**Excess Supply of Nutrients, Fungal Community, and Plant Litter Decomposition: A Case Study of Avian-Derived Excreta Deposition in Conifer Plantations<sup>1</sup>**

Takashi Osono *Center for Ecological Research, Kyoto University Japan* 

#### **1. Introduction**

172 International Perspectives on Global Environmental Change

Ytrehus, B.; Bretten, T.; Bergsjø, B. & Isaksen, K. (2008). Fatal Pneumonia Epizootic in Musk

Zingerle, V. (1999). Spider and harvestman communities along a glaciation transect in the

Italian Dolomites. *Journal of Arachnology,* 27, 222-228.

213-223.

ox (*Ovibos moschatus*) in a period of extraordinary weather conditions. *EcoHealth,* 5,

#### **1.1 Excess supply of nutrients and terrestrial ecosystems**

Human activities have greatly accelerated emissions of both carbon dioxide and biologically reactive nutrients such as nitrogen (N) to the atmosphere (Canfield et al., 2010), which cause environmental changes affecting ecosystem processes and biodiversity in forests. Excess supply of N of anthropogenic origin to forest soils, such as combustion of fossil fuels, production of N fertilizers, and cultivation of N-fixing legumes, is an example of such environmental changes often leading to a decrease of the rate of carbon dioxide evolution and decomposition (Fog, 1988; Berg and Matzner, 1997) and a concomitant increase in the amount of soil carbon stock (deVries et al., 2006; Zak et al., 2008). These changes are primarily attributable to the reduced activity of fungal ligninolytic enzymes that play crucial roles in the turnover of soil organic carbon and are known to be sensitive to N deposition (Sinsabaugh, 2010). However, such changes in the enzymatic activity are not consistently associated with changes in the abundance and diversity of fungi that are responsible for the activity (Waldrop and Zak, 2006; Blackwood et al., 2007; Hassett et al., 2009). This discrepancy merits further studies to examine the response of ecological and functional properties of fungal communities to excess supply of N and its consequences on the dynamics of carbon and N in forest soils.

The transfer of nutrients by waterbirds from aquatic to terrestrial ecosystems provides similar situations to the anthropogenic supply of nutrients because birds feed on fish in the aquatic zone and deposit their waste rich in nutrients to the terrestrial parts of their habitats. Such allochthonous input of N and other nutrients to terrestrial ecosystems can lead locally to substantial enrichment of soils and plants and alter food webs, nutrient cycling, and

<sup>1</sup>This manuscript should be cited as follows: Osono, T. (2011). Excess supply of nutrients, fungal community, and plant litter decomposition: a case study of avian-derived excreta deposition in conifer plantations, In: *Environmental Change*, S.S. Young and S.E. Silvern, (Ed.), 000-000, InTech, ISBN979-953- 307-109-0, Rijeka, Croatia

Excess Supply of Nutrients, Fungal Community, and Plant Litter

cormorant effects (Kameda et al., 2006).

Decomposition: A Case Study of Avian-Derived Excreta Deposition in Conifer Plantations 175

indicative of N saturation at the study sites exposed to bird colonization (Aber et al., 1998). Excreta-derived N was incorporated into not only soils but also aboveground tissues of plants, as indicated by natural 15N abundance as a natural tracer (Kameda et al., 2006). Because cormorants are piscivorous birds and one of the top predators in aquatic food webs, 15N of their tissues and excreta is markedly higher (i.e., 13 to 17‰) than those of N from precipitation and N fixation (-1 to 1‰). The data of 15N in soils and plants were used to construct 'N stable isotope map' of Isaki Peninsula (Fig. 1) showing the spatial patterns of

Fig. 1. Study sites, cormorant colony boundaries and the year of colony establishment, and nitrogen stable isotope map of Isaki Peninsula (IP) at Lake Biwa, Japan. The nitrogen stable isotope map shows the intensity and duration of cormorant colonization (Kameda et al.,

IP-T

T Spring 1999 Temporarily colonized for 3 months before cormorants were

A 1996-1999 Abandoned after 3 years of colonization; no cormorants D 1992-1996 Declined after 4 years of intensive colonization; no cormorants

Table 1. Study sites and descriptions of breeding colony of cormorants at Isaki Peninsula.

expelled by hunters; no cormorants thereafter

2006). See Table 1 for the description of study sites.

