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Conifer foliage can provide valuable data to geneticists for reforestation studies.

Many conifers have characteristic extractive compounds that give distinctive characteristics to the wood. When such compounds are under genetic control these can be used as taxonomic markers just as well as "good" morphological characters. This forms the basis of chemotaxonomy (also known as biochemical systematics). The advantage of the chemical approach is that one deals with discrete chemical structures that can be quantified. Often, groups of biosynthetically related compounds are involved which can provide information that is not available from morphological data. However, it is essential to work on the basis of botanical classification and work with well-identified plant material.

My interest in this field goes back to a short sabbatical with Prof Holger Erdtman at Stockholm, Sweden in 1958. Erdtman must be considered as the pioneer in conifer chemotaxonomy. His studies on the heartwood extractives of pines, cedars, and junipers from all over the world laid the foundations for this field [1].

The various terpenes found in conifer wood, bark, cambium, and leaves are extractives that are suited for such studies. Initially, only the few major terpenes found in wood or bark were studied [ 1, 2 1. The conifer leaf only are much more complex and contain many minor and dozens of trace components that could not be isolated and identified by fractional distillation or column chromatography.

At the prairie Regional Laboratory, now the Plant Biotechnology Institute, of the National Research Council at Saskatoon, we were instrumental in developing gas-liquid chromatography to the point where complex mixtures such as the steam-volatile essential oils, fatty acid esters, Waxes, and amino acid derivatives could be analyzed both qualitatively and quantitatively. I have applied this technique to a systematic study of the differences between, and variation within, the leaf or foliage oils of our northern conifer species.

Holger Erdtman. [1] defined chemotaxonomy simply as the study of characteristic chemical compounds in related, or supposedly related plants. Since the primary cell metabolites are common to an plants, one must turn to the secondary compounds, or so-called plants extractives, for markers that can show closer or more distant botanical relationships. Groups of biosynthetically related compounds, such as the flavonoids, terpenes, and alkaloids, are particularly well-suited. The basic requirements for a given group of chemical compounds to be effective in chemotaxonomic studies have been discussed in some detail [3].

Just as the botanist looks at the different organs of a plant, so too the chemist should study the components of different anatomical parts. However, the numbers of samples to be analyzed is high since many individual plants or trees from the same and different populations must be examined.

Whenever possible, the whole geographical range of a species should be sampled. Hence we usually have to confine ourselves to one particularly useful organ. Erdtman analyzed the heartwood which, being dead, no longer involves physiological or seasonal changes. Mirov [2] and co-workers studied the gum turpentines of most of the pines of the world. I chose the leaf oils, not only because of the ease of isolation of the volatile oil from a well-defined plant organ, but because they contain a,broad spectrum of terpenes [3, 4]. A.E. Squillace, and others [5], carried out chemotaxonomic studies with the cortical and xylem oils.

Terpenes are poisonous to the primary cell metabolism and hence they cannot be stored in the regular plant cells. In conifers, the terpenes are laid down in the specialized cells of resin canals or oil glands. Leaf or needle resin canals are not connected to those of the branch wood (xylem) or bark (cortex). Thus, by separating the leaves from the branchlets [3, 4] the terpenes of a distinct anatomical, and presumably biosynthetic unit can be analyzed.

The volatile terpenes can be recovered easily by steam-distillation or by extraction [3, 4, 6] Yields (based on fresh weight) are rather variable, from a low of 0.1% or less in some spruces to over 2% in cedars and junipers. Some precaution in collecting, transporting and storing conifer leaf samples is called for.

The advantage of steam-distillation is that only the more volatile components are recovered. These include the mono- and sesquiterpenes, and sometimes also diterpenes, volatile aromatic ethers of the safrole-eugenol type, some aliphatic hydrocarbons, alcohols, and esters, and the odd aromatic ester. A few artifacts may also be encountered. A great variety of the better known monoterpenes are present, but to date none of the menthol types(common in the mint family) have been found. Biosynthetic differences with other essential oil bearing plant families are also evident. We have also isolated new terpenes with unusual structures. All these compounds can be separated and quantified by gas-liquid chromatography (gc), but isolation and identification is time-consuming and often difficult. This has been much improved and speeded up considerably by the combined gc-mass spectral method [7]. In a typical survey of a given species, the amount of percentage data that is accumulated is very large. To handle such data, we have developed computerized numerical (multivariate) analyses that permit ranking of trees and/or populations [8, 9].

Our initial studies with foliage collected during late fall and winter showed that for a given tree the relative terpene percentages vary little with different aspects of the crown and needle age, provided a sufficiently large sample is taken [ 3, 4 1. Grafts or clones have the same composition as the parents, irrespective of the sites where these are growing. Thus, ecological factors per se do not affect the relative amounts, but the overall yields of oil may vary.

