Measuring Rates of Symbiotic Nitrogen Fixation by Alnus tenuifolia

Measuring Rates of Symbiotic Nitrogen Fixation by Alnus tenuifoliaAuthor(s): Michael D. Fleschner, C. C. Delwiche and Charles R. GoldmanSource: American Journal of Botany, Vol. 63, No. 7 (Aug., 1976), pp. 945-950Published by: Botanical Society of AmericaStable URL: http://www.jstor.org/stable/2441752 .

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Amer. J. Bot. 63(7): 945-950. 1976.

MEASURING RATES OF SYMBIOTIC NITROGEN FIXATION BY ALNUS TENUIFOLIA'

MICHAEL D. FLESCHNER, C. C. DELWICHE, AND CHARLES R. GOLDMAN

Department of Botany and Department of Soils and Plant Nutrition; Division of Environmental Studies, University of California, Davis 95616

A B S T R A C T Rates of nitrogen fixation were determined for alder (Alnus tenuifolia Nutt.) with the C2H2

assay and verified by the 15N method. Rates of C2H2 reduction obtained varied between 0.0342 and 10.9,umoles per gram of nodule tissue (fresh weight) per hour with a seasonal average of 2.70 Amoles g-1 hr1'. Expressed as nitrogen fixation rates these are equivalent to 0.0114, 3.63, and 0.9 Amoles g-1 hr1' of elemental nitrogen, respectively. Nitrogen fixation (15N) rates aver- aged 2.20 ,umoles N g-1 hr1I with a standard deviation of 1.14. The influence of pC2H2, PO2, temperature during incubation, length of incubation time, presence vs. absence of N2 in the incubation atmosphere, and amount of nodule biomass per sample were investigated.

CURRENT INTEREST in the variety of impacts that human activity has on the global environment indicates a need for more quantitative data on nutrient flow patterns. Delwiche (1970) pointed out the increasingly important role that industrial manufacture of fertilizer plays in the world's nitrogen cycle. He also discussed the lack of ac- curate estimates of the magnitudes of such natural processes as nitrogen fixation and denitrification.

Goldman (1961) concluded that primary production in Castle Lake, California, was partially supported by nitrogen input from a population of Alnus tenuifolia growing on the shoreline among numerous springs which flow into the lake. The high nitrogen fixation rate of alder, the high nitrate content of the springs, and their close proximity to the water facilitated the nutrient entry into the aquatic system more readily than more distantly located nitrogen fixers. Therefore, on the hypoth- esis that alder may have special influence on the water quality of Lake Tahoe the alder was selected for primary consideration, although a number of other nitrogen fixing species which we have studied in less detail exist in the area. The alder grows on riparian or wet meadow sites. The soils are usually young and poorly developed, sandy to gravelly in texture, and well aerated.

Hardy et al. (1968) stressed the practicability of the acetylene (C2H2) reduction assay for esti- mating nitrogen fixation rates under agricultural and laboratory conditions. The technique seems equally applicable to natural ecosystems although

IReceived for publication 29 July 1975. We are grateful to Drs. Robert Leonard, Jack Major,

Michael Barbour, and Charles Fleschner who gave many helpful suggestions and to Mrs. Meryllene Smith who patiently typed the manuscript revisions. This work was supported by NSF-RANN Grant #GI-22.

few quantitative data have been published regard- ing nitrogen inputs on the ecosystem level. Since 1966 when Schollhorn and Burris (1966) and Dill- worth (1966) demonstrated the ability of the nitrogenase enzyme system to reduce C2H2 to ethylene (C2H4), considerable application of -the technique has been made as a presumptive indicator of nitrogen fixation (Koch and Evans, 1966; Stewart, Fitzgerald, and Burris, 1967; Hardy et al., 1968). The C2H2 assay has been compared with other methods of assessing biological nitrogen fixation (Bergersen, 1970; Akkermans, 1971), and several simple and inexpensive procedures for its field application have been outlined (Hardy et al., 1968; Waughman, 1971). Wheeler (1969) discussed limitations of the C2H2 assay as a quantitative measure of nitrogen fixation and Hardy et al. (1968) and Waughman (1972) indicated several possible sources of error. Much of the effort of developing our technique, therefore, involved assessing the influence of various suspected sources of error on our results.

