| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Carleton College, Department of Biology, Northfield, Minnesota, USA 55057
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
RAPD markers were used to examine the degree of genetic variation within the putatively asexual basidiomycete fungus (Lepiotaceae: provisionally named Leucoagaricus gongylophorus) associated with the leaf-cutting ant species Atta cephalotes. We analyzed fungal isolates from ant nests in two geographically distant sites, two isolates from Panama and five isolates from Trinidad. Ten decamer primers were used to amplify total DNA from these seven fungal isolates, and RAPD banding patterns were compared. Genetic similarity among isolates was determined by pair-wise comparisons of the shared number of DNA bands on an agarose gel. There was considerable genetic variation among isolates of the symbiotic fungus even within sites. Pairs of fungal isolates from the two different sites shared an average of only 36% of the bands in their RAPD profiles, while pairs from the within sites shared an average of 72% of the bands. RAPD markers may be useful for further investigation of the genetic structure of the fungal symbiont within species of leaf-cutting ants.
Key words: Atta cephalotes, fungal DNA, genetic variation, leaf-cutting ants, Leucoagaricus gongylophorus (Lepiotaceae), population differentiation, RAPD markers, symbiosis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). These ants are obligately dependent on a symbiotic fungus (Weber 1972, Hölldobler and Wilson 1990, Mueller et al 2001), which is cultivated on leaf fragments brought into the nest by foraging workers. The fungus produces swollen hyphae (‘staphylae’ or ‘gongylidia’) that are harvested by the ants for food, particularly for feeding the larvae (Quinlan and Cherrett 1979, Bass and Cherrett 1995).
The symbiotic fungus of leaf-cutting ants generally lacks sexual structures, which has made species identification and nomenclature difficult (reviewed in Pagnocca et al 2001). Progress has been made recently in determining the phylogeny of both the leaf-cutting ants and their symbiotic fungi, based on interspecific comparisons of DNA sequences (Chapela et al 1994, Hinkle et al 1994, Wetterer et al 1998). The fungus is in the basidiomycete family Lepiotaceae (Mueller et al 1998, 2001), and the fungus associated with Atta cephalotes provisionally has been named Leucoagaricus gongylophorus (Fisher et al 1994).
The symbiotic fungus may have been propagated asexually by leaf-cutting ants for millions of years (Chapela et al 1994, Hinkle et al 1994, Judson and Normark 1996). The usual means of reproduction is clonal; founding queens of leaf-cutting ants initiate gardens in new nests with small bits of fungus carried from their natal nest (Weber 1972). However, there is some evidence that sexual structures can be formed by the fungus on rare occasions (Fisher et al 1994, Pagnocca et al 2001, Mueller et al 2001), which suggests that recombination may be possible.
There has been considerable interest in the genetic structure of clonal lineages (e.g., Jokela et al 1997, Weider et al 1999, Mark Welch and Meselson 2000), though information is available from only a small sample of species. To investigate genetic variation in the symbiotic fungus of the leaf-cutting ant Atta cephalotes, we used random amplified polymorphic DNA (RAPD) markers to differentiate fungal clones from different source nests. RAPD markers typically have high overall variability and so can be useful for detecting genetic differences within species (Williams et al 1990, Parker et al 1998, Sunnucks 2000). This technique has been used to investigate intraspecific genetic variation in several fungi (e.g., Fegan et al 1993, Moore et al 2001).
We compared RAPDs of fungal isolates from Atta cephalotes nests, both within and between two distant sites (Panama and Trinidad), to test the hypothesis that geographically distant populations will be more genetically distinct than nearby populations.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Each of the seven fungal extracts was amplified with ten different 10-base pair primers (supplied by Operon Technologies, Alameda, California): OPN01–CTCACGTTGG; OPN03–GGTACTCCCC; OPN14–TCGTGCGGGT; OPN15–CAGCGACTGT; OPN19–GTCCGTACTG; OPN20–GGTGCTCCGT; OPS02–CCTCTGACTG; OPS04–CACCCCCTTG; OPS06–GATACCTCGG; OPS07–TCCGATGCTG. PCR amplification was performed using RAPD Analysis Beads (Amersham Pharmacia Biotech, Piscataway, New Jersey). Amplification was conducted in a 25 µL volume containing 20 ng of genomic DNA, 25 pmols of a single RAPD primer, and one Ready-To-Go RAPD analysis bead. PCR conditions were as follows: 1 cycle at 95 C for 5 min followed by 45 cycles at 95 C for 1 min, 36 C for 1 min and 72 C for 2 min, with a final extension of 10 min at 72 C. The PCR products were separated electrophoretically on 1.5% agarose gels in TBE buffer (45.0 mM Tris-borate, 1.0 mM EDTA). DNA bands were visualized by staining with ethidium bromide (0.5 µg/mL), observed under UV light and photographed.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
|
Pair-wise comparisons of band sharing in the fungal isolates are shown in Fig. 2. The two isolates from Panama shared 79.6% of their RAPD bands. The five isolates from Trinidad shared an average of 72.1% of bands (range = 61.7–83.3%). In contrast, an average of 36.3% of bands were shared between pairs of isolates from the two different sites (range = 26.9–43.0%). Pairs of fungal isolates within sites shared significantly more bands than pairs between sites (t-test, 19 df, P < 0.001).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). Since Trinidad is an island, the fungus and ant populations there may have been isolated from mainland areas for many generations.
This is the only examination (of which we are aware) of genetic variation of a leaf-cutting ant fungus between distant sites. Pagnocca et al (2001) used RAPD markers on the fungus associated with the leaf-cutting ant Acromyrmex hispidus from a single location in Brazil, in order to demonstrate that fruiting bodies were identical to sterile mycelium. Bot et al (2001) used AFLP (amplified fragment length polymorphism) markers to compare the symbiotic fungus from nests of Acromyrmex octospinosus and A. echinatior from one location in Panama. The AFLP markers were effective in detecting fungal isolates from different nests, and provided evidence that there is occasional exchange of fungal strains between the two Acromyrmex species (Bot et al 2001).
