太白山锐齿栎树干基部两种蚂蚁的筑巢生态学及其蚁巢、体表真菌组成研究

贺 虹，张雅林，王云果，高智辉，魏 琮*

(1．西北农林科技大学林学院，陕西杨凌712100;2．西北农林科技大学植保学院，陕西杨凌712100)

Ants are widely distributed throughout the world，and their importance in ecosystems is well recognized(Hölldobler＆ Wilson，1990;Folgarait，1998)．Ants play important roles in predation(Wang et al．，1991;Youngs，1983)，nutrient flow(Pêtal，1998;Ohashi et al．， 2007)， herbaceous vegetation structure(Zhang et al．，2002;Hou et al．，2002)，and soil improvement(Boulton＆ Amberman，2006)．Because of their great abundance，functional importance，and the complex interactions they have with the rest of the ecosystem，ants are often used as bioindicators in environmental assessment programs and forest health(Andersen ＆ Sparling，1997;Underwood ＆ Fisher，2006)．

Some forest ants that nest in decayed wood and remnant stumps or in the cavities of living trees do not eat wood，but they remove wood fibers as they create galleries and tunnels，and their activities enlarge the cavities slowly and finally cause the trees decay and death(Wu ＆ Wang，1995;Filip，2001)．Meanwhile，like some termites，ants nest in decayed wood environments laden with potential pathogens，including maybe bacteria，fungi，nematodes，viruses，and protozoa that can thrive within the humid and temperaturecontrolled conditions of the nests(Friese ＆ Allen，1993;David et al．，1993;Rosengaus et al．，1998)．On the other hand，ants can also become dissemination media of some microorganisms(Harinikumar et al．，1994;Pagnocca et al．，2008)．

Mt．Taibai is the highest peak of the Qinling Mountains in Shaanxi Province，China，which provide a natural boundary between the North and South of the country;and also it is part of the boundaries of the Palaearctic and Oriental Regions in China where support a huge variety of plant and wildlife．We have found 43 ant species there from 1999 to 2003(Liu et al．，1999;He et al．，2000，2003;Wei et al．，2001a，2001b)，and two ant species were found recently nesting in the basal trunks of Quercus aliena var．acuteserrata，and their activities significantly affect the growth of the oaks and greatly diminish the quality of related forest products．In this paper，we investigated the nesting ecology and fungi composition in nests and on workers'exoskeletons of the two ant species，Formica gagatoides Ruzsky and Aphaenogaster sp．， and addressed the following questions in this report:How the activities of the two ants affect the host trees?What about their nesting ecology and forage behavior?How many fungi can be found in this special microenvironment?This research will bring us a step closer to understand how ants and fungi work together to improve the wood decay and decomposition in temperate forests．

1 Materials and methods

1.1 Study site

The study site，the Quercus natural secondary forest，is located in the Mt．Taibai National Nature Reserve(107°22'30″～ 107°51'40″N，33°49'30″～34°08'11″W)in Shaanxi Province，China．The dominant sight woods of Quercus Linn．is divided into three sub-forest types according to the vegetation and elevation(Niu，1990)，i．e．，Q．variabilis，Q．aliena var．acuteserrata and Q．liaodongensis respectively．The predominant sight woods of Q．aliena var．acuteserrata are located mainly between 1300-1800 m in altitude，and the young and old trees are mixed，which shows that they are in obvious developing stage．Other common trees include Populus davidiana，Toxicodendron verniciflua，Pinus armandi，Juglans cathayensis，Litsea tsinlingensis，Elaeagnus umbellata，etc．Typical understory woody plants mainly include Lespedeza buergeri which is dominant，and the following Campylotropis macrocarpa(Bge．)Rehd．The nesting ecology and feeding behavior of the two ant species were observed in the forests from May to September of 2008 and 2009．In order to investigate the two ant species'vertical distribution in the sight woods of Q．aliena var．acuteserrata，we recorded the occurrence of them in altitude of 1300 m，1400 m，1500 m，1600 m，1700 m and 1800 m respectively，from May to September 2008．Among some areas where investigations conducted，we investigated only the trees available due to the complicated or dangerous topography．

1.2 Fungi isolates and identification

Ant colonies were dug by using sterilized shovels from the cavities in the basal trunks of Q．aliena var．acuteserrata on 20 May 2009．Samples of nest materials including workers were put to sterile plastic bags and then taken back to laboratory．The following experiments were conducted inside a sterile laminar flow hood．

Ten one-gram samples of bigger nest materials of three colonies of each ant species were individually placed into Petri dishes containing sterile PDA with 1%streptomycin，respectively．The small nest materials(10 g)were added to 90 mL of sterile water and blended for 30 min．Serial dilutions of 10-2，10-3and 10-4were made for each sample，and 0．5 mL of each dilution was placed onto plates containing sterile PDA with 1%streptomycin;N=3 replicates/sample．

