Does past contact reduce the degree of mutualism in the Alnus rubra - Frankia symbiosis?

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<ul><li><p>Does past contact reduce the degree ofmutualism in the Alnus rubra Frankiasymbiosis?</p><p>John H. Markham and Chris P. Chanway</p><p>Abstract: Although most vascular plants have symbiotic relationships with soil microbes, and there is an extensivetheoretical literature on the evolution of mutualism, there has been little experimental examination of the evolution ofmutualism between plants and their microbial symbionts. We inoculated red alder (Alnus rubra Bong.) seedlings fromthree high- and three low-elevation populations with crushed nodule suspensions containing the nitrogen fixingbacterium Frankia from either the parent trees (familiar strains) or the other plant population sampled within the parentwatershed (unfamiliar strains). The inoculated seedlings were planted on three high- and three low-elevation sites.Growth was monitored over the second and third year following planting, after which the whole plants were harvested.The proportion of nitrogen derived from fixation was estimated from the ratio of stable nitrogen isotopes in theharvested leaves. On low-elevation sites, which had high soil nitrogen, plants with familiar Frankia strains were halfthe size and derived less fixed nitrogen from their symbionts compared with plants inoculated with unfamiliar Frankiastrains. On high-elevation sites, which had low soil nitrogen, the type of inoculum had little effect on plantperformance, although plants with familiar inoculum were consistently larger than plants with unfamiliar inoculum.These results suggest that the degree of mutualism in this symbiosis depends on environmental conditions and maydecrease with time.</p><p>Key words: coevolution, Frankia, Alnus rubra, mutualism, nitrogen fixation, symbiosis.</p><p>Rsum : Bien que la majorit des plantes vasculaires aient des relations symbiotiques avec des microorganismes dusol, et quil y ait une importante littrature thorique sur lvolution du mutualisme, il existe peu de donnesexprimentales sur lvolution du mutualisme entre les plantes et leurs symbiontes microbiens. Les auteurs ont inoculdes plantules daulnes rouges (Alnus rubra Bong.) provenant de trois populations de haute altitude et de troispopulations de basse altitude avec des suspensions de nodules broys contenant les Frankia soit des arbres parents(souches familires) ou soit dune autre population de plantes chantillonnes partir du mme bassin hydrographique(souches non familires). Les plantules inocules ont t plantes sur trois sites de hautes altitude et trois sites de bassealtitude. Les auteurs ont suivi la croissance au cours des deuxime et troisime annes aprs la plantation, aprs quoiils ont rcolt les plantes entires. Ils ont estim la proportion de lazote drive de la fixation en utilisant le rapportdes isotopes stables de lazote dans les feuilles rcoltes. Sur les sites de basse lvation, bien pourvus en azote, lesplantes ayant les souches familires de Frankia sont deux fois plus petites et drivent moins dazote partir de lafixation par leurs symbiontes, comparativement aux plantes inocules avec des souches de Frankia non familires. Surles sites en altitude, moins bien pourvus en azote, le type dinocule nexerce pas beaucoup deffet sur la performancedes plantes bien que les plantes avec linoculum familier soient toujours plus grandes avec les inoculums non familiers.Ces rsultats suggrent que les degrs de mutualisme dans cette symbiose, dpendent de facteurs environnementaux etpeuvent diminuer avec le temps.</p><p>Mots cls : covolution, Frankia, Alnus rubra, mutualisme, fixation de lazote, symbiose.</p><p>[Traduit par la Rdaction] Markham and Chanway 441</p><p>Introduction</p><p>Most vascular plants enter into symbiotic relationshipswith soil microbes, i.e., mycorrhizal fungi and in some casesnitrogen-fixing bacteria or actinomycetes (Douglas 1995).Because symbiotic relationships with soil microbes play akey role in plant nutrition and ecosystem processes (Mallochet al. 1980; Perry et al. 1989), their development and main-tenance are important ecological questions. Because symbi-otic microbes can improve the mineral nutrition of theirhosts and the plants can provide the microbes with a sourceof reduced carbon, these relationships are often assumed tobe mutualistic. However, there are known instances where</p><p>Can. J. Bot. 77: 434441 (1999) 1999 NRC Canada</p><p>434</p><p>Received August 1, 1998.