IP-D

C No colonization Never colonized by cormorants (control)

P 1997-2003 Presently colonized; cormorants abundant

Site Colonization Description

ecosystem processes in bird colonies (Mizutani and Wada, 1988; Anderson and Polis, 1999). In contrast, much less concern has been directed toward the diversity and activity of saprobic fungi in forest soils affected by excess supply of avian-derived N and the consequences for carbon sequestration in forest soils.

#### **1.2 Cormorant populations in lakeside forests in Japan**

The great cormorant, *Phalacrocorax carbo* L., is a colonial piscivorous bird that is distributed almost all over the world (Johnsgard, 1993). In Japan, the cormorants breed and roost in trees in riparian woods and feed on fishes in lakes, rives, and coastal areas (Ishida et al., 2003). The population of cormorants increased rapidly after the 1980s as the number of new colonies increased (Kameda et al., 2003). For example, there were no breeding records of cormorants between World War II and 1982 within the watershed of Lake Biwa, currently one of the main habitats of cormorants in Japan, whereas the population size increased rapidly in the 1990s to reach more than 17,000 during the breeding season from January to August in 2003 (Kameda et al., 2006). The increased populations have caused serious conflicts with fisheries and forests in their habitats (Kameda et al., 2003).

Isaki Peninsula (35°12'N, 136°5'E, 57 ha), located on the southeast side of Lake Biwa (Fig. 1) and covered with plantations of Japanese cypress (*Chamaecyparis obtusa* Sieb. et Zucc.), was selected for the present study. The mean annual temperature is 15.1°C and annual precipitation is 1,474.5 mm at the Hikone Weather station about 20 km from the Isaki Peninsula. After cormorant nests were first discovered at Isaki Peninsula in 1988, the area of the colony expanded from 1.3 ha in 1992 to 19.3 ha in 1999 and the number of nests from 30 to 40 in 1989 to 5,300 in 1999 (Fig. 1) to become one of the major habitats of the cormorants in the watershed of Lake Biwa (Fujiwara and Takayanagi, 1999). Five study sites were chosen on Isaki Peninsula, Sites C, T, P, A, D, which had the same vegetation composition but were in different stages of breeding colony establishment (Table 1). A study plot (50 50 m) was established at each site and used to study the effects of cormorant colonization on soils and vegetation.

#### **1.3 Responses of forest ecosystems to cormorant colonization**

During the breeding season, the input of bird excreta at Site P was estimated at 2.2 t/ha/month (Kameda et al., 2000). Because the excreta are rich in N (11.1% w/w on average) and other nutrients such as P and Ca, the excreta input was estimated to be the equivalent of 0.24 t/ha/month of excreta-derived N, which corresponds to about 10,000 times the ordinary input by precipitation (Fig. 2) (Kameda et al., 2000). In addition, litterfall input at Site P during the breeding season was estimated at 2.6 t/ha/month, which was 7 to 22 times greater than that at Site C (Fig. 2) (Hobara et al., 2001). This increase of litterfall at Site P was due to damage of the overstory by the cormorants. *Chamaecyparis obtusa* was one of the most heavily damaged tree species at forest stands colonized by the cormorants (Ishida, 1996b). A part of forest stands intensively colonized by the cormorants declined due to high mortality of *C. obtusa* (Site D; Fig. 2) (Fujiwara and Takayanagi, 2001).