RAPD markers have been useful in other investigations of genetic variation among geographically distant populations of fungi. Li et al (2001) compared RAPD markers in populations of western gall rust fungus (Endocronartium harknesii) collected across western and central Canada from two host pine species. Most of the genetic variation in E. harknesii occurred between the two host species, but within hosts there was more variation among geographically widespread locations than within locations (Li et al 2001). This is comparable to the pattern we observed. In contrast, Furnier et al (1999) found complete uniformity in RAPD markers in isolates of the butternut canker fungus (Sirococcus clavigignenti-juglandacearum) collected from sites across the east-central U.S. This lack of genetic variation could be the result of the recent introduction of a single strain of butternut canker into North America (Furnier et al 1999).
Our results demonstrate the utility of RAPD analysis in measuring genetic variation within the fungal symbionts of leaf-cutting ants. RAPD or other molecular markers (Parker et al 1998, Sunnucks 2000) could allow further investigation of the spatial genetic structure of this symbiotic fungus. Samples from a broad range of distant sites would be particularly interesting. RAPD markers may also help address such questions as the extent of recombination in the fungus (Mueller et al 2001, Pagnocca et al 2001) and the importance of horizontal transfer of the fungus between different ant nests (Bot et al 2001).
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Accepted for publication May 24, 2002.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Bot ANM, Rehner SA, Boomsma JJ., 2001 Partial incompatibility between ants and symbiotic fungi in two sympatric species of Acromyrmex leaf-cutting ants. Evolution 55:1980-1991[Medline]
Chapela IH, Rehner SA, Schultz TR, Mueller UG., 1994 Evolutionary history of the symbiosis between fungus-growing ants and their fungi. Science 266:1691-1694
Fegan M, Manners JM, Maclean DJ, Irwin JAG, Samuels KDZ, Holdom DG, Li DP., 1993 Random amplified polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae. J Gen Microbiol 139:2075-2081
Fisher PJ, Stradling DJ, Pegler DN., 1994 Leucoagaricus basidiomata from a live nest of the leaf-cutting ant Atta cephalotes. Mycol Res 98:884-888
Furnier G, Stolz AM, Mustaphi RM, Ostry ME., 1999 Genetic evidence that butternut canker was recently introduced into North America. Can J Bot 77:783-785
Hinkle G, Wetterer JK, Schultz TR, Sogin ML., 1994 Phylogeny of the attine ant fungi based on analysis of small subunit ribosomal RNA gene sequences. Science 266:1695-1697
Hölldobler B, Wilson EO., 1990 The ants. Cambridge, Massachusetts: Harvard University Press. 732 p
Jokela J, Lively CM, Fox JA, Dybdahl MF., 1997 Flat reaction norms and "frozen" phenotypic variation in clonal snails (Potamopyrgus antipodarum). Evolution 51:1120-1129
Judson OP, Normark BB., 1996 Ancient asexual scandals. Trends in Ecology and Evolution 11:41-46
Li C, Yeh FC, Hiratsuka Y., 2001 Random amplified polymorphic DNA variability among geographic isolates of western gall rust fungus in Canada. Can J For Res 31:1304-1311
Mark Welch D, Meselson M., 2000 Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange. Science 288:1211-1215
Moore A, Challen MP, Warner P, Elliott TJ., 2001 RAPD discrimination of Agaricus bisporus mushroom cultivars. Appl Microbiol Biotechnol 55:742-749[Medline]
Mueller UG, Rehner SA, Schultz TR., 1998 The evolution of agriculture in ants. Science 281:2034-2038
———, Schultz TR, Currie CR, Adams RMM, Malloch D., 2001 The origin of the attine ant-fungus mutualism. Quarterly Review of Biology 76:169-197[Medline]
Pagnocca FC, Bacci MJr., Fungaro MH, Bueno OC, Hebling MJ, Sant'anna A, Capelari M., 2001 RAPD analysis of the sexual state and sterile mycelium of the fungus cultivated by the leaf-cutting ant Acromyrmex hispidus fallax. Mycol Res 105:173-176
Parker PG, Snow AA, Schug MD, Booton GC, Fuerst PA., 1998 What molecules can tell us about populations: choosing and using a molecular marker. Ecology 79:361-382
Quinlan RJ, Cherrett JM., 1979 Role of fungus in the diet of the leaf-cutting ant Atta cephalotes (L). Ecological Entomology 4:151-160
Sunnucks P., 2000 Efficient genetic markers for population biology. Trends in Ecology and Evolution 15:199-203
Weber NA., 1972 Gardening Ants: the Attines. Philadelphia, Pennsylvania: American Philosophical Society. 146 p
Weider LJ, Hobaek A, Colbourne JK, Crease TJ, Dufresne F, Hebert PDN., 1999 Holarctic phylogeography of an asexual species complex I. Mitochondrial DNA variation in arctic Daphnia. Evolution 53:777-792
Wetterer JK, Schultz TR, Meier R., 1998 Phylogeny of fungus-growing ants (Tribe Attini) based on mtDNA sequence and morphology. Molecular Phylogenetics and Evolution 9:42-47[Medline]
Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV., 1990 DNA polymorphisms—amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18:6531-6535
This article has been cited by other articles:
 |
|
 |
|
  M. Gally, A. M. Ramos, D. Dokmetzian, and S. E. Lopez Genetic variability of Phytophthora sojae isolates from Argentina. Mycologia, November 1, 2007; 99(6): 877 - 883. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
