natural abundance as a possible marker of the ectomycorrhizal habit of trees in mixed African woodlands

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1 New Phytol. (1990), 115, natural abundance as a possible marker of the ectomycorrhizal habit of trees in mixed African woodlands BY PETER HOGBERG Department of Forest Site Research, Swedish University of Agricultural Sciences, S Umea, Sweden {Received 20 November 1989; accepted 27 February 1990) SUMMARY The partial contribution by fixed nitrogen in Ng-fixing plants can be estimated if the ^^N natural abundance of non-ng-fixing reference species, which derive their N from the soil, deviates from that of atmospheric Ng (the ^^N natural abundance method). Data from Tanzanian miombo woodland showed a significant difference (= ^^N%o) in ^^N abundance between ectomycorrhizal and vesicular-arbuscular (VA) mycorrhizal reference species. This finding casts doubt upon arbitrary selection of reference species, and also raises the possibility of using ^^N abundance as a marker of the ectomycorrhizal habit. It is suggested that the higher ^*N abundance of ectomycorrhizal tree species could result from differences in discrimination during uptake or from greater utilization of organic N. Key words: ^^N, ectomycorrhiza, nitrogen fixation. INTRODUCTION Large subhumid areas in East and South-Central Africa, with soils poor in nitrogen and phosphorus, are covered by deciduous miombo woodlands (Young, 1976; White, 1983). A variety of tree species of the genera Brachystegia, Isoberlinia and Julbernardia (Leguminosae: subfamily Caesalpinioideae) dominate in miombo. These, as well as some co-dominants, form ectomycorrhizas, while species forming vesicular-arbuscular (VA) mycorrhizas or potentially Ng-fixing root nodules plus VA mycorrhizas, are less abundant (Hogberg & Nylund, 1981; Hogberg, 1982; Hogberg, 1986a, b; Hogberg & Piearce, 1986). The precise roles played by root symbioses in miombo are not known. It is sometimes possible to determine the proportion of N fixed in Ng-fixing trees by using the ^^N natural abundance method (e.g. Shearer & Kohl, 1986; Virginia et al., 1988). This method requires that the ^^N abundance of non-ng-fixing reference species deviates significantly from the atmospheric abundance, and that reference and tested species extract N from the same soil sources and have similar phenology (Shearer & Kohl, 1986). The method overcomes several difficulties facing other methods, and is attractive to use in studies on deep-rooted trees (e.g. Shearer et al., 1983). Usually, soils are slightly enriched in ^^N (Shearer & Kohl, 1986). The deviations are minute. Results can be expressed in 8 ^^N (in %o) units: 8 ^^N = 1000 X (^sample~^standar(l)/^standard where R = mass 29/mass 28 and the standard is atmospheric Ng ( atom % ^^N, cf. Junk & Svec, 1958; Mariotti, 1983). Results of high precision ^^N determinations performed on leaf samples collected previously at five woodland sites in Tanzania (Hogberg, 1986 a) are reported here in an attempt to discover if there is a difference in ^^N abundance between ecto- and VA mycorrhizal species. This question, which has not been addressed before, is pertinent to the choice of reference species. MATERIALS AND METHODS Details about locations, climate, soils, vegetation and sampling strategy were reported previously (Hogberg, 1986 a). The tree species sampled were nodulating species as well as non-nodulating ecto- or VA mycorrhizal species occurring intermingled with them (Table 1). Leaves of five trees of each of 48 species-site combinations were sampled. Chemical analysis was performed as described by Haystead (1983). 32-2