The work reported here is part of an integrated study of nutrient cycling mechanisms and their relationship to water quality and land use in the Lake Tahoe basin of California and Nevada. The Ward Creek watershed, located in the northwest corner of the basin, was selected for detailed study. It is approximately 2500 ha in extent and is located around coordinates Lat. 390 7'N Long. 120? 14'W. A description of vegetation types in the Lake Tahoe basin which includes the Ward Creek area was prepared by Neilson (1973).

METHODS-The selection of sampling sites was based on our observations of the vegetation in the watershed. One large alder stand was selected for intensive study on the basis that it appeared to en-

945



946 AMERICAN JOURNAL OF BOTANY [Vol. 63

compass the alder's local range of habitat variability with respect to soil moisture and exposure to sunlight. Within the stand, sampling sites were chosen to include this range of variability. In addition to this primary investigation site, several alder populations in other parts of the watershed were sampled less intensively.

The technique used to assess rates of nitrogen fixation was the C2H2 reduction assay, with the 15N isotopic method serving as a check. Both methods required the recovery of nodules from the soil. We began the excavation of nodules by dig- ging a trench tangential to the basal stem cluster of the alder plants. The usual depth of excavation was about 30 cm, corresponding to the depth within which the majority of nodules occur. The excavation then proceeded inwardly toward the base of the plant. Soil was removed by first loosening it with the point of a knife and then carefully brushing it away from nodules with a small paintbrush. Akkermans (1971) determined that the primary nitrogen fixing activity is located in the tips of the nodule lobes and, accordingly, great care was exercised to prevent physical dam- age to these tips. The nodules were excised at their point of attachment to the root.

Excised nodules were placed in a 17-ml serum tube which served as an incubation chamber. It was necessary to split the larger nodules so that they would fit into the tube. This was done by carefully breaking the nodule branches at their bases so the tips would not be damaged. Differ- ences in external appearance exist between alder nodules, ranging from a light tan, unwrinkled sur- face to dark brown and wrinkled. Quantitative measurements were not feasible, and no positive correlations between C2H2 reduction rates and nodule appearance were established. It was our experience, however, that unusally high rates of reduction were observed in nodules at the light tan, unwrinkled end of the spectrum. Since the light tan nodules were found only occasionally they were not included in samples selected for comparative purposes.

Upon collection of about 1.5 g of nodule tissue per serum tube, each was sealed with a serum stopper and 1.7 ml of high purity C2H2 was injected from a syringe via a #25 hypodermic needle. This produced a pC2H2 of 0.1 atmosphere in the incubation chamber.

To maintain constant in situ incubation temperatures, a pile of loose soil was formed in a corner of the excavation and the incubation chambers were inserted to the level of the serum stopper. Using this technique, temperatures remained constant with 1 C over a 3-hr incubation period.

After a prescribed period, usually one hour, a 0.5-ml sample of gas was removed from the incubation tube with a #25 hypodermic needle and syringe. This sample was immediately transferred to an evacuated 1 7-ml serum tube for storage

6.0 0

w _ 4.5 :

z 0

0 0 0

0

W a 0 Z 0 ,- 1.5 0 j

0 0 08

0 0 2 3 4 5 6 7

HOURS

Fig. 1. Time course of C2H2 reduction rates. The circles represent the spread of individual values for seven replicate samples at five time intervals. The data points (circles) are located at the midpoints of the time intervals of sampling because they are average rates for those periods. The triangles connected by a line are the average rates for all samples of each interval. Rates are in ,jmoles C2H2 g-1 hr-1 fresh weight.

prior to analysis. The gas samples obtained during incubation were analyzed chromatographically within four days.

Acetylene and C2H4 were detected by separa- tion on a gas chromatograph (Varian Aerograph model 1200 coupled with a Varian Aerograph model 20 recorder). The apparatus was fitted with a hydrogen flame ionization detector and a 2.13-m stainless steel 0.32-mm diameter column packed with 80-100 mesh PoropakR R (Van Waters and Associates). The temperature pro- gram was isothermal: injector 64 C, column 54 C, detector 100 C. All gases used were purity grade from cylinders in which the following flow rates were used: H2-25 ml min-', 02-100 ml min'l, N2- 25 ml min-'.