In order to isolate the fungi associated with ant's exoskeleton，ten workers per colony were placed in sterile 1．5 mL Eppendorf tubes with 550 μL of a 0．1%Tween 80 solution and centrifuged at 300 Xg at 4℃ for 20 minutes．Following centrifugation;20μL，30μL，50μL of the supernatant were seeded respectively on plates containing sterile PDA with 1%streptomycin;N=3 replicates/sample(according to the method of Rosengaus et al．，2003)．

Plates were inverted and incubated at 25℃for 2-4 d，until small colonies were visible．Each colony was individually transferred by sterile loop to a new Petri dish containing PDA，incubated at 25℃ for 7 d until sporulation，and transferred by sterile loop to new PDA plates．The edges of the inoculated dishes were sealed with Parafilm to maintain humidity．Fungi identification was accomplished after growth mainly according to the morphology(Deng，1963;Dai，1979)．

2 Results

2.1 Nesting ecology of the two ant species

The two ant species that nest in the basal trunks of Q．aliena var．acuteserrata were identified as F．gagatoides Ruzsky and Aphaenogaster sp．The investigation of the occurrence of the two ant species showed that F．gagatoides was distributed in the forests dominated by Q．aliena var．acuteserrata from 1300-1700 m in altitude，while Aphaenogaster sp．from 1600-1800 m in altitude．In addition，F．gagatoides was more dominant than Aphaenogaster sp．，i．e．，occurrences of the former in all areas investigated were all higher than those of the latter(Table 1)．The percentage of ant-habitat trees is about 20．3%on average and varied in relation to altitudes，i．e．，from6%at least(in woods of altitude of 1800 m)to 38．5%at maximum(in woods of altitude of 1600 m)(Table 1)．

F．gagatoides could be found both on the ground and trees frequently，and it was one of the most abundant ant species found in the Qurcus forest．This species mainly feeds on the honeydew of aphids that induce insect galls on the slim branches of Q．aliena var．acuteserrata，and also were found to catch some small insect corpses and plant materials．The nests of F．gagatoides were usually very wet when dug(Fig．1a，b)，and sometimes there was much water remaining there．The sunken necrotic areas varied from about 20-60 cm in diameter and 40-60 cm in depth．

Table 1 The occurrence of two ant species in the woods of Q.aliena var．acuteserrata

Aphaenogaster sp．was found only in the cavities of the Qurcus trunk．They seldom forage outside of their nest，and sometimes they were found catching small insects．The nests of Aphaenogaster sp．were drier than those of F．gagatoides，and the entrances of their nests were about 15-40 cm in diameter and the sunken necrotic areas were about 30-50 cm in depth(Fig．1c，d)．

图1 锐齿栎树干基部蚂蚁巢穴Fig．1 Ant-habitat cavities of Quercus aliena var．acuteserrata

Workers of the two ant species make galleries by chewing moist wood to provide more room for the expanding colony，and their nesting activities indeed accelerate and increase the amount of wood decay in stems of the oaks．The damaged area on the main stem appears as sunken necrotic area from about 20-60 cm under and 20-30 cm above ground line respectively．This kind of disturbance damage，a strong biodisturbance occurring in the sight woods of Q．aliena var．acuteserrata，greatly affects the water and nutrition transport of trees and weaken the trees'vigor．In addition，the significantly damaged trees are structurally weakened and likely to break during windstorms or harvesting operations．Therefore，the ants'activities affect not only the growth of the oaks，but also the vegetation succession and nutritional recycle of sight woods of Qurecus．

2.2 Fungi isolated from ants'nest materials and the exoskeleton of workers

Ten fungal species were isolated from the nest materials and exoskeleton of the two ant species in total，including Trichoderma sp．，Fusarium nivals，Botrytis sp．，Cephalosporium sp．，Paecilomyces sp．，Penicillium resedanum，Gliomastix sp．，Leptographium sp．，Verticillium sp．and Aureobasidium sp．(Table 2)．Whereas，the fungal community was distinctly different in two ants'nest materials and on their workers'exoskeleton(Table 2，3)．For ant F．gagatoides，seven fungi were isolated from the nest materials，and four fungi from exoskeleton of workers which all could be found in the nest material．For ant Aphaenogaster sp．，we just isolated two fungi from nest material and three fungi from exoskeleton of workers respectively，and the fungal species composition between them showed clear difference(Table 2)．Trichoderma sp．was the most prevalent species among fungi in this survey，which was isolated from both the nest materials and exoskeleton of the two ant species(Table 3)．Aureobasidium sp．occured only on the exoskeleton of Aphaenogaster sp．，and Leptographium sp．was only found in the nest materials of Aphaenogaster sp．

Table 2 The fungal species isolated from the nest material and workers'exoskeleton of the two ant species

Table 3 The occurrence of fungi in nest material and workers'exoskeleton of the two ant species

3 Discussion

Ants utilize various substrates for nesting，including dead wood，stumps and living trees(Hölldobler＆ Wilson，1990)，and usually cause some damage to plants and woods，e．g．，leafcutter ants culture their fungi on freshly cut foliage and flowers，becoming major forest and agricultural pests(Currie et al．，1999);carpenter ants cut“galleries”into the wood grain to provide passageways for movement from section to section of the nest(Tripp et al．，2000)．In addition，some ants even live in the specialized organs of some plants'possesses forming famous symbiotic relation-ship，with the ants helping to protect the plants from other insect pests and in return receiving shelter(Edwards et al．，2007)．But for some other ants that just nest in trees，are occasionally noticed，though their nest activities can also affect the growth and product of trees．