The forest decline was also partly and indirectly attributable to changes in soil properties caused by excess supply of excreta-derived nutrients. A dramatic increase in inorganic N pools, a decrease in carbon to N ratio, and an increase in nitrification rate were observed in forest floor materials and in soils at Sites P and A (Ishida, 1996a; Hobara et al., 2001),

ecosystem processes in bird colonies (Mizutani and Wada, 1988; Anderson and Polis, 1999). In contrast, much less concern has been directed toward the diversity and activity of saprobic fungi in forest soils affected by excess supply of avian-derived N and the

The great cormorant, *Phalacrocorax carbo* L., is a colonial piscivorous bird that is distributed almost all over the world (Johnsgard, 1993). In Japan, the cormorants breed and roost in trees in riparian woods and feed on fishes in lakes, rives, and coastal areas (Ishida et al., 2003). The population of cormorants increased rapidly after the 1980s as the number of new colonies increased (Kameda et al., 2003). For example, there were no breeding records of cormorants between World War II and 1982 within the watershed of Lake Biwa, currently one of the main habitats of cormorants in Japan, whereas the population size increased rapidly in the 1990s to reach more than 17,000 during the breeding season from January to August in 2003 (Kameda et al., 2006). The increased populations have caused serious

Isaki Peninsula (35°12'N, 136°5'E, 57 ha), located on the southeast side of Lake Biwa (Fig. 1) and covered with plantations of Japanese cypress (*Chamaecyparis obtusa* Sieb. et Zucc.), was selected for the present study. The mean annual temperature is 15.1°C and annual precipitation is 1,474.5 mm at the Hikone Weather station about 20 km from the Isaki Peninsula. After cormorant nests were first discovered at Isaki Peninsula in 1988, the area of the colony expanded from 1.3 ha in 1992 to 19.3 ha in 1999 and the number of nests from 30 to 40 in 1989 to 5,300 in 1999 (Fig. 1) to become one of the major habitats of the cormorants in the watershed of Lake Biwa (Fujiwara and Takayanagi, 1999). Five study sites were chosen on Isaki Peninsula, Sites C, T, P, A, D, which had the same vegetation composition but were in different stages of breeding colony establishment (Table 1). A study plot (50 50 m) was established at each site and used to study the effects of cormorant colonization on soils and

During the breeding season, the input of bird excreta at Site P was estimated at 2.2 t/ha/month (Kameda et al., 2000). Because the excreta are rich in N (11.1% w/w on average) and other nutrients such as P and Ca, the excreta input was estimated to be the equivalent of 0.24 t/ha/month of excreta-derived N, which corresponds to about 10,000 times the ordinary input by precipitation (Fig. 2) (Kameda et al., 2000). In addition, litterfall input at Site P during the breeding season was estimated at 2.6 t/ha/month, which was 7 to 22 times greater than that at Site C (Fig. 2) (Hobara et al., 2001). This increase of litterfall at Site P was due to damage of the overstory by the cormorants. *Chamaecyparis obtusa* was one of the most heavily damaged tree species at forest stands colonized by the cormorants (Ishida, 1996b). A part of forest stands intensively colonized by the cormorants declined due

The forest decline was also partly and indirectly attributable to changes in soil properties caused by excess supply of excreta-derived nutrients. A dramatic increase in inorganic N pools, a decrease in carbon to N ratio, and an increase in nitrification rate were observed in forest floor materials and in soils at Sites P and A (Ishida, 1996a; Hobara et al., 2001),

consequences for carbon sequestration in forest soils.

vegetation.

**1.2 Cormorant populations in lakeside forests in Japan** 

conflicts with fisheries and forests in their habitats (Kameda et al., 2003).

**1.3 Responses of forest ecosystems to cormorant colonization** 

to high mortality of *C. obtusa* (Site D; Fig. 2) (Fujiwara and Takayanagi, 2001).

indicative of N saturation at the study sites exposed to bird colonization (Aber et al., 1998). Excreta-derived N was incorporated into not only soils but also aboveground tissues of plants, as indicated by natural 15N abundance as a natural tracer (Kameda et al., 2006). Because cormorants are piscivorous birds and one of the top predators in aquatic food webs, 15N of their tissues and excreta is markedly higher (i.e., 13 to 17‰) than those of N from precipitation and N fixation (-1 to 1‰). The data of 15N in soils and plants were used to construct 'N stable isotope map' of Isaki Peninsula (Fig. 1) showing the spatial patterns of cormorant effects (Kameda et al., 2006).

Fig. 1. Study sites, cormorant colony boundaries and the year of colony establishment, and nitrogen stable isotope map of Isaki Peninsula (IP) at Lake Biwa, Japan. The nitrogen stable isotope map shows the intensity and duration of cormorant colonization (Kameda et al., 2006). See Table 1 for the description of study sites.


