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

Does past contact reduce the degree ofmutualism in the Alnus rubra – Frankiasymbiosis?

John H. Markham and Chris P. Chanway

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 rubraBong.) seedlings fromthree high- and three low-elevation populations with crushed nodule suspensions containing the nitrogen fixingbacteriumFrankia 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 familiarFrankia strains were halfthe size and derived less fixed nitrogen from their symbionts compared with plants inoculated with unfamiliarFrankiastrains. 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.

Key words: coevolution,Frankia, Alnus rubra, mutualism, nitrogen fixation, symbiosis.

Résumé: Bien que la majorité des plantes vasculaires aient des relations symbiotiques avec des microorganismes dusol, et qu’il y ait une importante littérature théorique sur l’évolution du mutualisme, il existe peu de donnéesexpérimentales sur l’évolution du mutualisme entre les plantes et leurs symbiontes microbiens. Les auteurs ont inoculédes plantules d’aulnes rouges (Alnus rubraBong.) provenant de trois populations de haute altitude et de troispopulations de basse altitude avec des suspensions de nodules broyés contenant lesFrankia soit des arbres parents(souches familières) ou soit d’une autre population de plantes échantillonnées à partir du même bassin hydrographique(souches non familières). Les plantules inoculées ont été plantées sur trois sites de hautes altitude et trois sites de bassealtitude. Les auteurs ont suivi la croissance au cours des deuxième et troisième années après la plantation, après quoiils ont récolté les plantes entières. Ils ont estimé la proportion de l’azote dérivée de la fixation en utilisant le rapportdes isotopes stables de l’azote dans les feuilles récoltées. Sur les sites de basse élévation, bien pourvus en azote, lesplantes ayant les souches familières deFrankia sont deux fois plus petites et dérivent moins d’azote à partir de lafixation par leurs symbiontes, comparativement aux plantes inoculées avec des souches deFrankia non familières. Surles sites en altitude, moins bien pourvus en azote, le type d’inocule n’exerce pas beaucoup d’effet sur la performancedes plantes bien que les plantes avec l’inoculum familier soient toujours plus grandes avec les inoculums non familiers.Ces résultats suggèrent que les degrés de mutualisme dans cette symbiose, dépendent de facteurs environnementaux etpeuvent diminuer avec le temps.

Mots clés: coévolution,Frankia, Alnus rubra, mutualisme, fixation de l’azote, symbiose.

[Traduit par la Rédaction] Markham and Chanway 441

Introduction

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

Can. J. Bot.77: 434–441 (1999) © 1999 NRC Canada

434

Received August 1, 1998.

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: [email protected]

J:\cjb\cjb77\cjb-03\B98-227.vpMonday, August 16, 1999 8:57:13 AM

Color profile: DisabledComposite Default screen

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 plant–soil 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.

Plants from eight families have been observed to enterinto a symbiotic relationship with the nitrogen-fixing actino-myceteFrankia (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 withFrankia populationsnormally associated with them or withFrankia 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 ofFrankia 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 differentA. rubra – Frankiacombinations. We examined plant–microbe interactions be-tween high- and low-elevation populations within single wa-tersheds because there can be substantial plant geneticvariation betweenA. 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.

Materials and methods

Collection of experimental materialSeed and nodules were collected from one high- and one low-

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 peat–perlite mixture in April, 1992. Before planting,seedlings from each of the six collection populations were inocu-

lated with a suspension of crushed nodules from either the seed-ling’s 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 5°C.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.

Field proceduresA full set of crosses from each collection watershed was planted

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 plant–bacteria 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 withFrankiacollected 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.