2 484 P. Hogberg Table 1. Tree species sampled at Tanzanian woodland sites Species I II ni IV V Potentially Ng-fixing, confirmed VA mycorrhizal Acacia goetzei Harms "~ ~ A. nigrescens Oliv. _..j. -I- 4- A. nilotica (L.) Del A. polyacantha Willd Albizia versicolor (Welw. ex) Oliv. + Dalbergia melanoxylon Guill. Perr D. nitidula (Welw. ex) Bak Dichrostachys cinerea (L.) Wight & Arn Entada abbyssinica (Steud. ex) A. Rich Erythrophleum africanum (Benth.) Harms. + ~ Pericopsis angolensis (Bak.) van Meeuwen Pterocarpus angolensis DC. _^_ _-..^-.+ -l--(- P. rotundifolius (Sond.) Druce ssp. polyanthus Mend & E. P. Sousa Xeroderris stuhlmannii Mend & E. P. Sousa Non-Ng-fixing, confirmed or potentially VA mycorrhizal Bauhinia petersiana Bolle ~ ~ ^ Combretum colunum Fresen x x C. zeyherii Sond. x x Cussonia arborea (Hochst. ex) A. Rich _ x x x Diplorhynchus condylocarpon (Mull. Arg.) Pichon x x x x Markhamia obtusifolia (Bak.) Sprague _ -_ x x Pseudolachnostylis maprouneifolia Pax. x x x Sterculia quinqueloba (Garcke) K. Schum. x Terminalia sericea (Burch. ex) DC. x Non-Ng-fixing, confirmed ectomycorrhizal Brachystegia boehmii Taub. _. x x B. microphylla Harms x B. spiciformis Benth. x jfulbernardia globifiora (Benth.) Troupin x x x -, not sampled at the site; + + +, nitrogenase (C2H2-reduction) activity confirmed by test of nodules from the site; + +, nodules found at the site; +, reported to form nodules elsewhere (Allen & Allen, 1981); x, non-nodulated species sampled at the site. Table 2. ^^N abundance of leaf samples collected in May of different years at a woodland site (III) in Tanzania Species Symbiotic status A 15TSJ (0/ \ Brachystegia boehmii B. microphylla Julbernardia globifiora Pterocarpus angolensis Diplorhynchus condylocarpon Xeroderris stuhlmannii Dichrostachys cinerea VA Data from 1980 refer to composite samples from five trees, while other data are averages for five trees. There is no significant variation between 1981 and 1984, and no species xyear interaction (two-way analysis of variance: F^ gg, = 017 and /^g gg, = 1 48, respectively)., ectomycorrhizal; VA, VA mycorrhizal; NO, nodulated.

3 abundance and the ectomycorrhizal habit 485 v> (0 g ±0 0) J J I J I IV Figure 1. ^^N abundance (x ± 1 SE) of leaves of Tanzanian woodland tree species forming different types of root symbioses. D, ectomycorrhizal; O, VA mycorrhizal; #, nodulated and VA mycorrhizal. Horizontal lines show the mean for each group of species. RESULTS AND DISCUSSION Figures of 8 ^^N {%o) varied between 2 and -I-2-5 (Fig. 1), and 1 SE was always less than units {x = 0'23). As regards precision, it was reassuring that there was relatively little variation between years (Table 2). The values for ecto- and VA mycorrhizal species clearly differed: ectomycorrhizal species had <J units higher ^^N abundance (Fig. 1). This difference was larger than that between Ng-fixing and non-ng-fixing VA mycorrhizal species. At site III the number of species was enough to apply a Mann-W^hitney U-test (Siegel, 1956), which showed that the difference was significant (wi = 3, «2 = 6, U = 0: P = 0-012). There was also some variation between sites, but this did not obscure the difference between ecto- and VA mycorrhizal species (Fig. 2). Data for Ng-fixing species were normally distributed around a value slightly below 0 (Fig. 1). This indicates that a large proportion of their N was derived from the atmosphere and a small discrimination during N^ fixation (Shearer & Kohl, 1986). Obviously, in selecting reference species the mycorrhizal habit has to be taken into accourit. The difference between ecto- and VA mycorrhizal species may have a number of explanations: (i) ecto- and VA mycorrhizal species occupy different parts of the sites or root at different soil depths; (ii) VA mycorrhizal species carry out some internal discrimination which does not occur in ectomycorrhizal species; (iii) the two types of mycorrhizas discriminate differently against ^^N during uptake; (iv) ectomycorrhizal species utilize sources of soil N other than those available to VA mycorrhizal species. Of these, (i) is ruled out since the stands are truly mixed and the roots of the various species clearly (A (0 0) u X U) +2 - ± J Figure 2. Variation in ^^N abundance between sites. Lines connect species which occurred at more than one site. Site III differs (P< 0-001) from sites II and IV (two-way analysis of variance: -^1,24) ^3-5 and JFJ^ ^g^ = 81-8, respectively). Symbols as in Figure 1. mingle. Only four closely related (Caesalpinioideae) ectomycorrhizal tree species were samples (Table 1), which makes (ii) possible. However, they were compared with VA mycorrhizal species from eight families, including one from Caesalpinioideae, and the variations between these were comparatively small. With regard to (iii), there is a report that ectomycorrhizal pine had a ^^N abundance different from non-mycorrhizal (Bardin, Domenach & Chalamet, 1977), but a difference between ecto- and VA mycorrhizal species has not previously been reported. The fact that ectomycorrhizal species had 0-5 % (of dry mass) higher N concentration than VA IV

