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Ecology |
2Department of Biology, Southern Oregon University, 1250 Siskiyou Blvd., Ashland, Oregon 97520 USA; 3Department of Geology, Southern Oregon University, 1250 Siskiyou Blvd., Ashland, Oregon 97520 USA
Received for publication April 15, 2004. Accepted for publication September 30, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: ectomycorrhizas • heavy metals • morphotyping • mycorrhizas • Oregon white oak • Quercus garryana • serpentine soil • ultramafic
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Kruckeberg, 1984
, 1992
). Serpentine floras provide an opportunity to examine causes of endemism and rarity, as well as molecular mechanisms that have evolved to withstand heavy metals. Among serpentine-tolerant plants are species that accumulate heavy metals (Reeves et al., 1983
; Brooks, 1987
). The distinctive features of serpentine vegetation above ground may be mirrored in the ectomycorrhizal community below ground.
Contact between plants and soil is mediated by mycorrhizas, modified roots formed of fungi in close association with root tissues. Ectomycorrhizas, found on conifers and oaks, for example, have a fungal mantle that encases the root tip, a network of hyphae that grow between root cortical cells, and emanating hyphae that extend into the soil, thus expanding the surface area of the root (Smith and Read, 1997
). The fungal partner of the mycorrhizal symbiosis is in direct contact with soil solutions.
Several factors make it difficult to predict how mycorrhizas on serpentine soil will differ from those on soils derived from metamorphic rocks of different composition (Leyval et al., 1997
). Serpentine minerals may be toxic to some fungal species and thus select for a suite of resistant or tolerant fungal taxa different from those on nonserpentine soils. Mycorrhizas on serpentine soils may adsorb minerals by virtue of extensive hyphae or hyphal walls with adsorbant properties and thus protect trees from toxic concentrations of elements (Gadd, 1990
; Meharg and Cairney, 2000
). Alternatively, mycorrhizas may not contribute to metal tolerance, but may help overcome low soil fertility. Reduced soil fertility might promote mycorrhizal formation. In soils of low fertility, trees are more dependent on ectomycorrhizas for mineral nutrition (Smith and Read, 1997
). Some notable serpentine endemics and nickel hyperaccumulators, e.g., Thlaspi montanum L. var. siskiyouense P. Holmgren, are members of the Brassicaceae (Kruckeberg, 1984
; Brooks, 1987
; Heath et al., 1997
) and lack mycorrhizas.
As compared to adjacent metavolcanic and metasedimentary rocks, ultramafic serpentine soils contain higher magnesium, iron, nickel, chromium, and cobalt and have a higher magnesium-to-calcium ratio, but contain less silicon, calcium, potassium, sodium, aluminum, and phosphorus (Coleman, 1977
; Garcia, 1979
; Alexander, 1988
; O'Hanley, 1996
; Harper, 2003
). Thus serpentine or ultramafic soils are lower in fertility and may contain relatively high amounts of potentially phytotoxic elements.
Previous studies of mycorrhizas associated with conifers on serpentine soils found a lower diversity of ectomycorrhizal fungi. The number of fungal species as measured by macrofungal fruiting bodies collected on serpentine soils was about half that on nonserpentine soils in Washington (Maas and Stuntz, 1969
). Diversity of basidiocarps, mycorrhizas, and mycorrhizal inocula was lower at a serpentine outcrop than on nearby nonserpentine soils in Virginia (Sheets et al., 2000
).
Cenococcum geophilum Fr., a widespread, multihost ectomycorrhizal fungus, has been isolated from roots of Pinus virginiana P. Mill. on both serpentine and nonserpentine soils (Panaccione et al., 2001
). Molecular analyses indicate that C. geophilum on serpentine soils differs from that on nonserpentine soils, yet the serpentine isolates also differ among themselves, as do the nonserpentine isolates.
Vesicular-arbuscular mycorrhizas also occur in plant roots on serpentine soils. Roots of grasses and forbs on serpentine soils in California were abundantly colonized by vesicular-arbuscular mycorrhizas (Hopkins, 1987
). Four species of nickel hyperaccumulators in the Asteraceae were colonized by arbuscular mycorrhizal fungi on South African serpentine soils (Turnau and Mesjasz-Przybylowicz, 2003
).
