Distribution of three fungi colonising fallen Pinus sylvestris needles along altitudinal transects

Mycol. Res. 104 (9) : 1133–1138 (September 2000). Printed in the United Kingdom.

1133

Distribution of three fungi colonising fallen Pinus sylvestris needles along altitudinal transects

A. van MAANEN1, D. DEBOUZIE2 and F. GOURBIERE1 " Laboratoire d’Ecologie Microbienne du Sol, UMR CNRS 5557, UniversiteT Claude Bernard Lyon I. Bat 741, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France. # Laboratoire de BiomeT trie, GeT neT tique et Biologie des Populations, UMR CNRS 5558, UniversiteT Claude Bernard Lyon I. Bat 741, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France. E-mail : frgourbi!biomserv.univIlyon1.fr Received 25 June 1999 ; accepted 1 November 1999.

This work tested the hypothesis that the abundance of fungal species on coniferous needles is correlated to climatic factors at a regional scale. The proportions of Pinus sylvestris needles colonised by Lophodermium pinastri, Cyclaneusma minus and Verticicladium trifidum were examined along two altitudinal transects in 2 successive years. L. pinastri and C. minus colonisation increased with altitude, whereas that of V. trifidum decreased. The data are discussed in relation to the importance of climatic controls on species distributions.

INTRODUCTION Climatic factors and resource availability are important factors that control the geographical distribution and abundance of fungi (Arnolds 1997). Distributions of microfungi are less well characterised than those of macrofungi and, among microfungi, the distributions of saprotrophs are less well known than those of parasitic species (Arnolds 1997). Fungi which decompose needles of conifers are ideal candidates for studies of the role of climate in fungal biogeography. Firstly, many observed species are specific to coniferous needles and often restricted to one or few genera of conifers. Consequently, their potential distribution follows that of their resources and field sampling can be restricted to these specific resources. Secondly, the large natural geographic distribution of many coniferous species allows fungal species to be observed on the same resource over a large climatic range. Many authors have studied the composition of micromycota developing on coniferous needles in Europe, especially on Pinus species. Some studies are restricted to one site in England (Kendrick & Burges 1962, Kendrick 1963, Hayes 1965, Lehmann & Hudson 1977, Mitchell & Millar 1978, Mitchell, Millar & Minter 1978, Minter 1980) and France (Ponge 1991). Other studies listed fungal species on a larger scale : Czechoslovakia (Minter 1981a), Poland (Kowalski 1988a), Netherlands (Gremmen 1957, 1959, 1960), and Finland (Kujala 1950). Numerous individual species reports are available from mycological and phytopathological literature. Despite the large geographic and climatic ranges covered

by these studies, however, no clear correlation appears between climate and mycota composition, or the distribution of individual species. Most species are distributed widely through the area of resource availability. It can be hypothesised that the influence of climate is greater on abundance than upon presence or absence of fungal species. This paper deals with a simple question : does the abundance of fungal species on the needles of one coniferous species vary according to climatic conditions ? To answer this question, the abundance of three of the most widely distributed fungi which colonise fallen Pinus sylvestris needles were quantified along a regional climatic gradient. Abundance was estimated as the proportion of needles bearing fruiting bodies. Climatic changes were observed along altitudinal transects. Temporal and spatial stability of the observed distributions was tested by monitoring two transects in the same region for 2 successive years. Statistical methods were employed for analysis of the proportions. The species studied, Lophodermium pinastri, Cyclaneusma minus and Verticicladium trifidum (the anamorph of Desmazierella acicola), are specific colonizers of Pinus needles. They are present in most of the European sites studied, and each of them can reach high frequencies of colonisation. They differ in their life histories : the first two are weak parasites colonising living needles as endophytes but fruiting after abscission and litter fall (Kowalski 1988b, Choi & Simpson 1991). V. trifidum is a typical litter fungus colonising needles after litter fall and fruiting after L. pinastri and C. minus (Kendrick & Burges 1962).

