Toward a sampling strategy for vesicular-arbuscular mycorrhizas

[ 353 ] Trans. Br. mycol. Soc. 87 (3), 353-358 (1986)

Printed in Great Britain

TOWARD A SAMPLING STRATEGY FOR VESICULAR-ARBUSCULAR MYCORRHIZAS By LEONARD L. TEWS Department of Biology and Microbiology, University of Wisconsin, Oshkosh, Wisconsin AND

54901

R. E. KOSKE

Department of Botany, University of Rhode Island, Kingston, Rhode Island

02881

Nine species of VAM fungi were recovered from a barrier dune system in Rhode Island. It was determined, by using species increment curves, that 9-30 samples were necessary to find all nine species. In a corroborative study of a Virginia dune, 26-30 samples were necessary to recover all 18 species at that site. Using cumulative averages, it was determined in three dune studies that 30 samples would usually yield a mean spore density of ±20 %. Standardized estimation formulae indicated that the numbers of samples required were usually outside the range of practical sampling feasibility . Although natural communities have been extensively studied for the presence and distribution of vesicular-arbuscular mycorrhizal (VAM) fungi, there have been few systematic investigations regarding the validity of the sampling methods used . In maritime sand dunes, a variety of sampling methods have been employed (G iovannetti & Nicolson, 1983; Koske, 1975; Koske & Holvorson, 1981; Nicolson, 1960), mostly following the methods used in other VAM studies, but there is not yet a clear indication regarding how many samples are appropriate for determining the size and composition of a VAM community. Methods used to describe the community structure of hyphomycetes in soil (Gochenaur , 1978, 1984) have not been applied to the VAM fungi. METHODS AND MA TERIALS

The dune system of Moonstone Beach, Rhode Island was intensively investigated and the data compared to those of a previous study at the same site and a survey of VAM fungi of the dunes of Assateague Island, Virginia. Moonstone Beach, a 14 ha barrier dune typical of those of the northeastern coast of the U . S. (Olsen & Grant, 1973), was selected as the major site because it had been sampled extensively since 1978. The dune is bordered on the south by Block Island Sound and on the north by a lagoon that varies in salinity from o to 35 %. Three studies were carried out. The major study - M oonstone Beach, sampled Mar. 198]. Samples (30) were collected

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from the root zones of dune plants at collection

points which were determined previously by using a stratified random sampling method. Most samples (28) were taken from the root zone of the dominant plant of the area, American beach grass, Ammophila breuiligulata Fern. One was taken from a stand of bayberry, Myrica pennsylvanica Loisel, and another from the pond edge in a stand of Phragmites communis Trin. Since relationships of VAM fungi to higher plants or other species of VAM fungi had already been investigated (Koske & Halvorson, 1981), no such correlations were attempted here . Approximately 2 I of soil was removed from each collection point and placed in a plastic bag. Samples were stored at 10 °C until examined for VAM fungi within four months of collection. Each soil sample was subsampled by taking about ten portions of soil from different areas ofthe bag to fill a 25 ml vial. Spores were recovered by wet-sieving/ filtration, and were identified and counted as described previously (Koske & Walker, 1984). Subsampling was repeated two more times. A species list was then compiled for the sample (= bag), and the average spore density of each VAM species per bag was calculated by combining the data from the three subsamples. Moonstone B each, sampled I Apr. 1982. A 10 X 10 m plot in mid-dune with a fairly uniform, thick cover of A . breviligulata was selected for intensive random sampling. At 31 points, the top 10 em of soil was removed and a 10 ml sample was placed in a plastic bag and returned to the laboratory for spore recovery . To enumerate [he spores of Gigaspora species present in soil samples collected

Sampling strategy for V AM fungi

354

First five samples within 20%

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A cumulative average density curve for Gigaspora gigantea in the Assateague Island Study.

with syringes, 100 soil samples were wet-sieved through No. 40 and 60 mesh sieves. Spores retained on the No. 60 mesh seive were identified and counted. Assateague Island, sampled 15 Mar. 1982. Soil samples (30) were collected from the root zones of A. breviligulata and Solidago sempertnrens L., the dominant plant species growing at the dune crest and mid-dune near the southern end of the island. Samples were taken from representative areas but were not truly random. Spores were harvested by sieving/filtration. Spore density for use in the cumulative density curves (see below) was expressed as spores/voo g soil (d. w). For estimation of sample number by formula, the density figure was the actual density of spores in the three 25 ml samples (= 111 g). Spore densities of Glomus and Acaulospora species were not determined in either study of Moonstone, although frequency of recovery was. Comparison with previous studies in sand dunes indicated that the major objective of the study could be achieved without counting the spores of every species. Spore densities and frequencies of Glomus and Acaulospora were determined for the Assateague samples, and a portion of that information is presented in this publication. Determination of minimum number of samples Spore density. To determine the minimum number of samples needed to estimate spore density, two approaches were used: 1. Cumulative average density curves: Curves were prepared by selecting the spore density of individual samples in a random sequence and

