Microb Ecol (1987) 14:277-290

MICROBIAL ECOLOGY 9 Springer-Verlag New York Inc. 1987

Fungi, Leaves, and the Theory of Island Biogeography John H. Andrews, 1Linda L. Kinkel, ~Flora M. Berbee,~ and Erik V. Nordheim 2 tDepartment of Plant Pathology and ZDepartments of Forestry and Statistics, University of Wisconsin-Madison, Madison, Wisconsin 53706 USA

Abstract. Species dynamics of fungi (filamentous fungi and yeasts) on apple leaves were studied within the framework of the theory of island biogeography by following "immigration" and "extinction" patterns on individual apple leaf "islands" over time. Total fungi were censused on unmanipulated leaves collected throughout two seasons; filamentous fungi only were monitored additionally for several weeks in one season on newly created, axenic, model (seedling) islands introduced to the orchard, and on surface-sterilized, preexisting leaves. Analyses based on both the natural and the surface-sterilized systems showed that an equilibrium in species number was reached and turnover in species composition occurred in both. Immigration and extinction events were strongly related to number of species present on each island. The balance between immigration and extinction implies that species number on leaves and "real" (oceanic) islands is determined by a common mechanism, and emphasizes the need to regard leaf microbial communities as dynamic.

Introduction

Although the microbial ecology of the leaf surface (phylloplane) has been studied actively for about 15 years, as evidenced by the convening of four international symposia [e.g., 6], research is still largely descriptive. A question of considerable interest is whether or not species dynamics in a particular microbial environment such as the phylloplane could be described by a general model, especially one applicable to macroorganisms. There has been some speculation [1, 40], but virtually no experimental evidence that the theory of island biogeography might be a relevant framework. Leaves act much like islands for microbes from the time of their emergence in the spring until abscission in the autumn. MacArthur and Wilson [19, 20] proposed the theory of island biogeography in an attempt to synthesize and explain a mass of insular biogeographic data on plants and animals. Briefly, the theory or equilibrium model postulates that the biota of an island result from an equilibrium established when the immigration rate of species new to the island equals the extinction rate of previously established species. The immigration rate falls as the number of species present on the island increases because fewer of the arriving species are new. Concur-

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rently, the extinction rate increases because there are increasing numbers of species to become extinct and because, with more species present, the population sizes of each are lower, thus making extinction progressively more likely. Equilibrium occurs when the two rates are equal. It is dynamic in that although the species number stabilizes, composition changes with time (turnover). The equilibrium number is influenced by island area and distance from the colonizing source: larger islands are viewed as having a lower extinction rate, hence equilibrating with more species, compared to smaller islands; distant islands have lower immigration curves, and consequently fewer species at equilibrium than do islands near the source [for reviews see 4, 14, 22, 27, 31]. The theory stems from two empirical relationships, namely, a direct association between species number and area, and a constant species abundance relationship resulting in an association between number of species and number of individuals. Because of its apparent conceptual simplicity and general applicability to both "'real" (oceanic) and "virtual" (habitat patch) islands, the equilibrium concept rapidly became one of the most prominent and controversial in ecology. Its explanatory power, and generation of testable predictions, provided a mechanistic basis for longstanding observations for many organisms of a speciesarea association [e.g., 19, 20, 22]. The concept evoked numerous investigations, with species ranging from birds [11] to mammals [8], fish [35], protozoans [9], arthropods [25], marine epifaunal invertebrates [23], and plants [ 15]. Although not all of the studies were designed as tests of the theory, most that were have been subject to criticism [14, 18, 29]. It is generally agreed that the definitive experiments testing the theory have involved "defaunation" and subsequent recolonization by arthropods of mangrove [30, 32, 33, 41] or Spartina [25] islands in Florida. Both investigations showed that an equilibrium was reached eventually, and that turnover occurred. The original mangrove studies did not directly address the effects of area or isolation, although subsequently on other mangrove islands with segments removed, Simberloff [28] showed that species number increased with area alone independently of habitat diversity. For the Spartina islands, Rey [25] found a significant correlation between area and species number, but no effect of distance on immigration rates was demonstrable. Microbes on the phylloplane offer both advantages and limitations compared to macroorganisms in using the theory as an experimental framework. Leaves are accessible and easily manipulated and are replicated habitat patches with relatively simple communities compared to oceanic islands. Species dynamics of epiphytic microbes can be studied within a single growing season. However, sampling methods are necessarily incomplete and destructive, hence a single island cannot be tracked over time, and variability among communities from one island to the next on the same date must be taken into account. Sources of the species pool are diffuse, so the effects of distance cannot be tested. Arrival of immigrants is influenced by seasonal patterns in fungal reproduction. This report describes our attempt to examine microbial species dynamics on the phylloplane within the framework of island biogeography. For both filamentous fungi (molds) and yeasts we present evidence that a species equilibrium, with turnover, is established; however, the data do not support a speciesarea relationship.