© 1999 NRC Canada

Markham and Chanway 435



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 <2 mm fraction. The pH was measured on a1:1 soil water suspension and percent organic matter by loss on ig-nition according to McKeague (1981). N–P–K analysis was per-formed by Pacific Soil Analysis Inc. (Richmond, B.C.) accordingto the standard methods described in Page (1982). Nitrogen wasanalyzed as total nitrogen using a Technicon autoanalyzer, on asemimicro-Kjeldahl digest. Phosphorus availability was deter-mined colourimetrically using the ascorbic acid method, on a 1:10soil to Bray extract. Potassium was measured using a Perkin–Elmer atomic absorption spectrophotometer on a 1:5 soil to ammo-nium acetate extract. Soil on low elevation sites tends to be moredeveloped in this region, and on our sites (Table 2), total nitrogenwas significantly lower on the high-elevation (0.16 ± 0.03%;mean ± SE of the three sites) compared with the low-elevationsites (0.28 ± 0.06%).

Height and diameter measurements were made on a monthly ba-sis during the growing seasons on all plants, including neighboursstarting in May 1993. These data were converted to total dry massestimates for each plant using a linear regression of height × diam-eter2 to mass of harvested plants (r2 = 0.964) to estimate plantmass throughout the course of the experiment. Plants were har-vested between August 2 and 11, 1994. Because red alder, likemany other members of the Betulaceae family, sheds leaves inmidsummer, leaf litter was collected from around the base of eachplant and included as part of the leaf mass. For plants with neigh-bours, the leaf litter was assigned to each plant as a proportion ofthe stem mass of the plant, relative to the stem mass of all theplants in the plot (target plant and neighbours). The stems were cutca. 1 cm from the base of the plant, and the leaves and shoots werebagged separately. The roots were harvested by digging a trench,

with a 40-cm radius centered at the base of the target plant, to adepth of 40 cm. The remaining soil was then carefully workedaway from the roots. Any roots encountered during the digging ofthe trench that exceeded the trench boundary were dug until theroot was less than 2 mm in diameter and then pulled by hand fromthe soil. A comparison, using ANCOVA, of 16 plants on the sitewith the largest trees, which had their total root mass excavated, toplants with the roots harvested by the trenching method showed nosignificant difference in the root: shoot mass ratio.

Nitrogen fixationDifferences in ratios of naturally occurring nitrogen isotopes

were used to estimate differences in the proportion of fixed nitro-gen in the harvested leaf tissue. To reduce costs, plants with neigh-bours were not analyzed. The total dried leaf mass from each plantwas first crushed and mixed by hand, and a subsample (ca. 2 g)was then ground in a ball mill and sent for analysis ofδ15N (i.e.,the ratio15N:14N, relative to a standard sample) at the Departmentof Oceanography, University of British Columbia, as outlined byEhleringer and Osmond (1991). The percent nitrogen fixed (Ndfa)was calculated using the formula:

[1] Ndfa =−−

×X YX C

100

where X is the δ15N from a non-nitrogen-fixing plant,Y is δ15Nfrom a sample from an experimental plant, andC is theδ15N fromthe red alder when grown under N-free conditions (Domenach etal. 1989). Grasses and rushes were collected from around the per-imeter of each site (Holcus lanatusL. on the low-elevation sitesand mixture of aFescuespp. andJuncusspp. on the high-elevationsites) a week before the harvest of the experimental material. Asingle pooled sampled of leaves from each site was used for thenon-nitrogen-fixing plant sample (variableX) for that site. A har-

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436 Can. J. Bot. Vol. 77, 1999

Soil chemistry δ15N

Site El pH OM N P K Yield Alder Non-fix Ndfa Ndfa/na

Low elevationG 65 5.7 11.1 0.27 15.0 47 318±56 –1.26±0.12 –1.68 30±9 37±11G40 85 5.7 16.1 0.38 13.0 55 150±26 –1.14±0.12 –1.43 25±10 17±19K 250 5.6 13.1 0.18 10.0 19 86±22 –1.20±0.09 –2.93 67±6 31±4

High elevationH30 530 6.0 6.0 0.10 13.7 15 46±11 –1.06±0.13 –2.63 67±5 61±11H90 650 5.4 19.0 0.20 14.0 31 77±17 –1.13±0.07 –4.61 81±2 82±16H110 550 5.8 19.0 0.17 5.0 80 28±11