Table 1. Study sites and descriptions of breeding colony of cormorants at Isaki Peninsula.

Excess Supply of Nutrients, Fungal Community, and Plant Litter

and hence hyphal growth of basidiomycetes at Sites P and A.

CPA

Site

0

in Table 1. Data after Osono et al. (2002).

**2.2 Diversity and species composition of fungi** 

5000

10000

m/g dry material

15000

Decomposition: A Case Study of Avian-Derived Excreta Deposition in Conifer Plantations 177

to the decreased availability of carbon compounds owing to condensation of N-rich compounds (Osono et al., 2002). Söderström et al. (1983) also reported a decrease in microbial biomass after N fertilization in coniferous forest soils. The lower length of clampbearing hyphae (i.e., biomass of basidiomycetous fungi) at Sites P and A than at Site C might also have been due to a biochemical suppression of lignin-degrading enzymes of some fungi in the Basidiomycota caused by excess excreta deposition (Keyser et al., 1978; Fenn et al., 1981). This may have reduced competitiveness relative to that of other non-ligninolytic fungi

0

Fig. 3. Total hyphal lengths and lengths of clamp-bearing hyphae in dead needles and twigs of *Chamaecyparis obtusa* examined with an agar film method. needles; twigs. Sites are as

Fig. 4. A hypha with a clamp connection (arrow) observed under a microscope. Bar = 5 µm.

Secondly, species richness, diversity, and equitability of fungal assemblages associated with the dead needles and twigs were examined with a culture-dependent, surface disinfection method (Fig. 5). A total of 231 isolates of 70 fungal species were isolated from dead needles and twigs at Sites C, P, and A. Species richness (i.e., the number of species isolated) in needles was higher at Site A than at Sites C and P, but the species richness in twigs was

500

1000

1500

**Total hyphae Clamp-bearing hyphae**

CPA

Site

Fig. 2. Surface of the forest floor covered with dead twigs fallen at Site A (left), leaves of understory vegetation covered with excreta deposited at Site P (middle), and dead trees of *Chamaecyparis obtusa* in a declined forest stand at Site D (right).

#### **1.4 Purposes**

In this chapter I summarize a series of published papers reporting the effects of excess supply of N as avian excreta on fungal communities and plant litter decomposition in conifer plantations colonized by cormorants (Osono et al., 2002, 2006a, 2006b, unpublished data; Katsumata, 2004) to present a comprehensive picture of their relationships and to predict long-term patterns in the accumulation of dead plant tissues and excreta-derived nutrients on the forest floor. The following hypotheses are addressed. (i) The excess supply of nutrients affected the abundance, diversity, and species composition of saprobic fungal communities, as well as their nutrition and activity (Sections 2, 3, and 4). (ii) Such changes in fungal diversity and activity in turn affected the decomposition processes of dead plant tissues, such as needles, twigs, and stems (Section 5). (iii) Dead plant tissues abundantly supplied to the forest floor serve as reservoirs of excreta-derived N (Section 6). The studies explicitly demonstrate that the changes in fungal communities and decomposition of dead plant tissues had consequences regarding the long-term patterns of accumulation of carbon and N in soils of forest stands colonized by cormorants.

#### **2. Excreta deposition and fungal communities**

It is usually difficult to study both the biomass and the species composition of fungal assemblages simultaneously with any single method (Osono, 2007). Thus, fungal biomass and species composition were studied separately. Firstly, dead needles and twigs of *C. obtusa* were collected from the forest floor, and the length of hyphae in the tissues was examined with a direct observation method as a measure of fungal biomass and compared among forest stands with different histories of cormorant colonization (Osono et al., 2002). Twigs were defined as woody tissues with a diameter less than 0.5 cm.

#### **2.1 Fungal biomass in dead needles and twigs**

The total hyphal length was generally longer in needles than in twigs and was in the order: Sites C > P > A (Fig. 3), suggesting that the biomass of fungi was reduced in forest stands supplemented with excreta. The length of clamp-bearing hyphae, belonging to the Basidiomycota (Fig. 4), accounted for 10 to 11% of the total hyphal length at Site C but was reduced markedly at Sites P and A (Fig. 3).