All calculations of nitrogen fixation rates were made by applying the ratio of the recorded peak heights (C2H4: C2H2) to the initial amount of C2H2 injected into the incubation chamber. We assumed that the resulting figure represents the amount of C2H2 reduced to C2H4 by the nitrogenase enzyme system.

The biomass of each sample was measured on both a fresh and a dry weight basis (oven dried at 70 C). These figures were -combined with the C2H2 reduction rates which could then be expressed in micromoles of C2H2 produced per gram of nodule tissue per hour (/moles C2H4 g-1 hr'). This rate of C2H4 production was divided by the theoretical conversion ratio of three to obtain a rate figure representing nitrogen fixation.

Nodule samples were collected for the 15N analysis in the same manner as for the C2H2 method. They were placed in serum tubes which were then evacuated and an Ar-02-CO2 atmosphere reintro-



August, 1976] FLESCHNER ET AL.-NITROGEN FIXATION IN ALNUS 947

TABLE 1. Comparison of C2H2 reduction rates between samples in which the incubation atmosphere in one case contained air (first column) and in the other a mixture of 80% Ar, 18% 02, and 0.04% CO, (second column). Rates are in ,amoles C2H2 gH

hri fresh weight

Sample Air Ar-02-CO2

1 4.62 3.15 2 2.22 1.75 3 3.42 2.94 4 3.51 2.16 5 1.70 4.86 6 2.66 3.54

Mean 3.02 3.07

Standard deviation 1.05 1.10

duced. A gas mixture with a known excess frac- tion of 15N was injected into the serum tube and the nodules were incubated for eight hours before the reaction was terminated by exposing the nodules to air. Upon return to the laboratory, the plant tissues were subjected to micro-Kjeldahl analysis and the 15N content was determined with a mass spectrometer. The figure for percent atom excess of 15N thus obtained was used to calculate nitrogen fixed during the incubation time.

RESULTS-Figure 1 shows the variation of the C2H2 reduction rate of seven samples over a 10- hr period. The data points (circles) were plotted at the midpoints of the successive time intervals 0.5, 1, 2, 3, and 10 hr, because the rate figures are averages of each incubation period. The curve of the average reduction rate (triangles) tended to fall with increasing incubation time. The high rate observed in the first 0.5 hr period was due to one unusually active sample. Ignoring this one exception, the rates were roughly constant during the first two hours, then began a gradual steady decline throughout the remaining eight hours of incubation.

Atmospheric N2 is the natural substrate for the nitrogenase enzyme complex and will compete with C2H2 for the enzyme's active site. The relative concentrations of N2 and C2H2 may therefore have an important influence on results. Effects of the presence vs. absence of N2 in the incubation chamber are shown in Table 1. Six samples incubated in air were compared with six samples incubated in an atmosphere of 80 % Ar, 18 % 02, and 0.04 % CO2. The average C2H2 reduction rate of the samples incubated in the Ar-02-CO2 atmosphere (3.07 ymoles g1 hrF) was slightly higher than that of the samples incubated in air (3.02 ymoles g-l hr'l), but the difference was not significant at the 5 % level.

To determine if 0.1 atm of C2H2 was adequate to override N2 competition, five samples incubated

TABLE 2. Comparison of C2H2 reduction rates at two levels of C2H2 concentrations during incubation. The set number identifies paired samples. Rates are in Amoles C2H2 g` hr-' fresh weight

Set# 10% C2H2 20% C2H2

1 35.4 28.3 2 8.94 3.92 3 11.2 15.2 4 6.06 4.35 5 7.14 10.1

Mean 13.74 13.17


at 0.1 atm C2H2 were compared with five samples at 0.2 atm (Table 2). The average C2H2 reduction rate of the former (13.74 umoles g-1 hr-1) was very close to that of the latter (13.17 Amoles g-1 hr-1), and the difference was not significant at the 5 % level.

To assess effects of temperature, four pairs of samples were subjected to the following temperature regimes: during the first 0.5 hr of incubation one sample of each pair was incubated at a low temperature (between 13.5 and 16.0 C), the other sample at a higher temperature (between 20.5 and 22.0 C). A gas sample then was with- drawn from each for analysis. During the second 0.5 hr the temperature conditions of the samples were reversed. The C2H2 reduction rates for these two situations are compared in Table 3. Using the seven-degree temperature range from 14 to 21 C for the basis of calculation, we obtained a Qio value for the first 0.5 hr of 2.73, and for the second 0.5 hr of 4. 10.