Note: Planting site names refer to the name of the road nearest the site in the University of British Columbia Research Forest. Elevation(El) is metres above sea level. Soil organic matter (OM) and total nitrogen (N) are in percent by mass in the soil, phosphorus (P) andpotassium (K) are in ppm, and final mean (±1 SE) whole plant dry matter (yield) is in grams. The nitrogen derived from fixation (Ndfa) isexpressed as percentage of the total nitrogen in the plant. The non-nitrogen-fixing (Non-fix) plant species varied between planting sites.Ndfa/na is the proportion of fixed nitrogen relative to the proportion of biomass allocated to nodules.

Table 2. Description of planting sites.

Watershed

Chilliwack Haney Mamquam

Distance from ocean (km) 110 40 10Elevation (m) 100 700 65 540 110 650Latitude (N) 49°05′ 49°08′ 49°03′ 49°16′ 49°44′ 49°44′Longitude (W) 121°55′ 121°24′ 122°35′ 122°34′ 123°06′ 123°01′Site index (m at 50 years) 27.6 23.0 27.5 23.0 28.8 26.5Stand age (years) 16 18 24 15 23 17

Note: At each watershed, red alder seed and nodules were collected from one high- and one low-elevation population.

Table 1. Description of collection sites.



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 theδ15N for the non-nitrogen-fixing plants harvestedon that site. We assumed a value of –0.3 % for theδ15N of red al-der when growing under N-free conditions (variableC; 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 theδ15N 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.

Data analysisData from low- and high-elevation planting sites were analyzed

separately using the following factorial design with two levels ofblocking

[2]Y P I N PI I N PI N S

W e

i j k i j j k i j k l

m n mlkji

= + + + + + + + +

+

µ

( )

whereµ is the mean of all data,P is the parent plant source (highor low elevation),I is the source ofFrankia inoculum (familiar orunfamiliar), N is the presence or absence of neighbours,S is theplanting site, andW 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 andNdfa nay . Because only plants withoutneighbours were used in the15N analysis, the neighbour effect wasdropped from the model when analyzing the nitrogen fixation data.Analyses were performed using the software package JMP version2.0.2 (SAS Institute Inc.), which uses an effective hypothesis test(i.e., means model; Shaw and Mitchell-Olds 1993) in cases of un-balanced designs. In most cases, plant mortality resulted in unevensample sizes. Total mortality was 21% of all transplants with 55%of the mortality occurring on one planting site, H110. Seventy-seven percent of all mortality occurred during the winter as the re-sult of frost heaving pushing the roots from the soil. The propor-tion of mortality associated with plants from low-elevation parents,plants inoculated with familiar inoculum, or plants grown withneighbours was 52, 39, and 52%, respectively. We therefore con-sidered mortality to be independent of these treatments. The as-sumption of homogeneity of variance was tested by a visualexamination of the residuals and in cases of heterogeneous vari-ances, data were transformed before analysis.

Results

The inoculation treatments had a long-lasting and strongeffect on plant performance that was independent of plantsource and neighbour presence but varied between low- andhigh-elevation planting sites (Table 3). On low-elevationplanting sites, plants inoculated with unfamiliarFrankiastrains had a mean final yield, based on back-transformedmeans, that was about two times greater than plants inocu-lated with familiar strains (Fig. 1,p = 0.086 for the inocula-tion effect in the ANOVA using ln transformed final massdata). Although the presence of neighbours significantly re-

© 1999 NRC Canada


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duced final yield (p = 0.056) by an average of 46%, it didnot interact with any other treatment indicating that intra-specific competition did not affect the plant–microbe inter-actions. The doubling of size of plants inoculated withfamiliar Frankia strains was apparent within the first andsecond growing seasons, although high variability betweenplants within treatments made these differences nonsigni-ficant (p = 0.350 andp = 0.184 for the inoculation effect onthe first and second growing season data, respectively). In-oculation treatments had no significant effect on yearly rela-tive growth rates in 1993 and 1994 (data not shown). Thissuggests an immediate inoculation effect continuingthroughout the experiment. The increased growth in plantsinoculated with an unfamiliar inoculum was also reflected inthe nitrogen-fixation data. Plants inoculated with unfamiliarFrankia strains derived 1.6 (p = 0.027) times more nitrogenand 3.4 (p = 0.100) times more nitrogen per allocation tonodule mass than plants inoculated with familiarFrankiastrains (Tables 2 and 3).