Oaks also form ectomycorrhizas. Over 40 ectomycorrhizal fungal species, characterized by morphotype, were found in association with roots of Quercus garryana Hook. in southern Oregon (Valentine et al., 2002
, 2004
). In addition, Q. garryana roots also develop vesicular mycorrhizas as a small component of the total mycorrhizal infection (Valentine et al., 2002
).
Many Quercus species occur on serpentine soils worldwide (Hull and Wood, 1984
; Brooks, 1987
). Although Q. garryana is not common on serpentine soils in southern Oregon, it does occur on low-elevation serpentine soils, both as the tree form, Q. garryana Dougl. Ex Hook. var. garryana, and as the shrub form, Q. garryana var. breweri (Engelm.) Jepson. In the Klamath-Siskiyou bioregion of southern Oregon, serpentine rocks and soils occur as isolated outcrops and as extensive ophiolite zones (Wells, 1956
; Ramp and Peterson, 1979
; Alexander, 1988
).
The purpose of this study was to compare ectomycorrhizal diversity of Q. garryana growing on and off serpentine soils at three sites. We examined ectomycorrhizas directly and did not depend on collection of fungal fruiting bodies to estimate mycorrhizal diversity. In addition, two factors were held constant: (1) The ectomycorrhizas were on a single host species, Q. garryana, both on and off serpentine soil. (2) The differing soil types, serpentine and nonserpentine, were close to each other, minimizing microclimate differences. We hypothesized that roots on serpentine soil would have lower mycorrhizal diversity and a unique set of serpentine-specific fungi.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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for the Brickpile Ranch site, and Ramp and Peterson (1979)
for the Sexton Mountain and Eight Dollar Mountain sites. We verified by field inspection of sites that serpentine soils were derived from ultramafic or serpentine sources and that nonserpentine soils were derived from plutonic or nonserpentine metamorphic sources. All serpentine sites had components of serpentine soils with some variability. The Eight Dollar Mountain serpentine soil had no alluvial component. At Sexton Mountain, loose rocks were consistently serpentine, but some alluvial material had washed down from plutonic material above the serpentine bench. Brickpile Ranch was located in a drainage surrounded by nonserpentine metamorphic rocks and had greater soil development resulting from retention of alluvium and organic matter.
Soil and root sampling
For mycorrhizas, four soil samples per tree for each of four trees were collected along radii in the four cardinal directions at the canopy drip line. From February to June 2003, soil samples were extracted with a soil corer (2.5 cm diameter x 25 cm long) from the upper 15–20 cm of soil. Roots were washed and stored in water at 4°C before analysis.
Composites of eight subsamples per soil type at each site were dried and sieved. Soils were analyzed at DANR Analytical Laboratory, University of California, Davis, California, USA. Methods of analyses are described at http://danranlab.ucanr.org. Standard operating procedures used were pH from a saturated soil paste; soil nitrate and extractable ammonium by flow injection analyzer method; carbon by combustion gas analyzer method; extractable phosphorus by the Olsen method; exchangeable potassium, calcium, and magnesium by ammonium acetate extraction; and extractable micronutrients using diethylenetriaminepentaacetic acid (DTPA) for iron and total elements via inductively coupled plasma atomic emission spectroscopy for chromium and nickel.
Morphotyping
Ectomycorrhizal assessment and morphotype descriptions followed Agerer (1991)
, Goodman et al. (2002)
, and Valentine et al. (2002
, 2004)
. Color, shape, branching pattern, surface texture, hyphal structure and abundance, and mantle pattern were recorded. Mycorrhizal tips of each morphotype were counted. Micrographs of each morphotype and its mantle peel were taken with a Spot RT digital camera (Diagnostic Instruments, Inc., Sterling Heights, Michigan, USA) using Leica DMLB compound and MZ75 dissecting microscopes. Detailed descriptions of ectomycorrhizal morphotypes are listed in the Supplemental Data accompanying the online version of this article. Numbers of morphotypes at each site and soil category were compared by two-way ANOVA.
Morphotypes were categorized as dark or light and as fuzzy or smooth, with or without abundant emanating hyphae. Data were analyzed using a chi-square 2 x 2 contingency table (Brower et al., 1998
).