Fungi along altitude transects MATERIAL AND METHODS Sites Two altitudinal transects were studied in France, one in the Pilat Natural Park (Loire, c. 45m 25h N, 4m 35h E, alt. from 388–1360 m), and one in Arde' che (c. 45m 08h N, 4m 30h E, alt. 220–1244 m), 50 km away on the eastern edge of the Massif Central. Both transects had an altitudinal gradient of 1000 m over a distance of 50 km. Eleven sampling sites of Pinus sylvestris were identified along each transect, with 1 site per 100 m of altitude. The two transects were sampled in 2 successive years in the summer (June–September 1996 and June–July 1997). The sampling design was : 2 transectsi 11 altitudes (sites)i2 years, giving a total of 44 different samples. According to Kowalski (1988a), young Pinus sylvestris stands (0–15 years) have a different mycota from older ones. All the sampled stands were more than 25 years old to avoid this stand age effect. Rainfall data were obtained from 35 meteorological stations located in the same area. Temperatures were available from 20 stations. As the F1 needles examined in 1996 fell in 1994 (see below), the climatic data were analysed from 1994 to 1997, the period that could have affected the development of the litter microflora. Sampling method Each sample was a 20i20 cm square cut through all the litter layers. The variability in fungal colonisation of needles at each site was established from three samples removed from an area of approx. 1000 m# at each site. One hundred needles of the L layer (light brown needles) and 100 of the F1 layer (entire, dark brown needles) were taken from each sample at the laboratory. For the transect study, the three replicates were obviously pseudoreplications (Dixon & Garrett 1994, Crawley 1996). Indeed, if the unit sampled was the stand, true replications should have been taken from different stands at the same altitude on each transect each year. As a consequence, we pooled the observations of the three replicates to give one compound sample of 300 needles for transect analysis.

1134 needles compared to F1 type needles) and consequently L type needles cannot be used to estimate accurately the final frequency of colonised needles.

Statistical analysis We attempted to find a relationship between the frequency of fungal colonisation and altitude for each species. As the proportions (p) of colonised needles were always between 0 and 1 (0–100 % needles colonised), simple linear regression cannot be used. A logistic relationship with 0 and 1 as asymptotic values is more appropriate, if p exhibits monotonous variations between 0 and 1. This type of logistic relationship between p and a variable x is obtained by logit transformation in the generalised linear model (Crawley 1996) : log [ p\(1kp)] l axjb p l exp [axjb]\(1jexp [axjb]) The relationship between frequency of colonisation and altitude was studied with respect to two qualitative factors (transect and year). The relative contributions of altitude, year and transect were determined by comparing three models : (1) no explanatory variables, model 1 gave the general mean and total deviance (deviance in the generalised linear model is equivalent to variance in classical statistics) ; (2) only altitude as explanatory variable : model 2 gave one regression for each species, data pooled over the 2 years and 2 transects ; and (3) altitude as variable and transect and year as factors (complete model) : model 3 predicted a logistic relationship between colonisation and altitude for each transect i on year j (with parameters aij and bij) : pij l exp [aij xjbij]\(1jexp [aij xjbij])

(1)

Model adequacy was measured by the percentage of deviance explained. Deviance was estimated according to a binomial distribution of the sampling error (Crawley 1996). Significance was tested after correction for overdispersion using the scale parameter method (Crawley 1996). All analysis were performed using GLIM software (NAG 1993).

Mycological observations Species abundances were reported as the proportion of needles bearing fruiting bodies. The needles were examined under a dissecting microscope (i10–i50) for the presence of Verticicladium trifidum conidiophores, Cyclaneusma minus apothecia, Lophodermium pinastri ascomata (and conidiomata of its Leptostroma anamorph). In situ observations showed that needles senesced and fell in September of the third year. L type needles sampled in July of year ‘ n ’ fell in the autumn of year ‘ nk1 ’. F1 type needles sampled at the same date fell in the autumn ‘ nk2 ’. L. pinastri and C. minus have been studied on L type needles, since F1 type needles darken after 1 year in the litter, which makes their observation difficult. V. trifidum was studied on the F1 type needles. Indeed, although L type needles were colonised by V. trifidum, its fruiting was not achieved at the sampling dates (only 0–20 % of colonised

RESULTS Intrasite homogeneity Before pooling the three samples of each site, their homogeneity was tested using the χ# test independently for each species. Only 74 of the 132 triplicates were homogeneous (P
0n05). Heterogeneity was maximal for V. trifidum (homogeneous triplicates in only 34 % of samples, P
0n05), while 72 % of the L. pinastri and 61 % of the C. minus triplicates were homogenous (P
0n05). These values suggest that there were litter patches having statistically different low and high colonisations by a species. In this case, pooling triplicates gave more valuable estimations of the stand population than a single sample of 300 needles (Dixon & Garrett 1994, Garson & Moser 1995).

A. Van Maanen, D. Debouzie and F. Gourbiere

1135

Table 1. Mean frequencies of needles colonised by Verticicladium trifidum, Cyclaneusma minus and Lophodermium pinastri by year and transect. Mean altitudes of the transects : Arde' che 742 m, Pilat 848 m. Only the colonisation by C. minus differs significantly between transects (see text).