On high-elevation planting sites, overall plant size was re-duced and treatment effects were less pronounced comparedwith the low-elevation sites (Table 3). Plants inoculated withfamiliar Frankia strains tended to have a greater yield (abouttwo times, based on back-transformed means) than plants in-oculated with unfamiliar strains. However, there was also apossibly significant (p = 0.080) interaction between neigh-bour presence, parent source, and inoculum (based on ln-transformed final mass data) with the greatest inoculation ef-fects occurring in plants from low-elevation parents without,and plants from high-elevation parents with, neighbours.There was also a significant interaction between parentsource and inoculum (p = 0.038) for the proportion of nitro-gen derived from fixation. Plants from both parent types de-rived more nitrogen from fixation when inoculated withFrankia from the low-elevation population. Plants inocu-lated with familiarFrankia derived 1.5 times more nitrogenper investment in nodules (p = 0.077) and plants from high-

elevation parents derived 2.2 times more nitrogen per alloca-tion to nodules than plants from low-elevation parents, withno interaction between parent source and inoculum type.

There was a significant site effect for all dependent vari-ables on both high- and low-elevation planting sites (Ta-ble 2). On both low- and high-elevation planting sites therewas a positive relationship between yield and soil phospho-rus levels. On low-elevation sites, there was a negative rela-tionship between yield and Ndfa but a positive relationshipon high-elevation sites.

Discussion

Our results present a complex picture of red alder –Frankia interactions between populations distributed acrossrelatively short distances. The expression of these interac-tions varies with environmental conditions. Variation in theperformance of plants from the same population, grown onthe same site, and inoculated withFrankia from differentpopulations, confirms the existence of genetic variarion be-tween theFrankia populations. Similarly, variation in theperformance of plants from different populations, grown onthe same site, and inoculated withFrankia from the samepopulation, confirms the existence of genetic variation be-tween the plant populations. Within high-elevation sites,plants inoculated withFrankia from their parents showedthe greatest growth, supporting the idea of increased mutual-ism between red alder –Frankia populations, as they existnaturally. However, on low-elevation sites, where overallplant yield was greater and the inoculation effects were morepronounced, the red alder –Frankia interaction is more in-dicative of increased parasitism than mutualism. Although anumber of environmental conditions change across an eleva-tion gradient, one likely factor affecting the expression thered alder –Frankia interactions found here was soil nitro-gen. Low-elevation planting sites had almost twice as muchtotal nitrogen on average compared with high-elevation sites

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438 Can. J. Bot. Vol. 77, 1999

0

10

32 29

Low HighPlanting elevation

Tota

ldry

mas

s(g

)

25 26

200

300

Inoculum

familiar

unfamiliar

Fig. 1. Inoculation effect on mean final dry plant biomass. Values below bars are sample sizes. Error bars are 1 SD.



(Table 2), and since these low elevation sites likely hadhigher decomposition rates due the higher temperatures, theymost likely had more available soil nitrogen as well. Thissuggests the plant’s demand for symbiotically fixed nitrogen(and the need to form a symbiotic relationship withFrankia)was lower on the low elevation sites.