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Materials and M e t h o d s

Experimental Design In theory, the time course of island colonization can be followed by censusing or monitoring organisms of interest (1) from the natural origin of an island, (2) on newly created "model" islands, or (3) on sterilized preexisting islands [41]. We used all three approaches. The first, the "natural experiment" of ecologists [12], has advantages including high generality and the highest realism and temporal scale of the options available. Its major shortcoming is that variables cannot be controlled and, specifically in our system, that apple leaves emerging from buds are not sterile and the time of emergence varies. Hence, colonization could not be followed from its inception, and "replicate" leaves do not necessarily share the same immigration history. The second and third approaches typify the "field experiment category" [ 12] and offer some control of the experimental variables, high realism, but relatively low generality. Specifically, it is possible in theory to control the time at which leaf colonization begins. A potential concern, however, is the degree to which the newly created model and sterilized preexisting islands duplicate the unmanipulated leaf. For the first approach, samples were taken between April and October at 9 dates in 1981 and 11 dates in 1982 from a mature apple tree (Maluspumila var. Mclntosh) in an unsprayed experimental orchard near Arlington, Wisconsin. The epiphytic mycoflora and phenology of these leaf islands are relatively well known [2, 3]. The initial sample consisted of ten buds at the " d o r m a n t " stage of development and the second, of six small leaves (all were small at that date). Thereafter, samples were composed of the largest and the smallest leaf from each of three clusters and were coded as to origin. Each cluster was sampled on only one date. A single sample in time consisted of three large and three small leaves. Over the season, area (both surfaces) of the small leaves ranged from 1.2-9 cm 2 (av. 3.18 cm2), and the large leaves from 10.2-63 cm 2 (av. 26.9 cm2). A range in area was desired to test the effect of island size on the hypothesized equilibrium number. All samples were taken within a 1 m a volume of the tree, thus minimizing positional variability [3]. The leaves were transported to the laboratory in separate sterile plastic Petri dishes in an ice chest, and processed immediately. The system will be referred to as the "'natural field experiment." Second, uncolonized, model islands were produced to simulate their natural counterparts. Aseptically prepared apple seedlings (Kinkel and Andrews, unpublished) were introduced to a gridded platform within the canopy of an apple tree in the orchard described above. Sampling was conducted for 7 weeks during the 1983 growing season. Variability in time of emergence of the seedlings made island biogeographic analyses of these data difficult. Third, attached leaves on a mature apple tree (cv. Meiba) were surface-sterilized as described in [16] on August 1, 1983. Three treated leaves were sampled randomly at each of 11 dates from August 2, to September 21, 1983.

Laboratory Procedures Media. The

following media were tested in preliminary experiments to determine which was best for isolating diverse phylloplane molds and yeasts: (1) freshly prepared potato dextrose agar, PDA [38]; (2) prune agar (Difco Laboratories); (3) malt extract agar, MEA (Difco Laboratories); (4) full or half-strength yeast extract agar, YEA [2]; and (5) a bilayered medium of YEA above MEA. The media were amended with novobioein (125 mg/liter) to inhibit bacterial growth. PDA was selected and used thereafter because the colonies were easily visualized and because it enabled isolation of more "'types" (presumptive species; see below) and more individuals than the other media.

LeafProcessing. Leaves were prepared

for processing by removing petioles, recording fresh weight to 1 mg, and measuring area using a model LI-3100 area meter (LI-COR, Inc., Lincoln, Nebraska). A three-step procedure was used to isolate the organisms. First, each leaf was washed individually in 0.01 M phosphate buffer (pH 7.1) containing 0.01% Tween for 1 hour at 4~ on a reciprocal shaker at 180 cycles per min. The wash volume was standardized to leaf size and averaged 100

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J . H . Andrews et al.