Arde' che

Mean

47n8 53n4 50n6

60n1 40n0 50n0

53n9 46n7 50n3

30n2 34n4 32n3

41n7 45n2 43n4

36n0 39n8 37n9

19n1 27n6 23n3

20n0 22n7 21n4

19n5 23n2 22n4

Ardèche

100

100

90

90

80

80

70

70

60

60

50

50

40

40

30

30

20

20

10

10

0

0

100

100

90

90

80

80

70

70

60

60

50

50

40

40

30

30

20

20

10

10

0

0

100

90

90

80

80

70

70

60

60

50

50

40

40

30

30

20

20

10

10 0

0 400

600

800 1000 1200 1400

Altitude (m)

L

L

0·4

C C

0·2

0·2

Pilat 1997 0·8

V

Ardèche 1997 L

0·8

V

L

0·6

0·4

0·4

C

C

0·2

200

0·2

400

600

800 1000 1200 1400

200

400

Altitude (m)

600

800 1000 1200 1400

Altitude (m)

Fig. 2. Use of maximal logistic model (model 3) to describe distribution of the three species with altitude along each transect over the two successive years. V, Verticicladium trifidum ; C, Cyclaneusma minus ; L, Lophodermium pinastri.

D.F.

Deviance

V. trifidum

C. minus

L. pinastri

Model 1

43

Total explained

5027 0

2279 0

5226 0

Model 2 (jaltitude)

42

Residual explained

2479 51 %

1562 31 %

1242 76 %

Model 3 (jyears and transects)

36

Residual explained

1936 62 %

958 58 %

968 81 %

Scale parameter l 1.

Verticicladium trifidum 100

V

0·6

Table 2. Deviance explained by three different generalised linear models (see text).

Lophodermium pinastri Colonised needles (%)

0·8

0·6

0·6

Cyclaneusma minus

200

V

0·8

Colonised needles ( p)

Pilat

Pilat

0

Ardèche 1996

0·4

Transects

V. trifidum 1996 1997 Mean C. minus 1996 1997 Mean L. pinastri 1996 1997 Mean

Pilat 1996

0

200

400

600

800 1000 1200 1400

Altitude (m)

Fig. 1. Distribution of Cyclaneusma minus, Lophodermium pinastri and Verticicladium trifidum with altitude (#, 1996 ; $, 1997).

General trends The overall mean frequencies of occurrence of fungi in relation to year of sampling and transect location (Table 1) showed that the three species are important needle colonisers (50 % of

needles colonised by V. trifidum, 38 % by C. minus and 22 % by L. pinastri). V. trifidum frequency decreased markedly with increasing altitude (Fig. 1), from 80–90 % at 400 m to 0–10 % above 1200 m. The species was absent from some of the highest altitude samples. This was observed in 3 transectyears. The decrease is more irregular but remained significant in the Arde' che transect in 1997. In contrast, the colonisation of needles by C. minus and L. pinastri increased with altitude. L. pinastri showed similar patterns of distribution in the 4 transect-years (Fig. 1) : a strong increase in frequency, from 0–10 % at low altitude to 50–70 % of needles colonised above 800–900 m. The distribution of C. minus was less consistently influenced by altitude. Its frequency increased with altitude in Pilat over the 2 years. It exhibited a very irregular increase with altitude in Arde' che in 1996, and no significant correlation with altitude in Arde' che in 1997. Low altitude sites with high V. trifidum colonisation contrasted with high altitude sites having associations of L. pinastri and C. minus. The three species largely coexisted at intermediate altitudes and their frequencies reached similar values around 1000 m (Fig. 2).

Fungi along altitude transects 2000

96 97

2

96 Verticicladium

97

0

97

–2

96

b

97 96

–6

96 Lophodermium

–8 –10

Cyclaneusma

97 96

–4

–0·006 –0·004 –0·002

0

1994 1995 1996 1997

1800 Annual rainfall (mm)

4

1136

1600 1400

400 14 Mean annual temperature (°)

Role of altitude, transect and years

log [ pij\(1kpij)] l aij(xjα)jβ. Then, for x lkα : log [ pij\(1kpij)] l β, whatever the fungus, year, or transect. Consequently, all the species have the same frequency p at the altitude x lkα. In the present study, according to the observed correlation (α lk1010 and β lk0n54, Fig. 3), this situation is predicted at the altitude of 1010 m where the frequency of colonisation of the three fungi has a predicted value of 37 %. This fact is approximatively verified on Fig. 2, especially for Pilat 1997, where the frequency of the three species is the same around 1000 m.