Mutualism is often assumed to involve an initial invest-ment by both partners leading to future benefits, althoughother scenarios are possible (Connor 1995). If investmentsare made by both organisms, then it is possible for one or-ganism to cheat in the relationship by not providing an in-vestment, yet receiving a benefit. Game theory has predictedthat there is a strong selection for cheating whenever futureinteractions between partners becomes unlikely (Axlerodand Hamilton 1981). In this, as in other cross-inoculationstudies,Frankia were collected from mature trees. As red al-der stands mature there is often a reduction in the rate of ni-trogen fixation (Binkley et al. 1994) as the tree’s nitrogendemand decreases, and more nitrogen can be obtained fromthe soil. Because increased soil nitrogen can inhibit the for-mation of nodules (Granhall et al. 1983) the probability of abreakdown in the partnership will increase as stands mature,perhaps creating a situation where a less mutualistic interac-tion is favoured. Therefore, in mature red alder stands, lessmutualistic Frankia strains may have an advantage overmore mutualistic strains. Infection of seedlings by suchstrains would result in slower growing and, presumably,competitively inferior plants. Our data suggest that, for thisred alder –Frankia system, the partners must first coexistfor a less effective symbiosis to be expressed.

We predict that, in actinorhizal plant species in which thebreakdown of the relationship between plant and microbe isunlikely, plant performance will be best when plants havefamiliar Frankia as symbionts. Such situations would occurwhere the actinorhizal plant is part of the climax communityand has little effect on the nitrogen status of the soil. For ex-ample, in some boreal ecosystems,Alnusshrubs form a per-sistent part of the understory where their effect on soilnitrogen availability can be insignificant (Wurtz 1995).Dillon and Baker (1982) found differences in the effective-ness ofFrankia isolated from plants from different habitats.Myrica galeL. (sweet gale), which persists in bog communi-ties, which are generally low in available nitrogen, had thehighest acetylene reduction (AR) rate when inoculated withFrankia from Myrica pensylvanicaLoisel., compared withinoculation withFrankia from Comptonia perigrina(L.) J.Coult. or three alder species. This suggests increased mut-ualism with familiar Frankia in the genusMyrica. In thesame study plants were also inoculated withFrankia fromC. perigrina. This plant tends to be an early successionalspecies, so Frankia populations in the soil whereC. perigrina is found would have an increasingly unlikelyprobability of having it as a host as time goes by. Whenplants were inoculated withFrankia from C. perigrina thelowest AR rate occurred inC. perigrina, compared withM. galeand the three alder species. This suggests decreasingmutualism with familiarFrankia in the genusComptonia.

There is at least one other report ofFrankia from A. rubrahaving a depressive effect when inoculated ontoA. rubra.Tessier du Cros et al. (1984) found in one of two trials that

when Alnus glutinosa(L.) Gaertn.,Alnus cordata(Loisel.)Duby, andA. rubra were inoculated with a pure strain ofFrankia from A. rubra, two of threeA. rubra provenancesshowed significantly lower plant height as compared withuninoculated (but nodulated) plants, whereasA. cordataandA. glutinosaprovenances were unaffected by the inoculationtreatment. Cole et al. (1990) have also found that when redalder is planted on previous red alder sites, growth is stuntedcompared to red alder planted on previous Douglas-fir(Pseudotsuga menzeisii(Mirb.) Franco) sites. Although theyprovide data showing decreased soil pH and phosphorusavailability on the sites previously occupied by red alder,part of the decrease in growth could be due to differences inFrankia populations on the two sites.

Our results indicate a fine-scale interaction between alderhost andFrankia endophyte populations and suggests thatactinorhizal symbioses can exhibit a similar degree of spe-cialization between populations as is found in the legume–Rhizobium symbiosis. When similar types of cross-inoculation studies have been performed using the legumesymbiosis, performance of plants is generally found to begreatest when inoculated with the familiar bacterial geno-type (Lie et al. 1987; Chanway et al. 1989; Parker 1995).However, there is evidence for virulentRhizobiummutantsand resistant host plants (Djordjevic et al. 1987). Also, inef-fective Rhizobiumstrains have been found to be common insome field situations (Hagedorn 1978; Holding and King1963).