Oxygen levels in the incubation chambers could be expected to affect nodule respiration, which in turn would influence C2H2 reduction rates. Since the incubation chamber walls are rigid, the partial pressure of oxygen (pO2) is related to the amount of respiring nodule tissue of each sample. We compared the average C2H2 reduction rate for a 1-hr incubation period of eight samples, weighing less than 1.5 g fresh weight each, with eight samples which weighed about 3 g each. The average C2H2 reduction rate for the 1.5-g group was 9.09 ymoles g-l hr-l and that of the 3-g group was 8.46 ymoles g-l hr-l (Table 4).

It was observed (Fig. 1) that the C2H2 reduction rate began to decline after 2 hr of incubation. If the decline were related to the inhibition of respiration due to low PO2, addition of 02 should rejuvenate the reaction. Therefore, to assess ad- ditionally the effects of PO2 on C2H2 reduction, six samples were incubated for 3 hr, and then an additional 20 % 02 was injected to observe possible stimulatory effects. However, a sharp drop (relative to two control samples) in the C2H2 re-




TABLE 3. Comparison of five sets of two samples each incubated under two temperature regimes. The first sample of each set was incubated for the first 0.5 hr at low temperature, the second 0.5 hr at high temperature. For the second sample of each set (primed) the procedure was reversed. Low temperature ranged from 13.5 to 16.0 C; high temperature from 20.5 to 22.0 C (a range of 7 C between low and high temperatures was used to calculate QLo values). Acetylene reduction rates are in Amoles C2H2 g' hr' fresh weight

First 0.5 hr. Second 0.5 hr.

Sample # Temp. C2H2 red rate Temp. C2H2 red rate

1 low 8.46 high 7.83 1' high 17.9 low 1.58 2 low 7.77 high 6.63 2' high 14.7 low 1.72 3 low 2.18 high 1.64 3' high 3.27 low 1.34 4 low 0.384 high 0.810 4' high 1.28 low 1.51 5 low 0.993 high 1.47 5' high 0.648 low 0.258

Low temperature mean 3.96 1.28 High temperature mean 7.56 3.96 Qio value 2.73 4.10

duction rate occurred when the O2 was injected (Fig. 2).

Five samples of nodule tissue were subjected to analysis by using the 15N isotopic assay to serve as an independent check on the C2H2 reduction technique. The average rate of nitrogen fixation for the five samples was 2.20 ymoles N g-l hr-1 with a standard deviation of 1.14.

In addition to the various tests of assay technique described above, an average nitrogen fixation rate for the 1973 growing season (12 June- 20 October) was calculated by the C2H2 assay. Samples taken from beneath the snow in Novem- ber, February, and May showed no evidence of C2H2 reduction, and therefore we considered reduction activity to be negligible from November to June. One hundred thirty six samples collected during the period June to October between the hours of 10:00 AM and 4:00 PM were used in the calculation. The mean rate was 0.900 ,umoles N g-l hrl (fresh weight) with a standard deviation of 0.690. The values ranged from 0.0114 to 3.63 ymoles N g-l hr-'. Samples collected during the extreme ends of the growing season in Octo- ber and June had characteristically low fixation rates. If these are ignored as insignificant, then 90 % of the remaining samples fall within the range of 0.300 and 2.26 ,moles N g-l hr'.

DIscussIoN-Hardy et al. (1968), in studies of soybeans grown under agricultural conditions, described a cumulative curve for C2H2 reduction

TABLE 4. Differences in C2H2 reduction rates between two weight classes of nodule samples. Rates are in yImoles C2,H2 g' hr' dry weight

Sample # Nodule wt 3 g Nodule wt 1.5 g

1 10.9 13.8 2 6.93 4.41 3 3.93 3.78 4 4.59 8.85 5 8.91 3.12 6 10.7 17.3 7 10.7 14.6 8 10.9 6.81

Mean 8.46 9.09


which increased linearly up to about 1 hr and then leveled off sharply. The time course curve presented in Fig. 1 is expressed as a rate of C2H2 reduction; it contains a greater range of variability and there is no clear linear trend. We ascribe these differences to greater environmental and biological variability of nodule tissues in native alder stands as opposed to the more uniform conditions of a soybean field. The average annual nitrogen fixation rate data presented earlier and the data in Fig. 1 and 2 indicate that the variability falls roughly within a range of one order of magnitude. Akkermans (1971) encountered an even greater range of variability in his results. In the study of native vegetation in situ (the Ward Valley ecosystem) our results represent useful initial estimates of nitrogen fixation rates.