</p><p>J.H. Markham.1 Faculty of Forestry, University of BritishColumbia, 270-2357 Main Mall, Vancouver, BC V6T 1Z4,Canada.C.P. Chanway. Faculty of Forestry and Department of SoilScience, Faculty of Agricultural Sciences, University ofBritish Columbia, 270-2357 Main Mall, Vancouver,BC V6T 1Z4, Canada.1Author to whom all correspondence should be addressed.Present address: Biology Department, Douglas College,P.O. Box 250, New Westminster, BC V3L 5B2, Canada.e-mail:</p><p>J:\cjb\cjb77\cjb-03\B98-227.vpMonday, August 16, 1999 8:57:13 AM</p><p>Color profile: DisabledComposite Default screen</p></li><li><p>symbiotic soil microbes provide no apparent benefit to theirhost (Hagedorn 1978; Hahn et al. 1988; Baker et al. 1980),so the development and maintenance of mutualism in plantsoil microbe relationships is not assured. The use of gametheory has shown that mutualisms can be advantageous (andtherefore selected for) even if there is an immediate gain ina more selfish interaction (Axelrod and Hamilton 1981;Ferriere and Michod 1996; Nowak and Sigmund 1993). Theevolution of mutualism by this scenario requires that there issome probability of continued interaction between partnersand some ability to recognize or maintain a relationship withpotential partners. However, little experimental evidence ex-ists which examines the degree of mutualism between plantsand their symbionts under different conditions.</p><p>Plants from eight families have been observed to enterinto a symbiotic relationship with the nitrogen-fixing actino-mycete Frankia (Bond 1983) by forming root nodules thatencapsulate the microbe. In coastal British Columbia, red al-der (Alnus rubra Bong.) is the most common host ofFrankia. The presence of red alder on a site can result inlarge increases in soil nitrogen (Bormann et al. 1994). Weperformed a cross-inoculation experiment to determine if redalder populations performed best with Frankia populationsnormally associated with them or with Frankia populationsfound in other red alder populations. Our hypothesis wasthat coevolution of red alder populations with familiar (i.e.,indigenous) Frankia populations would lead to an increasein the degree of mutualism. Given the difficulties in deter-mining the fitness of Frankia in field experiments, our mainfocus was on plant performance. Plant performance was as-sessed by growing inoculated plants both with and withoutother red alder neighbours to determine if competitive inter-actions had any effect on the different A. rubra Frankiacombinations. We examined plantmicrobe interactions be-tween high- and low-elevation populations within single wa-tersheds because there can be substantial plant geneticvariation between A. rubra populations at this geographicscale (Ager et al. 1993). All wild red alder examined in thisregion have nodules (personal observation), and we made noattempt to control infection of experimental plants by wildFrankia strains after planting. Therefore, we were also inter-ested in determining how long an initial inoculation treat-ment would have an effect on planted seedlings.</p><p>Materials and methods</p><p>Collection of experimental materialSeed and nodules were collected from one high- and one low-</p><p>elevation population in each of three watersheds in southwesternBritish Columbia (Table 1). The low-elevation populations were allbelow 120 m, and the high-elevation populations were between540 and 700 m, the elevation limit of red alder in each of the wa-tersheds. Each population contained at least 15 reproductive indi-viduals, and pooled seed and nodule collections were made on atleast eight trees in each population. The age of each populationwas determined by taking cores at breast height from at least fiveof the dominant trees in each stand. Site index, an estimate of theheight of the dominant trees in a stand at age 50, was calculatedfrom standard tables (Harrington and Curtis 1986). Seed was col-lected in the fall of 1991 and germinated in a greenhouse on a ster-ilized peatperlite mixture in April, 1992. Before planting,seedlings from each of the six collection populations were inocu-</p><p>lated with a suspension of crushed nodules from either the seed-lings parent population (familiar inoculum) or from the other par-ent population (i.e., different elevation) in the same watershed(non-indigenous or unfamiliar inoculum). Nodules were collectedfrom the base of the seed trees in July 1992 and stored at 5C.Nodules were surface sterilized in 50% bleach for 5 min to ensurethat only microbes within in the nodule were present in the inoc-ulum. Using this procedure no surface contaminants were cultur-able when the nodules were placed on plates of tryptic soy agar for3 days. Nodules were crushed in distilled water with a sterile mor-tar and pestle. Seedlings were inoculated by injecting 1 mL of thecrushed nodule suspension into the rooting zone with a syringe. Toensure nodulation, seedlings were inoculated three times: 1 weekbefore transplanting (July 15, 1992), 3 weeks after transplanting(August 10), and in April 1993. For the first inoculation a suspen-sion of 16.7 mg fresh weight of nodules per millilitre was used(100 times the concentration used by Huss-Dannell 1991). For sub-sequent inoculations a concentration of 1.67 mg/mL was used. Toensure that variation in initial plant size had no effect on the varia-tion in plant performance between each treatment, the followingrandomization procedure was used. Prior to initial inoculation,seedlings from each population were sorted by height and plantsapproximately 1.5 times larger and smaller in height than the aver-age plant height were discarded. Plants were then randomly as-signed to each treatment. Plants were then grouped by treatmentsand any treatment that had mostly taller or shorter than averageplants had new plants assigned to them. Noninoculated seedlingsgrown in the same type of containers as the inoculated seedlingsdid not develop nodules.