Other cross inoculation studies performed using actino-rhizal species to date have been inconsistent in that no host–endophyte interactions have been detected (Dawson and Sun1981; Nelson and Lopez 1989; Mansour and Baker 1994;Prat 1989; Sougoufara et al. 1992) or when they were de-tected, they did not result from either consistently greater orlower effectiveness when plants were inoculated withFrankia from their own taxa (Carpenter et al. 1984; Dillonand Baker 1982; Weber et al. 1989). Our data suggest thatany pattern in effectiveness depends on the ecology of thepartners and the conditions plants are grown under. Cross-inoculation studies have tended to use single pure culturesfrom each host tested and grown the inoculated plants underlaboratory or greenhouse conditions. Although the use ofpure cultures has many advantages and reduces the possibil-ity of contamination by other microbes, the difficulty (and insome cases impossibility) of isolating and growing someFrankia strains in pure culture suggests that the use of purecultures introduces unwanted selection of strains (i.e., strainsthat are readily culturable are selected for). Use of a collec-tion of crushed nodules from a stand as opposed to a singlepure culture as an inoculum could have a number of effectson host–endophyte interactions. A number of strains havebeen found within a singleAlnus stand (Benson and Hanna1983) and even within a single nodule (Gardes and Lalonde1987). Using three different isolates on eightAlnusspecies,Prat (1989) found that, although one strain consistently re-sulted in greater growth when applied as a single strain, amixture of the strains produced greater plant growth thanany single strain inoculation. Therefore, it is possible thatthe crushed nodules used in this study had a greater effect onplant growth than single strains would have. Although the

© 1999 NRC Canada




use of more controlled environmental conditions may reducevariation within treatments, it may also prevent treatment ef-fects, like those found here, from being expressed.

Difficulty in manipulating and measuring the fitness ofsoil microbes has resulted in limited field experimentation.Frankia are slow-growing microbes. Until 1978 they couldnot be isolated and to date certain physiological groups havenot been isolated and direct isolation from the soil is gener-ally not possible (Lechevalier and Lechevalier 1990). Forthis reason they are not particularly amenable to field exper-iments. However, these bacteria do form discrete, perenialstructures on their hosts. In this experiment we inoculatedplants using a collection of nodules and assumed that theFrankia from those nodules would nodulate the test plantsand have a lasting effect on plant performance. Although wecan’t make any conclusions about the population dynamicsof the Frankia from the inoculum used, the strong inocula-tion treatment effects observed attests to the fact that our in-oculation procedure had long lasting effects on plantperformance. The data suggest the interaction between thepartners in this symbiosis depends on environmental condi-tions and our suggestion that, in some situations, there maybe selection for a less effective symbiosis implies that themaintenance of a mutualistic interaction requires a change inenvironmental conditions.

Acknowledgments

This study was supported by a Natural Sciences and Engi-neering Research Council of Canada (NSERC) operatinggrant to C.P.C. and a NSERC postgraduate scholarship toJ.H.M. Phil Burton, Brian Holl, and Roy Turkington madevaluable comments at various stages of the study. EllenMacDonald made valuable comments on the statistical anal-ysis.

References

Ager, A.A., Heilman, P.E., and Stettler, R.F. 1993. Genetic varia-tion in red alder (Alnus rubra) in relation to native climate andgeography. Can. J. For. Res.23: 1930–1939.

Axelrod, R., and Hamilton, W.D. 1981. The evolution of coopera-tion. Science (Washington, D.C.),211: 1390–1396.

Baker, D., Newcomb, W., and Torrey, J.G. 1980. Characterizationof an ineffective actinorhizal microsymbiont,Frankia sp. Eul1.(Actinomycetales). Can. J. Microbiol.26: 1072–1089.

Benson, D.R., and Hanna, D. 1983.Frankia diversity in an alderstand as estimated by sodium dodecyl sulfate – polyacrylamidegel electrophoresis of whole cell proteins. Can. J. Bot.61:2919–2923.

Binkley, D., Sollins, P., and McGill, B. 1985. Natural abundance ofnitrogen-15 as a tool for tracing alder-fixed nitrogen. Soil Sci.Soc. Am. J.49: 444–447.