Table 1. Hypothetical data set and calculations to illustrate method for calculating immigrations, extinctions, and within- and betweensample variability (species incongruence) Time T~ Leaf (L)

Time T2

Species present

Leaf (L)

Species present

1

A, B, C

4

A, B, F, G

2 3

A, B, D, E A,B,E

5 6

A,C,F A, B, C, G

Permutation .

Immigrations

Extinctions

2 1 1 2 2 2 2 2 2 1.78

1 1 0 2 3 2 1 2 1 1.44

L~ ~ L4 L~ ~ Ls L~ ~ L6 L2 ~ L4 Lz ~ L5 L 2 ~ L6 L3 ~ L4 L3 ~ L5 L3 ~ L6 Average

Between-sample variability

Within-sample variability T~

Lt ~ L2 Li ~ L3 Lz ~ L3 Average

3 2 1 2.00

L~ Lj L~ L2

~ ~ ~ ~

L4 I-.5 L6 L4

T2

L4 ~ L5 L4~L6 L5 ~ L6 Average

3 2 3 2.67

L2 ~ Ls L2 --, L6 L3 ~ L4 L3 ~ L5 L3 ~ L6 Average

3 2 1 4 5 4 3 4 3 3.22

ml. A tenfold dilution series to 10 -3 was prepared from the washings, and 8-10 plates o f PDA per dilution were spread with 0,1 ml inoculum per plate. These plates were incubated at 24~ for 5 days. Second, the leaves were removed from the buffer solution immediately after the wash period, blotted onto sterile filter paper, and the abaxial and adaxial surfaces uniformly imprinted once onto PDA [2]. These plates were incubated at 24~ for 5 days. Third, following imprinting, each intact leaf was incubated, abaxial surface down, on PDA at 12~ for 7-10 days.

Sampling Protocol. The total number of individuals removed from the leaf was estimated by tallying all colonies from the most suitable dilution of the series (with 40-100 colonies per plate) from the wash plates. The relative number of individuals of each species was estimated from a 200-colony subset from the plates with suitable dilution from each leaf at every date. Each plate was subdivided into segments on a grid. Starting with the first plate, one section was chosen randomly and all colonies in that sector sampled. Successive segments were sampled clockwise around the plate. Successive plates in that dilution were sampled similarly, if necessary, until 200 colonies (individuals) were removed. Fungi were transferred to short PDA slants by standard hyphal-tipping procedures; yeasts

Fungi, Leaves, and Island Biogeography

281

were suspended in buffer-tween, streaked on PDA plates, and then transferred to slants from resultant single colonies. To estimate total number of species, colonies from the 200-set were used, plus those visually different taken from unexamined segments and dilution series, together with those from the leaf imprint and the intact leaf incubation assays. All data (numbers of individuals, relative species dominance, and total species counts) were expressed for each leaf both on an absolute basis and per unit area. The isolates were grown in tubes under identical conditions and sorted into morphologically similar "types," i.e., presumptive species, at 7 and 14 days. Three tubes of each type were saved, if available; for rare species for which fewer than three tubes were available, all tubes were saved. This enabled ca. 1,400 isolates from each sample to be reduced to a more realistic subset of 50200 for identification using standard texts [5, 13, 17, 37]. Isolates that did not sporulate on PDA plates were grown on several mycological media under different light regimes to induce formation of identifiable reproductive structures. Remaining sterile mycelial cultures grown under identical conditions were sorted by colony and hyphal morphology into types that were treated as species. The sorting procedure was assessed by comparing all retained tubes (usually 3 per sample per type). Isolates of the same type proved consistently to be of the same species. This was further confirmed using "'blind" checks. Species were often split into several types. For example, on one sample date, Microsphaeropsis arundinis isolates were sorted into seven types based on slight differences in morphology and growth rate. Since representatives of each type were subsequently keyed to the proper species, this sorting difference was corrected and was inconsequential. To assess the effect of sample size on estimated species n u m b e r for molds, the n u m b e r of individuals examined from one leaf on the dilution plates was increased sixfold in blocks of 200 up to 1,200 by plating wash water form six aliquots. Additional species recovered from the imprint and intact leaf incubation assays were also tallied. The six "standard" samples of 200 colonies from wash plates plus additional species from other dilution plates and from the imprint and incubation assays yielded 23, 21, 19, 21, 23, and 19 species each, representing a total of 36 mold species. Of these, 15 species were represented by only 1 of the approximately 1,500 isolates. The repeated samples consistently recorded the presence of a group of c o m m o n species (15 species found in each of the 6 samples) along with a cluster o f " r a r e r " species. Thus, despite the inability of the standard sample to detect all species present (an achievement that seems highly unlikely even with samples of much greater size), the sampling does provide consistent community and species number estimates.

