, 1987; Morris, 1987; Silvertown, 1989). Trade-offs between offspring size and number are often masked by variation in parental size because larger organisms, having higher absolute allocation to reproduction, can increase both offspring size and offspring number at the same time (Stearns, 1992; Begon et al., 1996). Thus, interspeci®c scaling is a pervasive source of variation in offspring size in plants and animals (Stearns, 1992). In ¯owering plants, for instance, seed size is often correlated positively with fruit size and vegetative size (Primack, 1987; Thompson & Rabinowitz, 1989; Niklas, 1994). Compared to animals and plants, the evolutionary ecology of offspring size in the third kingdom of multicellular eucaryotes, i.e. fungi, has received much less attention (but see Ingold, 1971; Kreisel, 1984; Parmasto & Parmasto, 1987). As a structure whose function is to Correspondence: P. Meerts, Laboratoire de GeÂneÂtique et Ecologie veÂgeÂtales, Universite Libre de Bruxelles, ChausseÂe de Wavre 1850, B-1160 Bruxelles, Belgium. E-mail: [email protected]

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produce and liberate spores as ef®ciently as possible (Webster, 1980; PoÈder, 1983), there is little doubt that fruit-body (basidiocarp) morphology has been subjected to optimizing selection, particularly in Homobasidiomycetes (McNight & Roundy, 1991; PoÈder, 1992; PoÈder & Kirchmair, 1995). Basidiospores are unicellular, haploid propagules dispersed by air. This mode of dispersion, in addition to narrow habitat requirements, must result in high rates of density-independent mortality, a selective regime known to favour small offspring size (Stearns, 1992; Begon et al., 1996). In Agarics, i.e. Homobasidiomycetes with a stipe, a pileus (cap) and a hymenophore consisting of lamellae (gills), spore volume varies by about three orders of magnitude among species (Pegler & Young, 1971; Singer, 1975; Webster, 1980). In this paper, I test the simple hypothesis that spore size is correlated with basidiocarp size. I anticipate a positive correlation, on the grounds that basidiocarp growth occurs mostly through cellular swelling and spores are produced by unicellular sporocysts (basidia) (Webster, 1980; Oberwinkler, 1982) (Fig. 1).

Materials and methods The taxonomic groups considered are the Agaricales and the Russulales. Spore dimensions [length (L) and width

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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: hymenium; T: trama; B: basidium; B1: caryogamy; B2: meiosis; B3: spore formation.

(w)] and cap diameter are from the standard ¯ora of Moser (1983). Therein, spore and cap dimensions are reported as the two extremes of the species' normal variation range; typically, the range of values is about 10± 25% of the average value for spore dimensions (e.g. spore length: 10±12 lm) and 50±100% for cap diameter (e.g. 2± 4 cm). Cap size indeed shows large phenotypic plasticity depending on age and growth conditions (Webster, 1980). The average value of the two extremes of the normal variation range was used for subsequent analysis. Species for which only a single value was available (e.g. cap diameter `up to 3 cm') were not considered. Cap diameter was preferred over other basidiocarp size measurements because it is positively correlated with total hymenophore area and thus, with total spore number (PoÈder, 1983). Spore volume was calculated as that of a revolution ellipsoid: 4p/3 (L/2)*(w/2)2 (Gross, 1972). Two different approaches were then conducted. Phylogenetically independent contrasts (PICs) Phylogenetically independent contrasts (PICs) (Harvey & Pagel, 1991) were constructed by the following procedure. A three-fold difference in average cap diameter was chosen as the size difference criterion for discriminating between big and small mushrooms. This criterion was a compromise between maximizing the number of PICs included in the analysis and minimizing overlaps in the size range of small and big species. One big and one small species (as de®ned

above) were selected randomly from each of 54 genera showing suf®ciently large variation in cap size among species, from a total of about 95 genera comprising two species or more. The genera included were (total number of species in parentheses, round ®gures): Agaricus (67), Agrocybe (18), Amanita (36), Bolbitius (6), Calocybe (13), Camarophyllus (12), Clitocybe (94), Clitopilus (9), Collybia (33), Conocybe (30), Coprinus (92), Cortinarius (530), Crepidotus (25), Cystoderma (13), Cystolepiota (8), Entoloma (150), Flammulaster (19), Galerina (57), Gerronema (8), Gymnopilus (13), Hebeloma (53), Hohenbuehelia (12), Hygrocybe (57), Hygrophorus (46), Hypholoma (16), Inocybe (170), Lactarius (89), Lentinellus (9), Lepiota (52), Leucoagaricus (13), Leucocoprinus (15), Leucopaxillus (14), Lyophyllum (19), Macrolepiota (11), Marasmius (32), Melanoleuca (31), Micromphale (6), Mycena (130), Omphalina (31), Panellus, Pholiota (34), Pluteus (47), Psathyrella (100), Pseudobaeospora (4), Psilocybe (17), Rhodocybe (11), Ripartites (6), Russula (160), Squamanita (7), Stropharia (16), Tephrocybe (24), Tricholoma (67), Tubaria (11), Volvariella (16). Spore length, width and volume were then compared between big and small mushrooms by means of two-sample paired t-tests. Spore±cap correlations within genera Those 16 genera of Agaricales and Russulales comprising more than 50 species were used to investigate correlations 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 (Agaricus, 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 appropriate (w1 and w2) and spore volume was calculated accordingly. All data were transformed to natural logarithms before analysis. Associations between cap diameter, 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 mushrooms; these differences were, however, signi®cant only at a one-tailed error rate (mean values ‹ standard errors over 54 species, length: big 8.37 ‹ 0.41 lm, small 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, t ˆ 1.85 P ˆ 0.035) (Fig. 2). Compared to their 54 small counterparts, the spores of the large species were longer in 29 cases, wider in 35 cases and more voluminous in 34 cases. Correlations within individual genera The correlation patterns with cap diameter were very similar for all spore size measurements so that detailed results are presented only for spore volume (Table 1). At a tablewide error rate, spore volume was signi®cantly correlated with cap diameter in four of 16 genera (positive correlation in Agaricus, Coprinus and Cortinarius, negative correlation in Psathyrella).