1997

800 600

Fig. 3. Correlation between parameters a and b of the logistic model. b l k1010ak0n54 ; R# l 0n99. Squares, Lophodermium pinastri ; circles, Cyclaneusma minus ; triangles, Verticicladium trifidum. Open symbols, Arde' che ; black symbols, Pilat. 96–97, years.

which is equivalent to

1995

1000

0·002 0·004 0·006 0·008 0·01 a

log [ pij\(1kpij)] l aij xjαaijjβ

1996

1200

97

Altitude alone (model 2) explained 31–76 % of deviance according to fungal species (Table 2). Adding year and transect location, model 3 (Fig. 2) explained from 58 % (V. trifidum and C. minus) to 81 % (L. pinastri) of deviance. After correction for overdispersion, the increases in explained deviance obtained by adding year and transect factors (j5 % for L. pinastri, j11 % for V. trifidum and j27 % for C. minus) were significant only for C. minus. The 2 parameters aij and bij of the logistic regression log [ pij\(1kpij)] l aij xjbij for all species and factors were highly correlated (R# l 0n99) and clearly classified in a hierarchical way (Fig. 3) : (1) with respect to fungal species ; (2) according to transect factor, within species ; and (3) according to year within each transect-species. Regarding that correlation, and more generally assuming that bij l αaijjβ for each fungi (with α and β constant whatever the fungi, transect and year), the logistic regression becomes :

1994

13 12 11 10 9 8 7 6 5

1997 1994 1995 1996

1994 1995 1996 1997 0

200

400

600 800 1000 1200 1400 1600 Altitude (m)

Fig. 4. Climatic characteristics of the region studied. Variations of annual rainfall P (mm) and mean annual temperature T (mC) with altitude (m). Linear regression equations : 1994 : 1995 : 1996 : 1997 : 1994 : 1995 : 1996 : 1997 :

P l 760j0n53 alt P l 685j0n39 alt P l 696j0n54 alt P l 524j0n32 alt T l 14n5k0n0055 alt T l 13n8k0n0055 alt T l 13n0k0n0054 alt T l 14n0k0n0046 alt

R# l 0n72 R# l 0n54 R# l 0n73 R# l 0n59 R# l 0n99 R# l 0n98 R# l 0n99 R# l 0n97

n l 32 n l 34 n l 35 n l 35 n l 15 n l 18 n l 20 n l 20

years (Fig. 4). Linear regression explained 53–71 % of rainfall variance according to the year. Residuals from regressions were not correlated with altitude (R# l 0), but were correlated between years (R# l 0n45–0n58, P 0n0001). This shows that some stable differences occur between stations of similar altitude and suggests the existence of microclimates. The monthly rainfall distributions over the 4 years were synchronous in all stations (data not shown). In all stations rainfall peaked in autumn (September or November) and spring (April or May), and was minimal in late winter, early spring (March), and summer (June to September). The mean temperature decreased in an almost perfect linear manner with increasing altitude (R# l 97–99 %) (Fig. 4). No differences between transects were observed for rainfall or temperature. Between-year variations were similar along the two transects. DISCUSSION Role of altitude and other factors

Climatic data There was a linear increase in rainfall with altitude during the 4 years studied, but there was considerable variation between

The results confirm that regional climatic\altitudinal gradients influence the frequency of occurrence of the three widespread needle-decay fungi studied.