Binkley, D., Cromack, K., Jr., and Baker, D.D. 1994. Nitrogen fix-ation by red alder: biology, rates, and controls.In The biologyand management of red alder.Edited by D.E. Hibbs, D.S.DeBell, and R.F. Tarrant. Oregon State University Press,Corvallis, Oreg. pp. 57–72.

Bond, G. 1983. Taxonomy and distribution of non-legume nitrogen-fixing systems.In Biological nitrogen fixation in forest ecosys-tems: foundations and applications.Edited byJ.C. Gordon andC.T. Wheeler. Junk Publishers, The Hague, The Netherlands.pp. 55–87.

Bormann, B.T., Cromack, K., Jr., and Russell, W.O., III. 1994. In-fluences of red alder on soils and long-term ecosystem produc-tivity. In The biology and management of red alder.Edited byD.E. Hibbs, D.S. DeBell, and R.F. Tarrant. Oregon State Univer-sity Press, Corvallis, Oreg. pp. 47–56.

Carpenter, C.V., Robertson, L.A., Gordon, J.C., and Perry, D.A.1984. The effect of four newFrankia isolates on growth andnitrogenase activity in clones ofAlnus rubraandAlnus sinuata.Can. J. For. Res.14: 701–706.

Chanway, C.P., Holl, F.B., and Turkington, R. 1989. Effect ofRhizobium leguminosarumbiovar trifolii genotype on specificitybetweenTrifolium repensL. and Lolium perenneL. J. Ecol.7:1150–1160.

Cole, D.W., Compton, J., Van Miegroet, H., and Homann, P. 1990.Changes in soil properties and site productivity caused by redalder. Water Air Soil Pollut.54: 231–246.

Connor, R.C. 1995. The benefits of mutualism: a conceptualframework. Biol. Rev.70: 427–457.

Dawson, J.O., and Sun, S.H. 1981. The effect ofFrankia isolatesfrom Comptonia peregrinaandAlnus glutinosaon the growth ofAlnus glutinosa, A. cordataand A. incanaclones. Can. J. For.Res.11: 758–762.

Dillon, J.T., and Baker, D. 1982. Variation in nitrogenase activityamong pure-culturedFrankia strains tested on actinorhizalplants as an indication of symbiotic compatibility. New Phytol.92: 215–219.

Djordjevic, M.A., Gabriel, D.W., and Rolfe, B.G. 1987.Rhizobium: the refined parasite of legumes. Annu. Rev.Phytopathol.25: 145–168.

Domenach, A.M., Kurdali, F., and Bardin, R. 1989. Estimation ofsymbiotic dinitrogen fixation in alder forest by the methodbased on natural15N abundance. Plant Soil,118: 51–59.

Douglas, A.E. 1995. The ecology of symbiotic micro-organisms.Adv. Ecol. Res. No. 26. pp. 69–103.

Ehleringer, J.R., and Osmond, C.B. 1991. Stable Isotopes.In Plantphysiological ecology: field methods and instrumentation.Edited byR.W. Pearcy, J.R. Ehleringer, H.A. Mooney, and P.W.Rundel. Chapman & Hall, London, U.K. pp. 281–300.

Ferriere, R., and Michod, R.E. 1996. The evolution of cooperationin spatially heterogeneous populations. Am. Nat.147: 692–717.

Gardes, M., and Lalonde, M. 1987. Identification and sub-grouping of Frankia strains using sodium dodecyl sulfate –polyacrylamide gel electrophoresis. Physiol. Plant.70: 237–244.

Granhall, U., Ericsson, T., and Clarholm, D. 1983. Dinitrogen fixa-tion and nodulation byFrankia in Alnus incanaas affected byinorganic nitrogen in pot experiments with peat. Can. J. Bot.61:2956–2963.

Hagedorn, C. 1978. Effectiveness ofRhizobium trifoliipopulationsassociated withTrifolium subterraneumL. in southwest Oregonsoils. Soil Sci. Soc. Am. J.42: 447–451.