Calculations The terms immigration, extinction, turnover, and equilibrium for phylloplane communities required specific, operational definitions (below) because of the destructive sampling procedure. In all cases the individual leaf is viewed as the island; since the same leaf island cannot be tracked through time, methods had to be developed for comparing leaves sequentially and to account for variability among leaves within a sample and also between samples. Our definitions are an attempt to retain the spirit of those coined by MacArthur and Wilson [20]. Also, consistent with the theory, general patterns are more important than "absolute" numbers. Except where noted otherwise, data from the small and large leaves were combined. This protocol is validated in the Results section. To calculate immigration and extinction, leaves were considered individually; every leaf in a given sample on a given date was compared separately with every leaf in the immediately succeeding sample. Since six leaves were sampled at each date for the natural field experiment, there were 36 or 6 x 6 possible leaf pairs between adjacent samples. This resulted in 36 immigration and 36 extinction values calculated for each pair of adjacent samples. Average immigration and extinction values for all permutations between adjacent samples were then calculated as a function of the sampling time of the first sample in the pair of samples being considered. The rationale for this was to express events for any comparison in terms of the initial state of the island.

282

J . H . Andrews et al.

These calculations can be illustrated with a simple example using a hypothetical data set (Table 1). Assume there are three leaves in each of the two succesive time periods and a total o f seven species, A-G. Species A, B, C (only) are present on leaf I at time Tl- Comparing leaf 1 to leaf 4 there are two species, F and G, present on leaf4 but absent on leaf 1. These represent immigrations. The average number of such immigrations for the nine leaf pairs is 1.78. Accordingly, this is the overall estimate of immigration. Again, comparing leaf I to leaf 4 there is one species, C, present on leaf 1 and absent on leaf 4. This represents an extinction. The average across all leaf pairs is 1.44; this is the overall estimate of extinction. Equilibrium was considered a balance between the numbers o f immigration and extinction events, rather than as a balance between rates sensu MacArthur and Wilson [20]. Rate calculations are highly influenced by the length of time between samples, making it difficult to compare rates or interpret results from samples taken at varying intervals. The short microbial generation times in relation to the sample interval may result in an inability to measure real changes in immigration, extinction, or turnover rates. Three approaches to quantifying equilibrium were taken. Each tested for the presence of a "steadystate" in numbers of species per leaf. All approaches involved successive examination of samples starting with the last sample and moving backwards through time. The significance level taken for all analyses was a ~ 0.05.

Approach One Ho: M(xl) = M(xi+, + x . . . . . .

+ xi+z).

Equilibrium is concluded to exist at the time of sample x~ if the mean (M) number o f species per leaf in that sample is not significantly different from the overall mean number o f species per leaf in all later samples. Student's two (independent) sample t test was used to evaluate significance [36]. Thus, the first step is to compare the means o f the last two samples. If these are not significantly different, the mean o f the next preceding sample is compared with the mean of the last two samples. This procedure is continued backwards through time utilizing the next preceding sample until the null hypothesis i5 rejected, Equilibrium i~ saiO not to exigt at and before the point of first rejection.

Approach Two Ho: M(xi) = M(xi+,) Equilibrium is concluded to exist at the time of sample x~ if the mean number of species per leaf in that sample is not different from that in the immediately succeeding sample xi + ~. The two (independent) sample t test is used. As in approach one, the test begins with the last two samples and moves backward in time but considers only one pair at a time (e.g., I 1-10, 10-9, 9-8) as long as the null hypothesis is not rejected. The procedure terminates at the first rejection.