4 486 P. Hogberg mycorrhizal species indicates a more efficient uptake of this element (Hogberg, 1989). Recent work on temperate ectomycorrhizas has shown that they have the capacity to utilize complex organic N compounds, e.g. proteins (e.g. Abuzinadah, Finlay & Read 1986). This has not been found to be the case for VA mycorrhizal species. During decomposition and nitrification, enzymatic processes can discriminate against ^^N (e.g. Shearer & Kohl, 1986). A higher ^^N abundance could thus be due to the uptake of a mixture of organic and inorganic N rather than solely inorganic N. The most likely explanations are therefore (iii) and (iv). It should be possible by this method to distinguish between ectoand VA mycorrhizal species, and also to get a more detailed insight into their nitrogen nutrition. Further work is in progress. ACKNOWLEDGEMENT This study was funded by the Swedish Agency for Research Cooperation (SAR). I would like to thank Dr A. Haystead and staff for excellent analytical services. REFERENCES ABUZINADAH, R. A., FINLAY, R. D. & READ, D. J. (1986). The role of proteins in the nitrogen nutrition of ectomycorrhizal plants II. New Phytologist 103, ALLEN, O. N. & ALLEN, E. K. (1981). The Leguminosae : A Source Book of Characteristics, Uses and Nodulation. Macmillan, London and Basingstoke. BARDIN, R., DOMENACH, A.-M. & CHALAMET, A. (1977). Rapports isotopiques de Tazote II. Revue d'ecologie et de Biologie du Sol 14, 395^02. HAYSTEAD, A. (1983). Analysis of nitrogen isotope ratios by mass spectrometry. In: Soil Analysis: Instrumental Techniques and Related Procedures (Ed. by K. A. Smith), pp Marcel Dekker, New York. H6GBERG, P. (1982). Mycorrhizal associations in some woodland and forest trees and shrubs in Tanzania. New Phytologist 92, H6GBERG, P. (1986a). Nitrogen fixation and nutrient relations in savanna woodland trees (Tanzania). Journal of Applied Ecology 23, HQGBERG, P. (19866). Soil nutrient availability, root symbioses and tree species composition in tropical Africa: a review. Journal of Tropical Ecology 2, HOGBERG, P. (1989). Root symbioses of trees in savannas. In: Mineral Nutrients in Tropical Forest and Savanna Ecosystems (Ed. by J. Proctor), pp Blackwell, Oxford. HOGBERG, P. & NYLUND, J.-E. (1981). Ectomycorrhizae in coastal miombo woodland of Tanzania. Plant and Soil 63, HOGBERG, P. & PIEARCE, G. D. (1986). Mycorrhizas in Zambian trees in relation to host taxonomy, vegetation type and successional patterns. Journal of Ecology 74, JUNK, G. & SV, H. J. (1958). The absolute abundance of the nitrogen isotopes in the atmosphere and compressed gas from various sources. Geochimica et cosmochimica acta 14, MARIOTTI, A. (1983). Atmospheric nitrogen is a reliable standard for natural ^^N abundance measurements. Nature 303, (1983). SHEARER, G. & KOHL, D. H. (1986). Nj-fixation in field settings: estimations based on natural ^*N abundance. Australian Journal of Plant Physiology 13, SHEARER, G., KOHL, D. H., VIRGINIA, R. A., BRYAN, B. A., SKEETERS, J. L., NILSEN, E. T., SHARIFI, M. R. & RUNDEL, P. W. (1983). Estimates of N2-fixation from variations in the natural abundance of ^*N in Sonoran Desert ecosystems. Oecologia 56, SiEGEL, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, Tokyo. VIRGINIA, R. A., JARRELL, W. M., RUNDEL, P. W., SHEARER, G. & KOHL, D. H. (1988). The use of variation in the natural abundance of ^^N to assess symbiotic nitrogen fixation by woody plants. In: Stable Isotopes in Ecological Research (Ed. by P. W. Rundel, J. R. Ehleringer & K. A. Nagy), pp Springer Verlag, New York. WHITE, F. (1983). The Vegetation of Africa. Unesco, Paris. YOUNG, A. (1976). Tropical Soils and Soil Survey. Cambridge University Press, Cambridge.

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