In our research，we investigated two ant species nesting in the basal trunk of Q．aliena var．acuteserrata，and found that their feeding behavior and the internal environment of nests were clearly different．Their effects on the trees were not deadly quick，but their long-term effects on the trees will be profound and to be worth paying close attention．We also found ten cavities were empty but there the geminated seeds of Qurcus were found，but we don't know so far if this phenomenon has related to ants'activities．

We just use the traditional culture method with substrate PDA to isolate the fungi related with the nest materials and exoskeleton of the two ant species in this study，instead of using molecular procedures in the meantime，since we are aiming to determine preliminarily the difference of fungi composition of the nests and on exoskeletons between the two ant species．Therefore，the fungal species isolated are in all probability fewer than that actually．The difference of fungal species isolated from the nest materials of the two ant species may relate closely to ant species as well as their nests'internal environment．For example，the nests of F．gagatoides were very wet，and they seemed to be highly favorable for the growth and development of a variety of fungi(seven fungal species were isolated from the nest material，see Table 2)．But，by contrast，Aphaenogaster sp．inhabited relatively dry environment and didn't favor the growth of fungi(only two species were isolated from the nest material)．

The filamentous fungi found in our survey are all ascomycetes which could be commonly isolated from the soil and decaying wood and plant debris(Yao et al．，1998)．Of those，Paecilomyces，Verticillium and Fusarium may be entomophagous;however，most of the identified fungi are not pathogenic．The fungal species of the genus Trichoderma are often predominant components in various ecosystems，and some strains are promising biocontrol agents for plant disease due to fungi(Li et al．，2009)．In our survey，the massive occurrence of Trichoderma in both collections suggests that this fungus has a special role in ant nest，e．g．，it probably could help trees increase the resistance to ants'nesting activities and wood decay，or maybe ants utilize this fungus to suppress the growth of other fungi．The function of this fungus is not yet clear but they probably provide both individual and social protection against disease．

Leptographium are associated with，and often distributed by，one or more of a variety of bark-feeding，bark-boring，or wood-boring insects(Barnard ＆Meeker，1995;Lu ＆ Sun，2008)．Most of these fungi are carried by bark beetles and function to degenerate wood(Six＆ Klepzig，2004)．Therefore，the occurrence of this fungus in the ant nest found in our study probably also help ants to degenerate wood and facilitate the ants to enlarge their nest，and also may be harbored by the ants，as is very interesting and worthy to be studied further．

Insects are one of the main dissemination media of many fungi，and they can harbor several species of filamentous fungi and yeasts on their exoskeleton(Rosa et al．，2003)or specialized structures adapted to transport fungi(e．g．Mycangia)，as is the case of the bark beetle-fungal association(Six ＆ Klepzig，2004)．In all of these insect microbial associations hosts have an impact in the dispersal or survival of the associated microbes．Therefore，ants can also become the potential medium for dissemination of some fungi(Harinikumar＆ Bagyaraj，1994;Gracia-Garza et al．，1998;Ei-Hamalawi＆ Mense，1996;Pagnocca et al．，2008)，but most of their dispersing mechanisms are still quite unclear yet，except that in some leafcutting ants，in which microbes have been found in the fungus gardens(Currie et al．，1999;Santos et al．，2004;Rodrigues et al．，2008)，on the ant's exoskeleton(Little＆ Currie，2007)and in fungal pellets carried by nest-founding gynes(Quinlan ＆ Cherrett，1978)．We also isolated some fungal species from the worker's exoskeleton of the two ant species，but we are not sure whether they are parasitism or transient．

Ants employ several tactics as defenses against microbe and thereby influence fungal populations in ant nests(Veal et al．，1992;Currie et al．，1999;Zettler et al．，2002;Rodrigues et al．，2008)．But why some certain fungi can exist in nest，and not others?What are the roles performed by the various fungi found in nests and the worker's exoskeleton of the two ant species?Ants prefer to nest in wood maybe like some termites that has been“conditioned”by a variety of saprophytic fungi and can be attracted to such wood(Rosengaus et al．，2003)，and whether ants have mechanism of degrading wood and fungistatic activities similar to termites needs more research to verify．The results of our investigation indicate that some fungal species were widespread and prevalent in the ant nests，and they could have yet unknown roles in the ecosystem．Further research is required to determine the roles performed by the various fungi found in the nests of these two ant species，and molecular methods should be attempted to use to document more fungal species in the nest materials of ants．

Acknowledgement:We would like to express our sincere thanks to Prof．Zhou Shan-yi(Guangxi Normal University，China)and Prof．Xu Zheng-hui(Southwest Forestry University，China)for identifying the ant species Formica gagatoides Ruzsky，and also to Prof．Cao Zhi-min and Prof．Xue Quan-hong(Northwest A＆F University，China)for their valuable advice．

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