Approach Three Ho: b~=O Equilibrium is concluded to exist at the time of a given sample if the slope (bl) of the regression line including data from that sample and from all later samples is not significantly different from zero. A regression line Y = al + biX + e is hypothesized, where X is time (sample number), Y is the number of species on a leaf, a is the Y-intercept, and e is error. The F test [36] was used in evaluating significance o f the slope for samples over increasing increments o f time (e.g., samples I l and 10; samples 11, 10, and 9; samples 11, 10, 9, and 8, etc.). Again, testing was continued until the first rejection was found.

Fungi, Leaves, and Island Biogeography

283

The three approaches are sensitive to somewhat different attributes of equilibrium and, as such, are complementary. The t tests, especially those of approach two, are more sensitive to short-term changes, whereas the regression method focuses on long-term trends. Equilibrium was not declared unless supported by all three tests. These analyses were performed only for the filamentous fungi and total fungi data; the small range in the number of yeast species per leaf over time (either 8 or 9 species on every fully formed leaf) made such analyses inappropriate. Turnover (T) was calculated using the immigration (I) and extinction (E) values obtained by the method described above and was defined [11] as T = (I + E)/2. A key premise of the sampling was that fungal communities were more alike on the same date than on different dates, i.e., that variability in species composition among leaves within a sample was less than variability among leaves between distinct samples. Accordingly, "within" and "between" sample variability were defined and calculated as illustrated in the bottom portion of Table 1. For leaves Lz and L2 of sample Tt, there are three species--C, D, and E--that are found on one but not both leaves. This number, 3, is our measure of variability between leaves L~ and Lz. Similarly, values can be obtained for the other permutations L, ~ L3 and L2 ~ L3 which, when averaged across the three leaf pairs, give a value of 2.00. This is our within-sample variability for sample T~. The computation for sample T2 is analogous. For the between-sample variability, we consider the nine leaf pairs across the two samples. With respect to leaves L~ and L4, there are three species--C, F, and G--that are found on one but not both of the leaves. Averaging the nine leaf pair values results in a value of 3.22, our estimate of between-sample variability. (We note that our definition of between-sample variability leads to a value that is exactly twice the turnover rate.)

Results F o r t h e n a t u r a l field s y s t e m , t h e o b s e r v a t i o n s d e p i c t e d are f r o m the s e c o n d s e a s o n . T h e r e s u l t s were c o n s i s t e n t f r o m y e a r to year, b u t o u r d a t a base was larger a n d the m e t h o d o l o g y w a s i m p r o v e d for the s e c o n d year. M o s t o f the e v i d e n c e p r e s e n t e d here is f r o m the n a t u r a l field s y s t e m . T h e analyses e x c l u d e d s a m p l e 8 ( A u g u s t 11, 1982), a n o u t l i e r a p p a r e n t l y e x p l i c a b l e b y u n u s u a l air s p o r a c o n d i t i o n s o r i g i n a t i n g c o i n c i d e n t a l l y f r o m the h a r v e s t i n g o f a n a d j a c e n t field o f oats o n t h a t date. T h i s r e s u l t e d i n a n e x c e s s i v e l y large n u m b e r o f rare species o f m o l d s , i n c l u d i n g m a n y c o m m o n l y i s o l a t e d f r o m cereals a n d soil. D a t a f r o m the i n situ d i s i n f e s t a t i o n m o d e l h a v e b e e n d e t a i l e d e l s e w h e r e [ 16]. A s n o t e d earlier, d a t a f r o m t h e m o d e l (seedling) s y s t e m were difficult to a n a l y z e ; we d o n o t p r e s e n t these r e s u l t s here. W e p r e s e n t e v i d e n c e for v a l i d a t i o n o f t h e m e t h o d o l o g y first, f o l l o w e d b y t h e i s l a n d b i o g e o g r a p h i c a n a l y s e s .