Hahn, D., Starrenburg, M.J.C., and Akkermans, A.D.L. 1988. Vari-able compatibility of clonedAlnus glutinosaecotypes againstineffective Frankia strains. Plant Soil,107: 233–243.

Harrington, C.A., and Curtis, R.O. 1986. Height growth and siteindex curves for red alder. USDA For. Serv. Res. Pap. No.PNW-358.

Holding, A.J., and King, J. 1963. The effectiveness of indigenouspopulations ofRhizobium trifolii in relation to soil factors. PlantSoil, 18: 191–198.

Huss-Dannell, K. 1991. Influence of hostAlnusandMyrica geno-type on infectivity, N2 fixation, spore formation and hydro-genase activity inFrankia. New Phytol.119: 121–127.

Lechevalier, M.P., and Lechevalier, H.A. 1990. Systematics, isola-tion and culture ofFrankia. In The biology of Frankia and

© 1999 NRC Canada

440 Can. J. Bot. Vol. 77, 1999



actinorhizal plants. Edited by C.R. Schwintzer and J.D.Tjempkema. Academic Press Inc., Toronto, Ont. pp. 35–60.

Lie, T.A., Göktan, D., Engin, M., Pijnenborg, J., and Anlarsal, E.1987. Co-evolution of the legume–Rhizobiumassociation. PlantSoil, 100: 171–181

Malloch, D.W., Pirozynski, K.A., and Raven, P.H. 1980. Ecologi-cal and evolutionary significance of mycorrhizal symbiosis invascular plants (a review). Proc. Natl. Acad. Sci. U.S.A.77:2113–2118.

Mansour, S., and Baker, D. 1994. Selection trials for effective N2-fixing Casuarina–Frankia combinations in Egypt. Soil Biol.Biochem.26: 655–658.

McKeague, J. (Editor). 1981. Manual on soil sampling and meth-ods of analysis. 2nd ed. Canadian Society of Soil Science, Ot-tawa, Ont.

Nelson, D.L., and Lopez, C.F. 1989. Variation in nitrogen fixationamong populations ofFrankia spp. andCeanothusspp. in ac-tinorhizal associations. Biol. Fertil. Soils,7: 269–274.

Nowak, M., and Sigmund, K. 1993. A strategy of win–stay, lose–shift that outperforms tit-for-tat in the prisoner’s dilemma game.Nature (London),364: 56–58.

Page, A.L. (Editor). 1982. Methods of soil analysis. Part 2. Chemi-cal and microbiological properties. 2nd ed. Agronomy,9(2).

Parker, M.A. 1995. Plant fitness variation caused by different mut-ualist genotypes. Ecology,76: 1525–1535.

Perry, D.A., Amaranthus, M.P., Borchers, J.G., Borchers, S.L., andBrainerd, R.E. 1989. Bootstrapping in ecosystems. Bioscience,39: 230–237.

Prat, D. 1989. Effects of some pure and mixedFrankia strains onseedling growth in differentAlnus species. Plant Soil,113: 31–38.

Shaw, R.G., and Mitchell-Olds, T. 1993. ANOVA for unbalanceddata: an overview. Ecology,74: 1638–1645.

Sougoufara, B., Maggia, L., Douhoux, E., and Dommergues, Y.R.1992. Nodulation and N2 fixation in nine Casuarina clone –Frankia strain combinations. Acta Ecol.13: 497–503.

Tessier du Cros, E., Jung, G., and Bariteau, M. 1984. Alder–Frankia interaction and alder–poplar association for biomassproduction. Plant Soil,78: 235–243.

Weber, A., Sarsa, M.-L., and Sundman, V. 1989.Frankia – Alnusincana symbiosis: effect of endophyte on nitrogen fixation andbiomass production. Plant Soil,120: 291–297.

Wurtz, T.L. 1995. Understory alder in three boreal forests ofAlaska: local distribution and effects on soil fertility. Can. J.For. Res.25: 987–996.

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