For individual sites we calculated Simpson's diversity index, DS = 1 – [
ni(ni – 1)/N(N – 1)] where ni is the number of individuals in the ith species, and N is the total number of morphotypes, and Shannon's diversity index, H' = –pilog pi where pi is the proportion of the total number of individuals that belong to species i. In addition, maximum values of H' and DS were calculated based on even distributions of the actual numbers of morphotypes from each site. To compare pairs of soils or sites, we calculated Sørensen's index of similarity, CCS = 2c/(s1 + s2) where c is the number of species in common and s1 and s2 are the total number of species on two sites (Brower et al., 1998
). Two-tailed t tests were used to evaluate significance of differences between means.
Molecular methods
DNA was extracted from ectomycorrhizal tips using CTAB and amplified via polymerase chain reaction (PCR) using the fungal-specific primers ITS1-F and ITS4 (Gardes and Bruns, 1993
; Valentine et al., 2004
). PCR products were cut with restriction enzymes HinfI and TaqI, and the restriction fragments were separated on a 4% acrylamide gel. Gels were stained with ethidium bromide and photographed using a UV transilluminator and camera with Fotodyne imaging software. Gels were analyzed with ONE-Dscan software. Patterns of restriction fragment length polymorphisms (RFLPs) were used as characters to distinguish among similar morphotypes (see Data Supplement accompanying the online version of this article).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Species richness
Ectomycorrhizal morphotypes were abundant and diverse on serpentine and nonserpentine soils (Figs. 2–29, Table 2, Supplemental Data). Mantle peels (Figs. 10–13) and RFLP patterns (Supplemental Data) were used to distinguish among morphotypes that varied in form, pigmentation, and extent of emanating hyphae. No single form dominated. Some were single unbranched tips, others clustered (Figs. 9, 18, 28) or crowded with multiple morphotypes. All root tips were mycorrhizal. Pigmentation ranged from pale (Figs. 2, 22, 27) to very dark (Figs. 5, 15, 25, 26). Emanating hyphae varied from none, to short cystidial, to longer ones along the length of the mycorrhiza (Figs. 5, 7, 20). Others formed dense hyphal fans or mycelial strands (Figs. 4, 19, 23). Emanating hyphae were either the same color as the mantle (Figs. 5, 20) or white (Figs. 4, 19).
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Variation among sites
Although sites differed in total morphotypes and in the proportion of serpentine-specific morphotypes, neither total numbers of morphotypes nor numbers of soil-specific morphotypes differed significantly among sites (P = 0.95 and P = 0.82, respectively) or soils (P = 0.87 and P = 0.78, respectively; Table 2).
Dominant morphotypes varied among sites (see Data Supplement accompanying the online version of this article). At Sexton Mountain, the predominant morphotypes were SS07 (Fig. 26), SS24 (Fig. 23), and SS40 (Fig. 14). At Brickpile Ranch, SS36 (Fig. 22), SS52 (Fig. 15), SS59 (Fig. 18), and SS60 (Fig. 28) were abundant. At Eight Dollar Mountain, SS36 (Fig. 22) was abundant, as were SS78 (Fig. 19) and SS89 (not shown).
Diversity at all sites was similar, with no single morphotype dominating (Table 5). Neither Simpson's diversity index nor Shannon's diversity index differed significantly (P > 0.1) between serpentine and nonserpentine soils.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The Eight Dollar Mountain site on the Josephine ophiolite in the Klamath-Siskiyou bioregion of southwestern Oregon supports a classic serpentine flora on soils with levels of chromium and nickel sufficient for mining (Brooks, 1987
; Kruckeberg, 1992
). The relatively lower magnesium-to-calcium ratio in the well developed Brickpile Ranch serpentine soils may result from weathering (Brooks, 1987
). The Brickpile Ranch serpentine outcrop is located in a drainage surrounded by metamorphic rocks, but its high iron and significant chromium and nickel demonstrate a serpentine influence. At Sexton Mountain, the levels of chromium and nickel and a magnesium-to-calcium ratio greater than 1.0 support its inclusion as a serpentine site. On the serpentine soil with the greatest concentration of serpentine elements (Eight Dollar Mountain), the oak is Q. garryana var. breweri, a shrub form of Q. garryana. The other two sites with apparently greater alluvial influence supported Q. garryana var. garryana, the tree form. It is possible that Q. garryana grows on serpentine soil in areas where there are intrusions of nonserpentine materials, alluvial influences, greater weathering or greater soil development.