Methodology P l o t s o f species c o m p o s i t i o n a l differences ( i n c o n g r u e n c e o r v a r i a b i l i t y ) w i t h i n a n d b e t w e e n s a m p l e s for t h e n a t u r a l field e x p e r i m e n t (Fig. 1) a n d also for the d i s i n f e s t e d l e a f s y s t e m s u p p o r t e d the a s s u m p t i o n t h a t the f u n g a l c o m m u n i t i e s were m o r e alike o n t h e s a m e d a t e t h a n o n different dates. B e c a u s e t h e i n d i v i d u a l o b s e r v a t i o n s g o i n g i n t o t h e c a l c u l a t i o n s o f e a c h m e a n are n o t i n d e p e n d e n t , it is difficult to q u a n t i f y the difference b e t w e e n t h e c u r v e s i n Fig. 1. I m p o r t a n t l y , h o w e v e r , at e v e r y s a m p l i n g d a t e for the surface d i s i n f e s t e d s y s t e m a n d for e v e r y d a t e i n b o t h y e a r s o f t h e n a t u r a l field s y s t e m , t h e w i t h i n - s a m p l e v a r i a b i l i t y was a l w a y s less t h a n b e t w e e n - s a m p l e v a r i a b i l i t y .

284

J . H . Andrews et al. 24 22-

Fig. 1. Species compositional differences (incongruence) within (R (2) and between (A &) adjacent samples as illustrated by data for the natural field system, 1982. See text and Table 1 for method of calculation and reason for absence of error bars. Between-sample values are plotted vs the time of the first sample in each set of comparisons.

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Area Relationships and Island Biogeographic Analyses A species-area association was not detected in any o f the e x p e r i m e n t a l systems (Fig. 2 d e m o n s t r a t e s this for the natural system). T h e s e results justify pooling data f r o m the large a n d small leaf categories. T h e square o f the correlation coefficient between species n u m b e r a n d leaf area for all leaves for the natural field system was r 2 = 0.151 (P > 0.10). T h e r e was a significant individuals-area effect (Fig. 3). T h e correlation between n u m b e r o f individuals and leaf area for all leaves was r2 = 0.597 (P
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Fungi, Leaves, and Island Biogeography

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filamentous fungal individuals; r -~ = 0.394; P < 0.01 for the yeasts). Thus, the yeasts rather than the molds accounted for the significance o f the relationship. The time course o f colonization (Fig. 5) using all three definitions o f equilibrium show that stabilization (P > 0.05) in filamentous fungal and in total species counts was reached by the fifth and sixth samples, respectively (week 8, June 16; week 10, June 30). Thus, species counts did not change significantly from m i d - J u n e until the conclusion o f sampling. Further analysis showed that species o f filamentous fungi varied m o r e widely on the large than the small leaves, possibly because the f o r m e r were more variable in area and age than the latter. The m o l d c o m m u n i t y developing on the disinfested leaves reached an equilibrium o f a b o u t 12 species within 3 weeks o f hydrogen peroxide treatment. The n u m b e r o f yeast species was relatively uniform across dates. I m m i g r a t i o n and extinction expressed as a function o f species n u m b e r gave plots (Fig. 6) strikingly similar to the theoretical rate curves o f M a c A r t h u r and Wilson [20]. The equilibrium n u m b e r is a b o u t 24 species. O f these, 7 key

286

J . H . Andrews et al, Fig. 5. Number of fungal species (11-----~, including filamentous fungi (A A) and yeasts ( ~ - - - - O ) , isolated over time from apple leaves, natural field system, 1982. Time zero (sample 1) represents data from closed buds sampled on April 21. Points statistically at equilibrium by all three tests (P > 0.05) for filamentous fungi and total fungi connected by solid lines. Yeast data could not be analyzed statistically for reasons given in text. Data points at 16 weeks are outliers (see text). Vertical bars represent standard error.

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organisms were found in all 3 systems on virtually every leaf sampled, regardless of date: Aureobasidium pullulans, Alternaria alternata, Cladosporium clado-

sporioides, C. herbarum, Epicoccum purpurascens, Microsphaeropsis olivacea, and M. arundinis. Among the yeasts, which were examined in detail only for the natural field experiment, Cryptoccus albidus, C. laurentiL Rhodotorula glutinis, and R. rubra were present consistently. More than 100 additional fungal species were isolated, including numerous species isolated only once during the entire sampling sequence. Importantly, turnover did occur at equilibrium (Fig. 7), confirming that the observed stabilization was dynamic and not static. Expressed in terms of actual events, turnover in total fungi was about 8 of 24 species (33%) at equilibrium.