A. Van Maanen, D. Debouzie and F. Gourbiere Altitude explained 76 % and 51 % of deviance for L. pinastri and V. trifidum, and only 31 % for C. minus. Stand characteristics unrelated to altitude (e.g. age, density, site spatial heterogeneity and microclimate) and between-year climate variability appeared more important for C. minus colonisation than for those of the two other species. Comparison with large scale distribution Lophodermium pinastri and Cyclaneusma minus have been extensively studied as pathogens as well as saprobes (Butin 1973, Osorio & Rack 1980, Minter 1981b). L. pinastri is present in all sites studied in Europe and is reported from many European countries (Minter & Millar 1978, Minter 1981b). It can be hypothesized that L. pinastri is present in all Pinus stands in Europe. The present work shows, however, that L. pinastri colonisation exhibits regular quantitative variations in relation to local climatic gradient. C. minus is also widely distributed (Millar & Minter 1980) : Germany, France, Spain, Italy (Butlin, 1973), Czechoslovakia (Minter 1981a), Poland (Kowalski 1988a), Netherlands (Gremmen 1959), UK (Lehmann & Hudson 1977), Denmark, Switzerland (Millar & Minter 1980). It was not, however, reported in some site studies : in Scotland by Mitchell et al. (1978), in England by Hayes (1965) and in France by Ponge (1991). It was also not included in the list of fungi on coniferous hosts in Finland (Kujula 1950) and no report from Scandinavia is known to us (although it was cited as a needle endophyte at the northernmost limit of Pinus sylvestris distribution ; Helander, Sieber & Petrini 1994). It appears from the present study that the presence and abundance of C. minus cannot be predicted by climate alone at a regional scale. The decrease or absence of C. minus in northern Europe was not observed in the mountains of temperate Europe. The distribution of Verticicladium trifidum is less well known, but it is very widely distributed in Europe (it has been observed in all European sites studied). A preliminary survey of its distribution in Europe on different Pinus species (van Maanen & Gourbiere 1997) concludes that V. trifidum was not observed in northern Scandinavia and its abundance is maximal (near 100 % needles colonised) around a latitude of 45m N. The distribution of V. trifidum is, therefore, characterised by both a large latitudinal trend and local variations along a regional climatic gradient. V. trifidum colonisation decreases in cold climates along both latitude and altitude gradients. The local gradient we studied shows a negative correlation between temperature and rainfall (warm–dry to cold–wet gradient). Climate diversity at a larger scale (as in Europe) exhibits other local gradients with : (1) different mean values of annual temperature and rainfall ; (2) different correlations between the two factors ; and (3) various patterns of rainfall distribution over the year. Comparing the effect of such gradients on fungal distribution could be very helpful in unravelling the relationships between climate and the abundance of fungal species. The proportion of needles bearing fruit bodies is a useful measure for estimating the abundance of fungal species. The method we used was successful for describing species

1137 abundance (Kowalski 1988a), successional changes (Kendrick & Burges 1962, Mitchell & Millar 1978), effect of stand age (Kowalski 1988b) and effects of climatic factors (this work). In this paper, we show that the Generalised Linear Model provides a useful method for analysing the data and describing the general trends. The main bias in the direct observation method is the possibility that some needles were colonised but had not produced fruit bodies at the time of sampling. In Europe, C. minus produces its apothecia just after abscission from September to November (Kowalski 1988b, Choi & Simpson 1991). L. pinastri conidiomata (Leptostroma stage) appear from November to March, and the ascomata ripen later, in spring (Mitchell & Millar 1978, Minter & Millar 1980, Minter 1981b). Conidiophores of V. trifidum are produced in summer and autumn the year after litterfall and reach maximal abundance on F1 needles (Hughes 1951, Kendrick & Burges 1962). According to these phenological characteristics, sampling in early summer is appropriate for observing the final state of fruiting of L. pinastri and C. minus on L type needles, and of V. trifidum on F1 needles. A second bias is the possibility that some colonised needles never produce fruit bodies. Only isolation methods could establish the importance of this possible source of error.

Biological hypothesis The present work was not aimed at elucidating the factors that generate the observed correlations. Our results suggest, however, that rainfall or\and temperature may be responsible for the observed distributions. A first hypothesis is that each species has a specific optimum rainfall or\and temperature. A second hypothesis includes between-species interactions. As the three fungal species develop on the same resource (Pinus needles) in the same sites, we could not exclude the possibility that interactions occur between them or with other unstudied species. These interactions could be modified by climatic factors (Widden 1997). The observed correlation between parameters (Fig. 3) suggests that the relationships of the three species to altitude were not statistically independent. The dynamics of the three species could depend upon a common factor or be coupled by interactions. The stable coexistence of the three species around 1000 m (Fig. 2) could be related to optimal conditions of coexistence. A third hypothesis is that climatic factors modify needle quality and their ability to be colonized by each species. Pinus sylvestris populations and\or the chemical composition of needles could be different according to altitude and climate.

CONCLUSION This appears to be the first time that the effect of a regional climatic gradient on the abundance of litter decomposing microfungi has been clearly demonstrated. Further understanding of the effect of climate on the distribution of fungal species at large spatial scales requires more detailed study of the underlying ecological processes and controls on these species distributions.

Fungi along altitude transects A C K N O W L E D G E M E N TS We thank Me! te! ofrance for providing climatic data. This work was conducted as part of the European Communities GLOBIS (Global Change and Biodiversity in Soils) programme.

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