Species richness
Oak mycorrhizas on serpentine soils are abundant and diverse. Oak ectomycorrhizal diversity on serpentine soils is greater than that on alluvial soils derived from volcanic rocks in southern Oregon (Valentine et al., 2004
). This contrasts with studies on conifers, based on fruiting bodies and mycorrhizas, in which diversity of mycorrhizal fungi was lower on serpentine soils (Maas and Stuntz, 1969
; Sheets et al., 2000
). The relatively higher diversity of ectomycorrhizas associated with oaks on serpentine soils may result from climatic differences. Our sites differed from the conifer sites cited above in that they were in a region with a Mediterranean climate and extreme seasonal drying of soils.
Soils polluted with heavy metals from industrial wastes are extreme examples of low-fertility soils. On such sites, fungal responses vary. Some asexual forms of Ascomycota increased while others decreased (Gadd, 1990
). Soils high in heavy metals may select tolerant species or genotypes. In greenhouse-grown plants, nickel (100 µg/g) reduced ectomycorrhizal colonization of Quercus rubra L. by Suillus luteus (Fries) S. F. Gray (Dixon, 1988
). Mycorrhizas of Scots pine with zinc-tolerant genotypes of Suillus continued to take up phosphorus at control levels even when zinc levels were high (Adriaensen et al., 2003
). This protection is in the form of a mycorrhiza that maintains normal nutrient uptake in the presence of heavy metal contaminants.
Properties of serpentine mycorrhizal morphotypes
Dark morphotypes were not significantly more abundant on serpentine than on nonserpentine soils. Although melanin in cell walls can adsorb heavy metals (Gadd and de Rome, 1988
), these pigments appear to play a minor role in selection of serpentine mycorrhizal morphotypes. The percentage of morphotypes with abundant hyphae was not consistently higher on serpentine than on nonserpentine soils. Abundant hyphae are not correlated with development of serpentine morphotypes. Neither dark morphotypes nor abundance of hyphae indicates the serpentine level of a soil. It appears that elements such as iron, nickel, and chromium do not select for mycorrhizas with these physical characteristics.
Specificity to soil type
Many morphotypes were unique to serpentine soils. These are possible serpentine endemics and may tolerate or resist heavy metals. Another set of ectomycorrhizal morphotypes occurs on nonserpentine soils only, indicating species that avoid serpentine soil. A third group of species found on both serpentine and nonserpentine soils have the ability to survive and the potential to infect oaks in diverse soil types.
The proportion of serpentine-specific ectomycorrhizas with Q. garryana (43%) is greater than that reported by Maas and Stuntz (1969)
, who found that 19% of 212 fungal fruiting bodies occurred only on serpentine soils, 63% only on nonserpentine soils, and 18% on both soil types. However, different tree species occupied their serpentine and nonserpentine sites, so the proportion of potentially similar morphotypes would be lower.
Most morphotypes were found at only one site. One morphotype was found in all three serpentine soils. Cenococcum geophilum, an asexual ascomycete, was found at all six sites (serpentine and nonserpentine), as it is in most ectomycorrhizal communities world wide.
Variation among sites
Mycorrhizal species richness on serpentine soils is similar to that on nonserpentine soils, indicating that serpentine soils do not inhibit mycorrhizal formation. This may be the result of microclimate similarities such as temperature, precipitation, wind, soil influences, and available inocula. Beta diversity, the change in species composition with distance, remained relatively constant across the scale of this project. The value of Sørensen's index of similarity for adjacent sites was comparable to that among nonserpentine sites.
Our findings of rich ectomycorrhizal diversity on serpentine soils contrast with those of Maas and Stuntz (1969)
and Sheets et al. (2000)
. Several factors in our study contributed to the contrast between our results and those cited. We examined ectomycorrhizas directly and did not use collections of fungal fruiting bodies to estimate mycorrhizal diversity. This allowed us a more direct estimation of mycorrhizal species richness and diversity without depending on fruiting body production. We found no fungal fruiting bodies on most sites, so for our sites, fungal fruiting body production would have been a poor predictor of ectomycorrhizal diversity.
We conclude that oaks on serpentine soils have abundant mycorrhizal diversity with a significant fraction of fungal species found only on serpentine soils. The serpentine properties of low nutrient status combined with the seasonal dryness of Mediterranean climates may promote or support mycorrhizal diversity and abundance and balance the potentially toxic effects of heavy metals.