calculating the average density when each additional sample is included. This approach produces the type of configuration shown in Fig. 1. The minimum number of samples required to achieve an acceptable level of accuracy was determined by noting how many samples had to be averaged before the cumulative average curve levelled out. Two values were taken from the curve: (a) the minimum number of samples that results in 5 sequential cumulative averages being within ± 10 % of the mean of all 30 samples, (b) for those being within 20 % of the mean. In both cases, the 5 sequential samples are used to estimate the levelling-off portion of the curve. A minimum of eight curves was drawn for each VAM species from each of the three studies. A random-number computer program was used to randomize the sequence of sample data values. At the point on each cumulative density curve where the curve levelled out, i.e, the fifth consecutive sample, the standard error of the mean was calculated. This value was expressed as a percentage of the mean. 2. Estimation by formula: A number of mathematical formulae for estimating the number of samples required to achieve a certain degree of accuracy are available. The one selected for this study is that suggested for data that are not normally distributed (Southwood, 1978) (as VAM spore densities are not). Spore count data better fit the logarithmic or the negative binomial distributions (Anderson et al., 1983; St John & Keske, 1984; Walker, Mizo & McNabb, 1982). The formula is: 1

1

-+-

N=X

k E2 '

L. L. Tews and R. E. Koske where i = mean of ten preliminary samples, N = number of samples required, k = a measure of dispersion, E = desired accuracy: (E = 0'1 indicates standard error will be 10 % of the mean). i and k values for each VAM species and for each of the three study areas were calculated for the first ten, randomly selected at least eight times so that a representative range of i and k values could be obtained. The value k was estimated by one of two methods (Southwood, 1978): (1)

(N) = k log (1log --;;;: +~) ,

where N = total no. of samples in the preliminary sampling (10 in this case), no = no. of samples from which the organism(s) were absent, i = mean density of first 10 samples (note: density = spores/u i i g for studies no. 1, no. 3, spores/15 g, for study no. 2). (2)

e;

k=-S2_,

-x

where i = mean of first 10 samples, S2 = variance of first 10 samples. Method one was used whenever possible, but the formula may not yield a solution if frequency and density are very low. Species richness. To determine the minimum number of samples needed to estimate species richness at a site, species increment curves (Gochenaur, 1978) were drawn. Data from samples were selected in random order to draw curves. Eight such increment curves, each with a different random sequence of samples were plotted for the major study site and for the Assateague site. RESULTS

Nine species of V AM fungi were isolated during the major survey (Table 1). Based on the cumulative density curves, the minimum number

355

of samples required to estimate mean spore density within ± 10% (of 30 samples) ranged from 14 to greater than 30, and depended greatly upon the sequence in which the samples were processed (Table 2). For an accuracy of the mean within ±20%, eight to greater than 30 samples were required, also depending upon the sequence of sample processing. Examination of 29 samples ensured the ± 20 % accuracy for all species/sites examined except for G. gigantea from study 1. By the 30th consecutive sample (in one of the 8 sequences of samples processing), the cumulative mean of this species had levelled off to within ± 20 % of the mean for only 4 consecutive samples. The seven other sequences levelled off before the 30th sample. When the minimum number of samples was estimated by the formula using a standard error of the mean equal to 10% of the mean (an accuracy often desired for biological studies), the value ranged from 46 to 2182 samples. Depending upon the 10 samples used for the calculation, a great range of estimates of sample number could be obtained. For example, one grouping of 10 samples from Moonstone Beach predicted that 260 samples would give a standard error of the mean of 10 % of the mean for Gigaspora calospora. A second grouping of 10 samples estimated the required sample number for the same level of accuracy as 2182 samples. Only when the formula method was used with a standard error of 50 % of the mean as an acceptable level of accuracy was the number of samples to be analyzed feasible. Even then, for species occuring at low frequencies or densities, the minimum number of samples exceeded 40. The actual standard errors of the mean (as a percent of the mean) calculated from the cumulative density curves were in the range of 15-62 % for curves that levelled out within ± 10% of the mean and 17-87 % for curves that levelled out within ± 20 % of the mean. Findings from studies 2 and 3 corroborated study 1, and only a few examples are

Table 1. V A fungi isolated from Moonstone Beach in order of their respective frequencies" Species of VA fungi

Percent frequency

Acaulospora scrobiculata Trappe Gigaspora gigantea (Nicol. & Gerd.) Gerd, & Trappe G. persica Koske & Walker ined. Glomus fasciculatum (Thaxter) Gerd. & Trappe G. pustulatum (Koske & Friese) Walker & Dalpe Gigaspora erythropa Koske & Walker

77 67 5°

G. calospora (Nicol. & Gerd.) Gerd. & Trappe

20

Glomus aggregatum Schenck & Smith G. occultum Walker

17

33 3° 30

3

* No of samples from which a fungus is isolated/total no. of samples x 100.