This provided evidence that during the dormant season the relative amounts of terpenes are under strict genetic control. Tree-to-tree variability was found to be low, intermediate, or rather high, depending on species or variety. Most important, from the chemotaxonomic point of view, each conifer tree produces its own terpene pattern or "fingerprint" year after year.

Results

Six of our northern conifers have very low intra- and interpopulation variability. Black spruce (Picea matiana) has virtually the same terpene composition (main components: 40-60% bornyl acetate; 10-20% camphene, etc; for the many minor and trace components see our publications) throughout its natural range from Newfoundland to northeastern British Columbia and the Yukon Territory. The botanically closely related red spruce (P. rubens) has a similar leaf oil pattern with only minor quantitative differences. These are large enough to distinguish it from black spruce, but the hybrids and backcrosses from the zone of introgression in northeastern regions could not be distinguished.

Equally low variability was found in the two Thuja species, which are characterized by high percentages of thujone (65-80%) and isothujone (7-10%). Eastern white cedar can be distinguished from westrnm red cedar mainly by its higher content of fenchone (5-10% and 0.1-0.50% respectively). The latter species showed very little variation in the terpene percentages throughout its range, and only by applying sophisticated numerical analyses could very minor regional differences be asigned [9].

The western ponderosa pine (Pinus ponderosa) also has a leaf oil with low variability (40-65% [beta]-pinene, 10-20% [alpha]-pinene, 5-15% car-3-ene, etc.) and no regional differences were found within its range. But consistent qualitative and quantitative differences were found between it and the north-central variety P. ponderosa var. scopulorum. Low variability was also found in the leaf oil of the coastal variety of lodgepole pine, the socalled shore pine (P. contorta var. contorta), which contains mainly -phellandrene (50-75%). This contrasts with very large variations and several different terpene patterns in the interior lodgepole pines (var. latifolia, murrayana, and bolanden). Where the shore and lodgepole pines intergrade, intermediate patterns were found [10].

Several conifer species have medium variability in their leaf oil patterns and in some of these we found consistent geographical differences. Thus, white spruce (P. glauca) has an eastern and a western pattern (15-40% and 35-65% camphor, 15-35% and 10-20% bornyl acetate, 5-10% each camphene, etc., respectively).

We found a gradient in the western populations with the highest percentage of camphor being found in northeastern British Columbia. in balsam fir (Abies balsamea) we also found an eastern and western terpene type (20-45% and 30-55% [beta]-pinene, 5-35% and only a trace of car-3-ene, etc., respectively) and we could detect the introgression with interior subalpine fir (A. bifolia) in central Alberta. Medium variation was also found in the leaf oils of a limited number of samples of the eastern hemlock (Tsuga canadensis).

We studied in considerable detail the leaf oil compositions of the interior and coastal Douglas-fir varieties (Pseudotsuga menziesii var. glauca and var. menziesii resp.). The interior variety showed comparatively low variability 20-30% camphene 20-30% bornyl acetate, 10-15% [alpha]-pinene, etc.) and there is only a small quantitative gradient in some of the minor terpenes from northern to more southern populations.

In contrast, the coastal variety has two rather variable terpene patterns (type A: 25-55% [beta]-pinene, 8-15% [alpha]-pinene, etc.; Type C: up to 10-20% each of terpinen, terpinolene, sabinene and terpinen-4-ol, etc.). These intermix freely in many coastal regions.

It becomes more complex

To make the situation even more complex, the coastal and interior varieties introgress in the coastal mountains. Because of the unique terpene patterns it is possible to study this intermixing in a quantitative manner. We found intermediates at wet sites in the interior as well as in the coastal ranges. This has implications for creosote and other industrial impregnation of logs, etc., where the wood of the coastal variety is penetrated much more readily than that of the interior variety.

An equally complex situation is encountered with the interior spruce of British Columbia, which involves white spruce in the north and mainly Engelmann spruce (P. engelmannii) in the south and at higher elevations in the Rockies. These two species introgress extensively and many different crosses and backcrosses can be found in the central regions. We found Engelmann spruce to have one of the most variable terpene patterns of all conifers, yet it differs sufficiently from that of western white spruce so that intermediates can be distinguished.

There are also a number of species with a high tree-to-tree variation, yet the means of 10 to 15 trees each from different populations are similar within such a species. In Sitka spruce leaf oil (18-35% myrcene, 10-25% piperitone, variable amounts of camphor, [beta]-phellandrene, etc. and two unusual hemiterpene esters) the means of northern, central and southern west Coast populations were similar. The introgression with white spruce (Skeena valley, Haines area) is readily discernible. Grand fir (A. grandis), Pacific silver fir (A. amabilis), the coastal (A. lasiocarpa) and interior subalpine fir (A. bifolia) also have high intrapopulation variations, but little or no geographic differences in population means were found.