The results of our investigations of the competition between N2 and C2H2 for the nitrogenase active site are consistent with those of Hardy et al. (1968) and Akkermans (1971). Hardy esti- mated the Michaelis constant of C2H2 to be only 5 % that of N2 based on partial pressures. Ak- kermans found that although the C2H2 concentra- tion may be critical at low levels, at concentrations as high as 10 % there was no measurable competition with N2 for the nitrogenase active site. Our results showed no difference between 0.1 and 0.2 atm of C2H2, and the presence of N2 in the incubation chamber had no measurable effect on the C2H2 reduction rate. We therefore chose 0.1 atm as the more convenient level with which to work.

There is a marked effect of temperature on the rate of CGH2 reduction. Hardy et al. (1968) presented figures which can be used to calculate a Oto of 4.98 between the temperatures of 15 and 20 C. Our Qio values range from 2.7 to 4.1 for a comparable range of temperatures. Ideally, temperature conditions should be identical when carrying out assays where results of more than one sample are to be compared. Alternatively, by in- troducing an extra step to calculate QOo values, a temperature adjustment can be made so that results can be compared.



August, 1976] FLESCHNER ET AL.-NITROGEN FIXATION IN ALNUS 949

6.0

0

1- 4.5 A 0

i-T3,0 ~~ 0 Z A~~~ o'o 7 . 3.0 A 0i o

A A

O A A w j5 A A A

N

0 A

0 1 2 3 4 5 HOURS

Fig. 2. Time course of C2H2 reduction rates of two groups of samples. Triangles represent six replicates (open are individual values; closed are the averages) which were given an additional 20% 02 after 3-hr incubation. The circles represent two control samples which did not receive additional 02. Data points are at the midpoint of the time intervals sampled because they represent average rates for those periods. Rates are in ,umoles CT2H g-' hrl fresh weight.

Our results produced no evidence that pO2 was a critical factor when the C2H2 technique was used as described. The addition of 0.2 atm of 02 after three hours of incubation produced no stimulatory effect on the reduction rate. The sharp decline that did result (Fig. 2) indicates that 02 levels at the end of three hours were sufficiently high to produce concentrations greatly in excess of the normal atmosphere when O2 recharge took place. Such conditions have been observed to in- hibit the nitrogenase catalyzed reaction (Becking, 1970).

Increasing the biomass of nodules in each sample from 1.5 to 3 g had no measurable effect on C2H2 reduction rates in a 1-hr incubation period. This indicates that nodule respiration was not a limiting factor. The selection of a constant 1.5 g- nodule biomass sample was therefore based on convenience in nodule collection rather than on factors of incubation pO2 and nodule respiration.

The mean values for nitrogen fixation obtained by the C2H2 method were in agreement with those obtained by the isotopic 15N technique. The value of 2.20 umoles N g-l hr-1 falls well within the range of values obtained by the C2H2 technique (0.01 14 to 3.63). Only five samples were analyzed via the 15N method. We present these figures to indicate that the two techniques yield results that are generally comparable, but we did not feel that the data were sufficient to develop our own C2H2 reduction, N2 fixation conversion ratio. The ease and low cost of the C2H2 assay favor its use, es- pecially in studies which require extensive field sampling.