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FOOTNOTES |
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4 Author for correspondence: E-mail: southworth{at}sou.edu
. Fax: 541-552-6415
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Agerer R. 1991 Characterization of ectomycorrhizas. Methods in Microbiology 23: 25-73
Alexander E. B. 1988 Morphology, fertility, and classification of productive soils on serpentinized peridotite, California, USA. Geoderma 41: 337-351
Brooks R. R. 1987 Serpentine and its vegetation. Dioscorides Press, Portland, Oregon, USA
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Dixon R. K. 1988 Response of ectomycorrhizal Quercus rubra to soil cadium, nickel and lead. Soil Biology and Biochemistry 20: 555-559[CrossRef]
Gadd G. M. 1990 Metal tolerance. In C. Edwards [ed.], Microbiology of extreme environments, 178–210. McGraw-Hill, New York, New York, USA
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Garcia M. 1979 Petrology of the Rogue and Galice Formations, Klamath Mountains, Oregon: identification of a Jurassic Island arc sequence. Journal of Geology 86: 29-41
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Kruckeberg A. R. 1992 Plant life of western North American ultramafics. In B. A. Roberts and J. Proctor [eds.], The ecology of areas with serpentinized rocks: a world view, 31–73. Kluwer Academic Publishers, Dordrecht, Netherlands
Leyval C. K. Turnau K. Haselwandter 1997 Effect of heavy metal pollution on mycorrhizal colonization and function: physiological, ecological and applied aspects. Mycorrhiza 7: 139-153
Maas J. L. D. E. Stuntz 1969 Mycoecology on serpentine soil. Mycologia 61: 1106-1116
Meharg A. A. J. W. G. Cairney 2000 Co-evolution of mycorrhizal symbionts and their hosts to metal-contaminated environments. Advances in Ecological Research 30: 69-112
O'Hanley D. S. 1996 Serpentinites: records of tectonic and petrologic history. Oxford University Press, London, UK
Panaccione D. G. N. L. Sheets S. P. Miller J. R. Cumming 2001 Diversity of Cenococcum geophilum isolates from serpentine and non-serpentine soils. Mycologia 93: 645-652[CrossRef][ISI]
Ramp L. N. V. Peterson 1979 Geologic map of Josephine County, Oregon, 1979. Bulletin 100, Geology and mineral resources of Josephine County, Oregon. Department of Geology and Mineral Industries, State of Oregon, Salem, Oregon, USA
Reeves R. D. R. M. MacFarlane R. R. Brooks 1983 Accumulation of nickel and zinc by western North American genera containing serpentine-tolerant species. American Journal of Botany 70: 1297-1303[CrossRef][ISI]
Sheets N. L. J. R. Cumming S. P. Miller D. G. Panaccione 2000 Diversity of ectomycorrhizal fungal communities and Cenococcum geophilum populations from serpentine and nonserpentine soils. Phytopathology 90: S71 (Abstract)
Smith S. E. D. M. Read 1997 Mycorrhizal symbiosis. Academic Press, London, UK
Turnau K. J. Mesjasz-Przybylowicz 2003 Arbuscular mycorrhiza of Berkheya coddii and other Ni-hyperaccumulating members of Asteraceae from ultramafic soils in South Africa. Mycorrhiza 13: 185-190[CrossRef][ISI][Medline]
Valentine L. L. T. L. Fiedler S. R. Haney H. K. Berninghausen D. Southworth 2002 Biodiversity of mycorrhizas on Garry oak (Quercus garryana) in a southern Oregon savanna. In R. B. Standiford, D. McCreary, and K. L. Purcell [eds.], Proceedings of the fifth symposium on oak woodlands: oaks in California's changing landscape. USDA Forest Service General Technical Report PSW-GTR-184, 151–157. Also available at http://danr.ucop.edu/ihrmp/proceed/valentine.pdf, accessed 20 September 2004
Valentine L. L. T. L. Fiedler A. N. Hart C. A. Petersen H. K. Berninghausen D. Southworth 2004 Biodiversity of ectomycorrhizal fungi associated with Quercus garryana. Canadian Journal of Botany 82: 123-135[ISI]
Wells F. G. 1956 Geologic map of the Medford Quadrangle, Oregon–California. Map GQ-89. U.S. Geological Survey, Washington, DC, USA
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