The reduced fungal biomass at Sites P and A was possibly attributable to the inhibitory effects on fungal growth of excreta rich in ammonia, uric acid, and salts (see Section 4.1) and

Fig. 2. Surface of the forest floor covered with dead twigs fallen at Site A (left), leaves of understory vegetation covered with excreta deposited at Site P (middle), and dead trees of

In this chapter I summarize a series of published papers reporting the effects of excess supply of N as avian excreta on fungal communities and plant litter decomposition in conifer plantations colonized by cormorants (Osono et al., 2002, 2006a, 2006b, unpublished data; Katsumata, 2004) to present a comprehensive picture of their relationships and to predict long-term patterns in the accumulation of dead plant tissues and excreta-derived nutrients on the forest floor. The following hypotheses are addressed. (i) The excess supply of nutrients affected the abundance, diversity, and species composition of saprobic fungal communities, as well as their nutrition and activity (Sections 2, 3, and 4). (ii) Such changes in fungal diversity and activity in turn affected the decomposition processes of dead plant tissues, such as needles, twigs, and stems (Section 5). (iii) Dead plant tissues abundantly supplied to the forest floor serve as reservoirs of excreta-derived N (Section 6). The studies explicitly demonstrate that the changes in fungal communities and decomposition of dead plant tissues had consequences regarding the long-term patterns of accumulation of carbon

It is usually difficult to study both the biomass and the species composition of fungal assemblages simultaneously with any single method (Osono, 2007). Thus, fungal biomass and species composition were studied separately. Firstly, dead needles and twigs of *C. obtusa* were collected from the forest floor, and the length of hyphae in the tissues was examined with a direct observation method as a measure of fungal biomass and compared among forest stands with different histories of cormorant colonization (Osono et al., 2002).

The total hyphal length was generally longer in needles than in twigs and was in the order: Sites C > P > A (Fig. 3), suggesting that the biomass of fungi was reduced in forest stands supplemented with excreta. The length of clamp-bearing hyphae, belonging to the Basidiomycota (Fig. 4), accounted for 10 to 11% of the total hyphal length at Site C but was

The reduced fungal biomass at Sites P and A was possibly attributable to the inhibitory effects on fungal growth of excreta rich in ammonia, uric acid, and salts (see Section 4.1) and

*Chamaecyparis obtusa* in a declined forest stand at Site D (right).

and N in soils of forest stands colonized by cormorants.

**2. Excreta deposition and fungal communities** 

**2.1 Fungal biomass in dead needles and twigs** 

reduced markedly at Sites P and A (Fig. 3).

Twigs were defined as woody tissues with a diameter less than 0.5 cm.

**1.4 Purposes** 

to the decreased availability of carbon compounds owing to condensation of N-rich compounds (Osono et al., 2002). Söderström et al. (1983) also reported a decrease in microbial biomass after N fertilization in coniferous forest soils. The lower length of clampbearing hyphae (i.e., biomass of basidiomycetous fungi) at Sites P and A than at Site C might also have been due to a biochemical suppression of lignin-degrading enzymes of some fungi in the Basidiomycota caused by excess excreta deposition (Keyser et al., 1978; Fenn et al., 1981). This may have reduced competitiveness relative to that of other non-ligninolytic fungi and hence hyphal growth of basidiomycetes at Sites P and A.

Fig. 3. Total hyphal lengths and lengths of clamp-bearing hyphae in dead needles and twigs of *Chamaecyparis obtusa* examined with an agar film method. needles; twigs. Sites are as in Table 1. Data after Osono et al. (2002).

Fig. 4. A hypha with a clamp connection (arrow) observed under a microscope. Bar = 5 µm.

### **2.2 Diversity and species composition of fungi**

Secondly, species richness, diversity, and equitability of fungal assemblages associated with the dead needles and twigs were examined with a culture-dependent, surface disinfection method (Fig. 5). A total of 231 isolates of 70 fungal species were isolated from dead needles and twigs at Sites C, P, and A. Species richness (i.e., the number of species isolated) in needles was higher at Site A than at Sites C and P, but the species richness in twigs was

Excess Supply of Nutrients, Fungal Community, and Plant Litter

and different methodologies used for fungal isolation.

excreta.