The leaf off of coastal subalpine fir (35-60% [beta]-phellandrene, 15-20% [alpha]-pinene, 3-17% limonene, etc.) differs greatly from that of the interior subalpine fir (13-32% bornyl acetate, 10-30% [beta]-phellandrene, 8-23% [beta]-pinene pinene, 5-21 % limonene, 7-16% camphene, etc.) and their introgression can also be studied quantitatively. These differences were so large as to lend strong support to the old botanical classification into A. lasiocarpa and A. bifolia (11).

The same type of variability was found in the leaf oils of western hemlock, Tsuga heterophylla (15-30% myrcene, 13-25% [beta]-phellandrene, 5-20% [alpha]-pinene, 6-13% cisocimene) and mountain hemlock, T. mertensiana (4-40% car-3-ene, 12-32% a-pinene, 9-23% [beta]-phellandrene, 2-17% limonene, etc). In these two species the means from coastal and interior populations were similar, but in the latter there were consistent differences between northern and southern populations.

We also investigated the foliage oils of some North American junipers. The junipers of the subsection Sabina pose fascinating taxonomic problems. The red (Virginian) juniper grows from southern Ontario and Quebec south as far as Texas, where it overlaps the natural range of the Ashe juniper. Some botanists reported that these two junipers hybridize and introgress, but our terpene data (and subsequent micromorphological data) showed conclusively that this case; the gross morphological similarities were due to geographic variation in the red juniper. In contrast, the accepted introgression between the western creeping and Rocky Mountain junipers was clearly evident in the terpene data. Juniper oils are often rich in sesquiterpenes and aromatic ethers.

In the jackpine, Pinus banksiana, we found high tree-to-tree variability and some differences in population means. Several regional types were present, and the introgression with lodgepole pine in central Alberta could be discerned.

With the larches one is faced with the problem that their leaves are deciduous. Thus, the time for collecting is rather short, viz from mid to late summer when leaf elongation has stopped to just before they turn yellow and physiological changes as well as losses occur. Also, only a single year's growth can be examined. Thus, we looked also at the branch oils. Their terpene composition was as rich as, and fairly similar to those of the leaves and I suggested that the latter may be more suitable for further studies [12].

Conclusions

Our surveys have shown that conifer foliage is a rich, but often highly variable source of terpenes. in several species one particular terpene may occur in 50 to 80% amounts. But the high cost of collecting conifer branches in Canada does not make this a commercially attractive source of industrially useful terpenes. Hence, I have concentrated more on biological applications of our data. This has led to close and fruitful cooperation with various forestry organizations. Our data are not only of concern to the botanist, but to the geneticist, in reforestation, and they can provide data for studies of host-insect interactions. The extensive deforestation in many areas of North America has eliminated natural populations to a point where complete distribution and variation studies of different conifer species are no longer possible. Provenance trials at different forestry stations and plots can fill in the gaps, provided these cover the entire natural range of a given species. Just as the loss of species in the tropics deprives us of potentially valuable chemicals, so we may loose intermediate northern conifer types that are particularly well suited for the site at which they have evolved, and their contribution to e.g. population diversity, growth characteristics, or disease and insect resistance will never be known.

References

[1.] Holger Erdtman. Chemical Plant Taxonomy. (T. Swain, editor), Academic Press, Inc. New York, pp. 89-125 (1963). [2.] Nicholas T. Mirov. The genus Pinus Ronald Press, New York. (1962), and Composition of the gum Turpentines of Pines U.S. Forest Service Technical Bulletin No. 1239 (1961). [3.] Ernst von Rudloff, Biochemical Systematics and Ecology, 2, pp. 131-167 (1975). [4.] Ernst von Rudloff, Recent Advances in Phytochemistry, 2, pp. 128-162 (1969). [5.] A.E. Squillace, Modem Methods in Forest Genetics (J.P. Miksche, editor) Springer Verlag, pp. 126-157 (1976). [6.] John F. Manville, personal communication. [7.] Robert P. Adams, Michael Granat, Lawrence L. Hogge and Ernst von Rudloff, journal of Chromatography, 17, pp. 75-81 (February 1979). [8.] Martin S. Lapp and Ernst von Rudloff, Canadian Journal of Botany, 60, pp. 2762-2769 (1982). ]9.] Ernst von Rudloff, Martin S. Lapp, and Francis Yeh, Biochemical Systematics and Ecology, 16, pp. 119-125 (1988). [10.] Ernst von Rudloff and Martin S. Lapp, Canadian Journal of Forest Research, 17, pp. 1013-1025 (1987). [11.] Richard S. Hunt and Ernst von Rudloff, Taxon, 28, pp. 297-305 (1979). [12.] Ernst von Rudloff, Journal of Natural Products, 50, pp. 317-321 (1987).

Suggested reading

1. Jeffrey B. Harbome Comparative Biochemistry of the Flavonoids. Academic Press, London (1967). 2. Robert P. Adams. identification of Essential Oils by Ion Trap Mass Spectrometry Academic Press, Inc., New York (1989).
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Author:von Rudloff, Ernst
Publication:Canadian Chemical News
Date:Apr 1, 1992
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