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Table 2. Comparison of minimum number of samples needed to estimate spore density using density curves and formula Av. density curves

S.E.M.

Study site 1. Moonstone Beach

2. Moonstone Beach 3. Assateague Island

Species G, gigantea G, persica G. erythropa G. calospora G, gigantea G, persica G. gigantea G, oerrucosa

Av. sp, density"

(% of mean)

Freq. (%)b

7'3 2'3 1'2 0'4 66'7 8'3

27'7 3°'7 36'9 37'1 15'5 3 1'6

77 53 33 23 97 33 73 83 5° 80

±10% 17 to > 30 c d 14 to> 30 21 to > 30 26 to >30 16 to> 30 22 to >30 20 to >30 17 to >30 24 to >30 16 to> 30

S.E.M.

(%)

27-37e 27-46 37-49 37-40 15-20 3 1-40

±20% 16-730 10-29 20-29 15-27 12-25 11-24

Calc. by formula to achieve S.E.M. (% mean) (%)

28-41 e 32-54 38-51 4 1-55

10% 124- 3141 18 to > 23 325-1174 260-2182

17-27 33-40 25-40 19-35 38-87 33-54

46--90 160-1105 108-419 89-342 257-5 64 58-57 8

S.E.M.

25'S 26-34 9-25 18-24 17'4 9-25 5"3 G,fulgida 11'4 2'9 36-62 19-27 A. scrobiculata 8-28 22'9 24-47 3°'7 a Sporea/ too g soil (d.w.) calculated for the 30 samples. b Percent or samples from which a species was recovered. o Figures are the lowest and highest no. of samples that needed to be taken in eight random sequences of sample processing that resulted in at the mean ± 10% of the mean. d Only 30 samples were analyzed. If the curve had not levelled off such that the last five consecutive samples did not result in means within or 30 samples, > 30 is indicated. e Range of standard errors of the mean (expressed as percent of mean) achieved in levelling off of eight random sequence curves. 1 Range of required sample numbers to achieve a S.E.M. equal to 10 % of the mean. Based on eight random draws of ten samples each. 5'4

20% 3 1--'79 46-181 81-293 65-546 14-22 40-276 27-105 22-86 64-141 15-144

50% 5- 13 7-29 13-47 10-87 2-4 6-44 4- 17 4-14 10-23 2-23

a levelled-off curve ± 10 % of the mean

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L. L. Tews and R. E. Koske presented (T able 2). Fewest samples were required to accurately estimate the spore density of G. gigantea in study 2, where it occurred with high frequency (97 %) and average density (66' 7 spores/coo g) and a low standard error of the mean. For determining species richness by random selection of samples to draw increment curves, it was found that a minimum of 9-30 samples had to be examined to recover all nine species from the major study. In the Assateague Island survey, a minimum of 2~-"30 samples had to be examined to recover all 18 species . Ninety percent of the species (16) was found after 15-18 samples.

DISCUSSION

It is axiomatic that the more samples are taken, the greater the number of species that can be isolated from a natural community. Nevertheless, how does an investigator decide the point at which more effort will yield only minimally more information? Large numbers of samples suggested by mathematical formulae are often useless to the practicing microbial ecologist (Table 2). Currently microbiologists are searching for guidelines to sampling numbers based upon a more pragmatic approach. For example, Gochenauer (1984) found, when sampling the penicillia of the soils of an oak-birch forest, that a single month's study of 30 samples yielded more than the broad outlines of the structure of the penicillial flora. Data from the present study of two sites (M oonstone Beach and Assateague Island) indicate that 30 samples may be adequate to estimate species richness. While a single sampling cannot give an indication of yearly fluctuations in spore density, species richness can probably be determined fairly accurately (Walker et al., 1982 ). The dunes at Moonstone Beach have been sampled intensively since 1978 for VAM fungi. Yet 30 samples in a single month yielded 9 species or nearly all of the VAM fungi ever isolated from this dune. In recent intensive study of three root zones of the beach grass of Moonstone (Friese, 1984), including 144 samples, viable spores of only one additional VAM species, Gigaspora pellucida, were isolated. When numbers of species of VAM fungi in this study are compared to those of other more intensive studies in Iowa (Walker et al., 1982), they are similar. A total of ten species was isolated from a dry, sandy upper terrace along the Des Moines River and 12 species from another site, an old meadow with Nordaway silt loam . In another study of the VAM fungi along a single transect of