Decomposition: A Case Study of Avian-Derived Excreta Deposition in Conifer Plantations 179

A few studies have examined the effects of bird colonization on soil fungal assemblages. Ninomiya et al. (1993) and Schoenlein-Crusius et al. (1996) observed no difference in fungal diversity between soil affected by the presence of birds and control soil, which contrasted with the results of the present study. Osono et al. (2002) summarized previous studies on the effects of ornithologenic and anthropogenic eutrophication on the diversity of soil saprobic fungal assemblages and found that the response was variable depending on the study. The inconsistency of the eutrophication effect on fungal diversity suggests that factors other than nutrient addition may also affect the diversity, such as the amount and/or form of nutrients added, time after fertilization, physical and chemical properties of soils,

Clear differences were found for the patterns of occurrence of 11 major fungal species among the sites (Fig. 6). *Penicillium montanense*, *Geniculosporium* sp., and *Marasmius* sp. dominated at Site C were decreased at Sites P and A. Koide and Osono (2003) reported a similar result that an udentified species of *Geniculosporium* was isolated from leaf litter of *Camellia japonica* at Site C but not at Site A. This contrasted to *Sordaria* sp., *Chaetomium* sp., Discomycete sp., an unidentified arthroconidial species, and *Fusarium solani*, which showed marked increases at Site P in both needles and twigs. Arthroconidial sp. and *F. solani* also occurred frequently at Site A, as did *Trichoderma viride*, *T. hamatum*, and *Penicillium* sp. The absence of a ligninolytic basidiomycete *Marasmius* sp. from twigs at Sites P and A was consistent with the decrease in clamp-bearing hyphae (Fig. 3) and may have been due to enzymatic suppression by excessive inorganic-N or N-rich compounds in these sites as discussed above. *Sordaria* sp. is considered to be a coprophilous species associated with bird

In summary, the abundance of basidiomycetes (Fig. 3) and the relative frequency of ligninolytic *Marasmius* sp. (Fig. 6) were reduced at presently colonized (Site P) and abandoned forest stands (Site A), possibly due to excess supply of nutrients in excreta, such as N. To verify this possibility, effects of excreta addition on fungal growth and

Utilization of cormorant-derived N by fungi was demonstrated by investigating the natural 15N abundance in fruit bodies of litter- and wood-decomposing fungi collected in the study sites. 15N enrichments in plant tissues, forest floor materials, and mineral soils due to excreta deposition were demonstrated in the cormorant colonies at Isaki Peninsula (Section 1.3; Fig. 1), which was associated with such processes as trophic enrichment through aquatic food webs and ammonia volatilization from soils (Kameda et al., 2006). Using natural 15N abundance as a natural tracer thus makes it possible to test whether fungi utilized excreta-

The 15N values of fruiting bodies at Site C were 0.1 to 1.5‰ on average and at similar levels to that in precipitation at the vicinity of the study sites (Fig. 7) and were within the range for saprobic fungi previously reported (e.g., Kohzu et al., 1999; Trudell et al., 2004). 15N was significantly (generalized linear model, 2=39.0, P<0.001) different among Sites C, P, and A and was significantly (2=15.4, P<0.001) higher in litter- than in wood-decomposing fungi (Fig. 7). Mean 15N values of fruiting bodies were in the order: Sites A > P > C for both litterand wood-decomposing fungi (Fig. 7). 15N of dead needles, forest floor materials, and woody debris were also higher at Sites P and A than at Site C, and fruiting bodies of fungi

decomposition was examined under pure culture conditions in Section 4.

**3. Utilization of excreta-derived nutrients by fungi** 

derived N in the colonized forests.

similar among the sites. Diversity index was higher in twigs than in needles and was higher at Site A than at Sites C and P. Equitability was higher in twigs than in needles and in the order: Sites A > P > C in both needles and twigs.