</p><p>Field proceduresA full set of crosses from each collection watershed was planted</p><p>onto each of three high- and three low-elevation sites near the cen-ter of the region of the collection populations. Therefore, therewere three replicate cross-inoculation treatments (one from eachcollection watershed) planted onto each of three planting sites ateach elevation, for a total of nine replicates per planting elevation.To determine if the relationship between the different plantbacteria crosses had any effect on the competitive ability of theplants, a second complete set of crosses was also planted withthree alder seedlings surrounding each target seedling, on each site.The neighbouring seedlings for each target seedling were from thesame population as the target seedling and inoculated with Frankiacollected from around each planting site using the same proceduredescribed above. All planting sites were located within the Univer-sity of British Columbia research forest near Haney, B.C. (Ta-ble 2). The planting sites were all disturbed forest sites, a typicalhabitat of red alder. All the sites had been previously dominated byconiferous forests and harvested. A bulldozer was used to clear allvegetation from the sites and remove the organic soil layer in earlyJuly 1992. Plots were laid out using a 2-m spacing on each site. Toensure a homogeneous rooting environment, 30 cm diameter 30 cm deep holes were dug at each plot and the soil sieved througha 1-cm mesh back into the holes. Seedlings were dibbled into thecenter of the hole. Planting took place from July 21 to 23, 1992,with treatments randomly assigned to the plots. Plants were ap-proximately 4 cm tall at the time of planting, and a subsample ofunplanted, inoculated seedlings had nodules while uninoculatedplants had none. Neighbours were three plants on the north, south-west, and southeast side of the target plants, planted at a distanceof 8 cm from the base of the target plant. For the first month afterplanting, seedlings were watered, and any dead plants were re-placed with pre-inoculated plants kept in the greenhouse. Duringthe experiment weeds within the planting sites were mowed on upto a weekly basis using a gas powered grass trimmer. Also, all veg-etation within the diameter of the tree crown was removed fromaround the base of the plants on a monthly basis.</p><p> 1999 NRC Canada</p><p>Markham and Chanway 435</p><p>J:\cjb\cjb77\cjb-03\B98-227.vpMonday, August 16, 1999 8:57:15 AM</p><p>Color profile: DisabledComposite Default screen</p></li><li>Soil samples were collected from the planting sites in May 1993for chemical analysis. Approximately 100 cm3 of soil was col-lected to a depth of 10 cm at a point 50 cm south of each plot.Samples from each site were bulked, air dried, and chemical analy-sis performed on the </li><li><p>vest of nonexperimental plants showed that these species occupiedthe same rooting zone as the experimental alders. On one site,H110, no appropriate non-nitrogen-fixing plants were available, sodata from this site were excluded from the analysis. The percentnitrogen fixed (Ndfa) by plants from each remaining site was cal-culated using the d 15N for the non-nitrogen-fixing plants harvestedon that site. We assumed a value of 0.3 % for the d 15N of red al-der when growing under N-free conditions (variable C; publishedin Binkley et al. 1985). To determine the amount of benefit plantsreceived from the different inocula, we also divided Ndfa by theallocation to nodules (na, as a percent of the total plant mass). Be-cause the planting procedure disturbed the soil, and we used pub-lished data for the d 15N value for red alder when growing undernitrogen free conditions, the Ndfa values can only be used for rela-tive comparisons between treatments. Also, comparison of Ndfavalues between planting elevations must be viewed with caution,because of the use of different non-nitrogen-fixing plants on high,compared to low, elevation planting sites.</p><p>Data analysisData from low- and high-elevation planting sites were analyzed</p><p>separately using the following factorial design with two levels ofblocking</p><p>[2] Y P I N PI I N PI N SW e</p><p>i j k i j j k i j k l</p><p>m n mlkji</p><p>= + + + + + + + +</p><p>+</p><p>m</p><p>( )</p><p>where m is the mean of all data, P is the parent plant source (highor low elevation), I is the source of Frankia inoculum (familiar orunfamiliar), N is the presence or absence of neighbours, S is theplanting site, and W is the collection watershed. All factors arefixed except for the blocked effects, watershed and planting site.Dependent variables (Y ) used in the analysis were final plant mass,mass estimated at the end of the first (1992) and second (1993)growing seasons, Ndfa and Ndfa nay . Because only plants withoutneighbours were used in the 15N analysis, the neighbour ef...</p></li></ul>


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