Discussion Why are evolutionary changes in cap size only weakly correlated with changes in spore size in Agarics? A ®rst likely explanation might lie in the fact that larger spores do not consistently have a higher ®tness in fungi, owing to aerodynamic constraints. Theoretical aspects of particle transfer by air indicate that spore size is negatively correlated with dispersability and positively correlated with impaction ef®ciency (Whitehead, 1969; Dix & Webster, 1995). Thus, small spores are less prone to being trapped by obstacles before reaching a suitable substrate because they tend to follow the airstream around a potential collecting object (Whitehead, 1969; Ingold, 1971). The relationship between offspring size

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

Fig. 2 Phylogenetically independent contrasts of spore volume between big and small mushroom species (i.e. at least a threefold difference in cap diameter) in each of 54 genera of Agarics. Each point represents one pair of species. The diagonal line represents equal spore size for big and small mushrooms.

and ®tness might therefore be quite different than in animals and plants and the optimal spore size might conceivably vary depending on way of life. For instance, mushrooms which colonize twigs and living leaves tend 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 smaller spores in forest-¯oor species, but this does not Table 1 Pearson correlation coef®cients (r) between cap diameter and spore volume in the 16 largest genera of Agarics in Europe. n = number of species considered. Coef®cients in bold type remain signi®cant when a tablewide error rate is applied. ***P < 0.001, **P < 0.01, *P < 0.05, ns not signi®cant. n Agaricus Clitocybe Coprinus Cortinarius Entoloma Galerina Hygrocybe Hygrophorus Inocybe Lactarius Lepiota Mycena Pluteus Psathyrella Russula Tricholoma

57 80 74 142 73 50 47 43 40 46 43 119 44 89 96 57

r 0.688*** 0.169 ns 0.292** 0.310*** )0.063 ns )0.172 ns 0.363* )0.220 ns 0.269 ns )0.254 ns 0.269 ns 0.090 ns 0.042 ns )0.324** 0.141 ns 0.081 ns

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seem to have ever been tested. The positive correlation between spore size of small and large species across genera (Fig. 2) does support the idea that spore size tends to be canalized within lineages. Secondly, unlike plant fruits and animal body cavities, basidiocarps are open, not closed, structures in which total spore mass represents only a limited proportion of the energy devoted to reproduction (Webster, 1980). These peculiarities might loosen the constraint imposed by basidiocarp size on spore size. Clearly, fungal spores are not homologous either structurally or functionally of plant seeds and animal eggs. Surprisingly, however, the pattern of correlation of spore size with basidiocarp size is strongly lineagespeci®c. What might be the mechanism underlying the positive correlation found in four genera? At the intraspeci®c level, spore±cap correlations have been circumstantially reported on several occasions (e.g. Hanna, 1926; Watling, 1975; CleÂmencËon, 1979). Buller (1922, 1924) already noted that hairs on the pileus and spores of 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 (reviewed in Wells & Wells, 1982; Chiu & Moore, 1996) might offer a mechanistic explanation to these observations. It is now generally accepted that Agarics primarily 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 produced by unicellular meiosporocysts (basidia). A tight correlation between basidium volume and total spore volume per basidium does exist in Basidiomycetes (Corner, 1948; PoÈder, 1986), indicating that spore size is strongly constrained by basidium size. The size of hyphal articles therefore appears as a possible mediator of the spore±cap correlation. Two conditions must be ful®lled for cell size to generate a positive correlation between spore and basidiocarp size: (i) interspeci®c differences in cap size must be due, to a signi®cant extent, to differences in the magnitude of hyphal swelling during basidiocarp growth and (ii) hyphal swelling must be coordinated throughout the basidiocarp. Based on the results of this study, it would appear that only a few Agaric genera do ful®l both aforementioned conditions. Oberwinkler (1982) recognized two fundamental types of basidia, namely `non in¯ating' basidia and basidia that strongly expand apically during hymenophore development. He cites three genera as examples of the second category (Agaricus, Coprinus, Russula). He also comments that `very often, those species forming basidia that expand apically form basidiocarps in which the hyphae of the trama, subhymenium and hymenium also show similar secondary expansion. Possibly this feature is associated with the capacity of basidiocarps to undergo rapid expansion under optimum environmental conditions.' It is striking that two of the three genera cited as having in¯ating basidia showed a positive cap±spore correlation

(Agaricus, Coprinus). In Russula, basidium size is probably uncoupled from cap size because specialized cells (`sphaerocysts') account for most of the expansion process of the basidiocarp (Reijnders, 1963). The timing of the hyphal in¯ation process, relative to basidium formation (see Hammad et al., 1993), might also be of importance in the evolution of cap±spore correlations. Speci®cally, if the charging of the basidium with protoplast, which sets the upper limit of total spore volume (Corner, 1948), is achieved before the onset of the expansion process, there would be limited opportunity for a correlation to evolve. The morphogenetic basis of the negative cap±spore correlation in Psathyrella is, however, less clear and would deserve further investigation. In conclusion, the results reveal unsuspected differences between genera of Agarics in the pattern of covariation of spore and basidiocarp size, the ecological and evolutionary signi®cance of which remains to be elucidated. On the whole, the factors which govern spore size evolution in fungi need further investigation.

Acknowledgments J. Rammeloo and A. De Kesel commented on an earlier draft of the manuscript. This paper is dedicated to the memory of P. Heinemann.

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