TABLE 5. Some published rates of nitrogen fixation obtained by workers using different species and techniques. Rates have been converted to units of ,umoles N g-1 hr-1 (fresh weight) where necessary, using 3 moles C2H2 per 1 mole N conversion ratio of C2H2 reduction to nitrogen fixation

Method Worker Species (C2H2 or 15N) Rate

Delwiche et al. various Cea- 15N varied between (1965) nothus species 0.0106 and 0.212 Stewart et al. A lnus rugosa C2H2 1.5 (1967) Waughman Hippophae (1972) ramnoides

young plants C2H2 8.6 old plants C2H2 3.5

Akkermans Alnus glutinosa C2H2 varied between (1971) < 0.33-73.0 Dalton and Alnus crispa C2H2 1.7 Naylor (1975)

For a basis of comparison it is useful to look at other reported rates of nitrogen fixation described in the literature. Although to our knowledge no rate measurements have been made on the species Alnus tenuifolia, comparisons with other species and habitats are useful in establishing a frame of reference. Table 5 summarizes various rates reported by other workers. Results are reasonably consistent among different researchers, in that they are in the same order of magnitude, and are cor- roborated by different techniques.

Goldman and Armstrong (1969) showed nitrogen to be a major factor limiting primary productivity of phytoplankton in Lake Tahoe. It appears to be one of the chief factors limiting productivity in the terrestrial environment as well. A knowledge of the amounts and pathways of nitrogen flux through these systems is important to our understanding of their dynamic aspects. It seems reasonable to assume that the relatively large amounts of nitrogen potentially available via symbiotic nitrogen fixation must play an important role in such ecosystem characteristics as biomass, productivity, site fertility, successional trends, and climax structure. The measurement of nitrogen fixation rates presented here is the es- sential first step in quantifying the alders' contribution to the nitrogen cycle.

LITERATURE CITED

AKKERMANS, A. D. L. 1971. Nitrogen fixation and nodulation of A lnus and Hippophae under natural conditions. Doctoral dissertation, Rijksuniverseteit, Leibig.

BECKING, J. H. 1970. Plant-endophyte symbiosis in non-leguminous plants. Plant Soil 32: 611-654.

BERGERSEN, F. J. 1970. The quantitative relationship between nitrogen fixation and the acetylene reduction assay. Aust. J. Biol. Sci. 23: 1015-1025.




DALTON, D. A., AND A. W. NAYLOR. 1975. Studies on nitrogen fixation by Alnus crispa. Amer. J. Bot. 62: 76-80.

DELWICHE, C. C. 1970. The nitrogen cycle. Sci. Am. 223: 136-146.

, P. J. ZINKE, AND C. M. JOHNSON. 1965. Nitro- gen fixation by Ceanothus. Plant Physiol. 40: 1045- 1047.

DILLWORTH, M. J. 1966. Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteuria- num. Biochim. Biophys. Acta 127: 285-294.

GOLDMAN, C. R. 1961. The contribution of alder trees (Alnus tenuifolia) to the primary productivity of Castle Lake, California. Ecology 42: 282-294.

, AND R. ARMSTRONG. 1969. I. Productivity. Primary productivity studies in Lake Tahoe, Cali- fornia. Verhandlungen International Association of Theoretical and Applied Limnology 17: 49-71.

HARDY, R. W. F., R. 0. HOLSTEN, E. K. JACKSON, AND R. C. BURNS. 1968. The acetylene-ethylene assay for nitrogen fixation: laboratory and field evalua- tion. Plant Physiol. 43: 1185-1207.

KoCH, B., AND H. T. EVANS. 1966. Reduction of acetylene to ethylene by soybean root nodules. Plant Physiol. 41: 1748-1749.

NEILSON, J. A. 1973. Lake Tahoe basin vegetation II: Natural vegetation zones. Davis, Calif., Univ. of Calif., Institute of Govern. Affairs.

SCHOLLHORN, R., AND R. H. BuRRIs. 1966. Study of intermediates in nitrogen fixation. Fed. Proc. 25: 710.

STEWART, W. D. P., G. P. FITZGERALD, AND R. H. BURRIS. 1967. In situ studies of N2 fixation using the acetylene reduction technique. Proc. Nat. Acad. Sci. 58: 2071-2078.

WAUGHMAN, G. J. 1971. Field use of acetylene reduction assay for nitrogen fixation. Oikos 22: 111-113.

. 1972. The effect of varying oxygen tension, temperature and sample size on acetylene reduction by nodules of Alnus and Hippophae. Plant Soil 37: 521-528.

WHEELER, C. T. 1969. The diurnal fluctuation in nitrogen fixation in the nodules of Alnus glutinosa and Myrica gale. New Phytol. 68: 675-682.



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Measuring Rates of Symbiotic Nitrogen Fixation by Alnus tenuifolia