357

Moonstone Beach, six species were isolated (Koske

et al., 1981). Our results regarding spore density are not as clear as those for species richness. The non-random distribution of spores in soil is the underlying cause for the large variation in spore density between samples. Such variation demands that a large number of samples be counted to achieve low standard errors of the mean . Great variation between samples has been noted in other reports, e.g . Anderson et al. (1983), Koske et al. (1981), McGraw & Hendrix (1984) and Walker et al. (1982). Anderson et al. (1983) investigated the effects of sampling intensity and plot size in an effort to determine the minimum size for describing both the plant community and the V AM community in a prairie site. They were able to achieve a standard error of the mean of less than 10 % (of the mean ) only when they intensively sampled a 0'5 x 0'5 m plot, combining 75 corings into 25 samples for spore counts. Less intensive sampling or sampling of larger areas resulted in standard errors of the mean similar to those recorded in the present study. Because estimation of the required sample number is so dependent upon the range and variation of the first ten samples used to calculate the values of k and x for use in the formula, it is difficult to recommend what values of E (Standard error of mean or percent of mean) should be used in the formula. Clearly it would depend upon the aim of the study and the resources of the investigator. The authors thank Carl Friese and Keith Killingbeck for helpful comments; Don Tiller, Refuge Manager, Trustom Pond Wildlife Refuge, for his permission and cooperation; and Pamela Fielden and Peter Marx for their help in collecting. REFERENCES

ANDERSON, R. C., LIBERTA, A. E., DICKMAN, L. A. & KATZ, A. J. (1983). Spatial variation in vesiculararbuscular mycorrhiza spore density. Bulletin of the Torrey Botanical Club

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519-525.

FRIESE, C. F. (1984). The distribution and dispersal of sporesof VAM mycorrhizal fungi in a sand dune. MS Thesis, Universityof Rhode Island, Kingston. GIOVANNETTI, M. & NICOLSON, T . H . (1983). Vesiculararbuscular mycorrhizas in Italian sand dunes. Transactions of the British Mycological Society 80, 552-557.

GOCHENAUR, S. E. (1978). Fungi of a Long Island oak-beech forest. I. Community organization and seasonable occurence of the opportunisticdecomposers of the A horizon. Mycologia 70, 975--994. GoCHENAUR, S. E . (1984). Fungi of a Long Island oak-birchforest II. Populationdynamics and hydrolase patterns for the soilpenicillia. Mycologia 76, 218-231.

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Sampling strategy for V AM fungi

KOSKE, R. E. (1975). Endogone spores in Australian sand dunes. Canadian Journal of Botany 53, 668-672. KOSKE, R. E. & HALVORSON, W. L. (1981). Ecological studies of vesicular-arbuscular mycorrhizae in a barrier sand dune. Canadian Journal of Botany 59,1413-1422. KOSKE, R. E . & WALKER, c. (1984). Gigaspora erythropa, a new species forming arbuscular mycorrhizae. Mycologia 76, 250-255. MCGRAW, A. C. & HENDRIX, J . W. (1984). Host and soil fumigation effects on spore population densities of species of endogonaceous mycorrhizal fungi. Mycologia 76, 122-131, MILLER, O. K. (1983). Eetomycorrhizae in the agaricales and gasteromycetes. Canadian Journal of Botany 61, 909-9 16. NICOLSON, T. H . (1960). Mycorrhiza in the gramineae. II. Development in different habitats, particularly sand

dunes. Transa ctions of the British My cological S ociety 43, 132-145. OLSEN, S. B. & GRANT, M. J. (1973). Rhode Island's barrier beaches. Maritime Technical Rep ort no. 4, vol. 1, Coastal Resources Center, University Rhode Island, Kingston. ST JOHN, T. V. & KOSKE, R. E. (1984). Statistical treatment of endogonaceous spore counts. Proceedings of the 6th North American Conference on Mycorrhizae, 15-29 June, 1984, Bend, Oregon. SOUTHWOOD, T. R. E . (1978). Ecological Methods with Particular Reference to the Study of Insect Populations, znd Edn. John Wile y and Sons : New York . WALKER, C., MIZE, C. W. & McNABB, H . S. jr (1982). Populations of endogonaceous fung i at two locations in central Iowa. Canadian Journal of Botany 60 , 2518-25 29.

(R eceived for publication 8 November 1985)