Fungi, Leaves, and Island Biogeography

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5-

Fig. 7. Turnover of total

4-

fungal species on apple leaves, prior to (-- -- --) and at ( ) equilibrium. Natural field system, 1982 (sample 8 excluded).

32I0

Time (weeks)

24

Discussion

T h e essence o f this work is that the species d y n a m i c s o f the phylloplane fungal c o m m u n i t y can be partially described by an island model. T w o m a j o r postulates o f the theory, equilibrium and turnover, were sustained as was a mechanistic basis o f the theory, namely, that equilibrium results from balanced immigration and extinction. Rarely have these events been d o c u m e n t e d [14]. One key pred i c t i o n - t h a t larger islands should have m o r e species--was not met. Thus, the theory is at best an imperfect model for our findings. Absence o f an area effect may be an artifact in that an association indeed exists but went undetected, or it m a y be real. In the f o r m e r instance, because o f the phenology o f apple leaf d e v e l o p m e n t , smaller spur leaves m a y actually be older than the larger, in which case they might be expected to carry a relatively higher microbial burden, possibly negating an area effect in the natural field experiment. Although this explanation could not apply for the in situ disinfestation, in which all leaves were treated concurrently, area differences a m o n g those leaves (about threefold), or a m o n g leaves in the seedling experiment (about sixfold), m a y not have been sufficient to d e m o n s t r a t e an area effect. It is well known from macroecology that a particular range o f island sizes (and associated habitat diversity) m a y be insufficient to show a speciesarea association [39] or that small, unstable islands m a y have area-independent extinction rates [20]. On the other hand, area m a y not be limiting for leaf microbes as evidenced by the fact that only a small proportion (ca 1-10%) o f temperate leaf surfaces is colonized [21 ]. W h y this is so is open to question, but it m a y reflect physical stress, including removal o f propagules by wind and rain, such that abiotic factors d o m i n a t e before any area limitation (competition) can be realized. Significantly, a species equilibrium was reached. N o censusing m e t h o d in macro- or microbial ecology is complete. The epiphytic fungal c o m m u n i t y is apparently c o m p o s e d o f relatively few a b u n d a n t species and m a n y o f m o d e r a t e or rare occurrence, not unlike m a n y c o m m u n i t i e s

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ofmacroorganisms [24]. Our sampling protocol has undoubtedly missed species ("cryptoturnover") [18], perhaps more so than the nondestructive methods used for macroorganisms. Nevertheless, between-sample always exceeded within-sample variability. Additionally, no attempt was made to discriminate transient from resident species ("pseudoturnover") [18]. These terms, defined arbitrarily, are controversial in both macro- [18] and microbial [26] ecology. A strong precedent for including both types of species in censuses is the work by Simberloffon recolonization of the Florida Keys, in which 134 of 236 (57%) colonist species were extinguished within two sampling periods [30]. In our study, a biological rationale is that transients, even if quiescent as spores, may have a marked impact on the evolving community, for example, by serving as a reservoir of nutrients to be tapped by resident competitors [7], or by releasing antibiotics [ 10]. A potential criticism of island studies is that they may demonstrate nothing more than a normal pattern of movement within populations [18], in other words the scale of the system is inadequate to depict biologically meaningful immigration or extinction. If too small, few colonization events and rapid turnover would be expected. (The analogy used is that of a robin becoming "extinct" each time it leaves a particular apple tree) [34]. If the system is too large, it would be essentially static. A priori the leaf is a reasonable biological island since it presents a habitat of suitable dimensions, insularity, and diversity for microbes. Finally, even for situations in which intra-population movement is presumably considerable [23], the island concept can be useful. Importantly, the trends in aggregate across two experimental systems, two orchard sites, and three seasons were consistent. Despite some differences from the conventional island model, fungal species dynamics in this insular microbial community follow that depicted for macroorganisms on oceanic islands. Does this suggest some universal basis for community assembly? Testing of our results for other phylloplane microflora, and for microbes in other patchy habitats, await further work. Acknowledgments. This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; by NSF Grant DEB 8110199 to J. H. Andrews; and by an NSF graduate fellowship to L. L. Kinkel. We thank S. Hirano, J. Handelsman, and C. Smith for reviewing the manuscript; Michael Huebschmann and Peter Crump for help with the data processing; and J. Boston and G. Carlson for laboratory assistance.

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