Fig. 5. Diversity of fungal assemblages in dead needles and twigs of *Chamaecyparis obtusa*. needles; twigs. Sites are as in Table 1. Species richness (S) equals to the total number of species. Simpson's diversity index (D) and equitability (E) were calculated as: D = 1/∑ P*i*2, E = D/S, where P*i* was the relative frequency of the *i*th species in each fungal assemblage (Osono et al., 2002).

Fig. 6. Relative frequency (%) of major fungal species in dead needles and twigs of *Chamaecyparis obtusa* (Osono et al., 2002). Black bar, needles; open bar, twigs. Sites are as in Table 1.

similar among the sites. Diversity index was higher in twigs than in needles and was higher at Site A than at Sites C and P. Equitability was higher in twigs than in needles and in the

**Species richness Diversity index Equitability**

CPA

CPA

**Arthroconidial sp.**

*Fusarium solani*

<sup>20</sup> *Trichoderma viride*

CP A

*Penicillium* **sp.**

*Trichoderma hamatum*

0

0

20

0

0

20

0

20

0

20

0.5

1

Site

Fig. 5. Diversity of fungal assemblages in dead needles and twigs of *Chamaecyparis obtusa*. needles; twigs. Sites are as in Table 1. Species richness (S) equals to the total number of species. Simpson's diversity index (D) and equitability (E) were calculated as: D = 1/∑ P*i*2, E = D/S, where P*i* was the relative frequency of the *i*th species in each fungal assemblage

*Sordaria* **sp.**

CPA

*Chaetomium* **sp.**

**Discomycete sp.**

Site

*Chamaecyparis obtusa* (Osono et al., 2002). Black bar, needles; open bar, twigs. Sites are as in

Fig. 6. Relative frequency (%) of major fungal species in dead needles and twigs of

0

0

0

0

20

20

20

40

10

20

order: Sites A > P > C in both needles and twigs.

CPA

0

0

Frequency (%)

0

0

Table 1.

20

CPA

*Marasmius* **sp.**

*Geniculosporium* **sp.**

20

20

40

(Osono et al., 2002).

*Penicillium montanense*

10

20

30

A few studies have examined the effects of bird colonization on soil fungal assemblages. Ninomiya et al. (1993) and Schoenlein-Crusius et al. (1996) observed no difference in fungal diversity between soil affected by the presence of birds and control soil, which contrasted with the results of the present study. Osono et al. (2002) summarized previous studies on the effects of ornithologenic and anthropogenic eutrophication on the diversity of soil saprobic fungal assemblages and found that the response was variable depending on the study. The inconsistency of the eutrophication effect on fungal diversity suggests that factors other than nutrient addition may also affect the diversity, such as the amount and/or form of nutrients added, time after fertilization, physical and chemical properties of soils, and different methodologies used for fungal isolation.

Clear differences were found for the patterns of occurrence of 11 major fungal species among the sites (Fig. 6). *Penicillium montanense*, *Geniculosporium* sp., and *Marasmius* sp. dominated at Site C were decreased at Sites P and A. Koide and Osono (2003) reported a similar result that an udentified species of *Geniculosporium* was isolated from leaf litter of *Camellia japonica* at Site C but not at Site A. This contrasted to *Sordaria* sp., *Chaetomium* sp., Discomycete sp., an unidentified arthroconidial species, and *Fusarium solani*, which showed marked increases at Site P in both needles and twigs. Arthroconidial sp. and *F. solani* also occurred frequently at Site A, as did *Trichoderma viride*, *T. hamatum*, and *Penicillium* sp. The absence of a ligninolytic basidiomycete *Marasmius* sp. from twigs at Sites P and A was consistent with the decrease in clamp-bearing hyphae (Fig. 3) and may have been due to enzymatic suppression by excessive inorganic-N or N-rich compounds in these sites as discussed above. *Sordaria* sp. is considered to be a coprophilous species associated with bird excreta.

In summary, the abundance of basidiomycetes (Fig. 3) and the relative frequency of ligninolytic *Marasmius* sp. (Fig. 6) were reduced at presently colonized (Site P) and abandoned forest stands (Site A), possibly due to excess supply of nutrients in excreta, such as N. To verify this possibility, effects of excreta addition on fungal growth and decomposition was examined under pure culture conditions in Section 4.
