METAL ANALYSIS AND R4NDOM AMPLIFIED POLYMORPMC DNA CHARACTERIZATION OF JACK PINE (Pinus banksiana)

POPULATIONS FROM THE SUDBURY REGION

by Wayne Stéphane Granon

A thesis submitted in partial hlfiliment of the requirements

for the degree of Master of Science in Biology

School of Graduate Studies Laurentian University

Sudbury, Canada November, 1998

a Copyright by Wayne Stéphane Gratton 1998.

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To Seanna and rny parents, without whorn this would not have been possible

Abstract

Present day levels of metals in soi1 and jack pine (Pinus banksiana) needles taken near

smelters in Sudbury, Canada and fiom uncontaminated sites were measured by ICP-MS.

Significantly bigher levels of cadmium, copper, nickel and Iead in needles were observed

near Sudbury smelters compared to control sites. In contrast, zinc concentrations were

significantly Iower. Significant negative correlations between copper and zinc (r=-0.63,

ps0.05 ), nickel and zinc (r-0.69, ps0.05) and Iead and zinc (r-0.70, p d . 0 5 ) in jack pine

needles suggest possible antagonistic interactions. Analysis of soil samples indicated higher

accumulations of cadmium, cobalt, copper, nickel and lead in the fust 5 cm of soil

decreasing with soil depth Observation of tree cores collected near Sudbury smelters

reveded distinct sensitive ring patterns for years 196 1 - 1962, i 966- 1967 and 197 1 for most

cores.

Random arnplified polymorphic DNA ( M D ) analysis of jack pine trees fiom Sudbury and

uncontarninated sites indicated low levels of polymorphisms within and among populations.

Six out of eleven primers screened did not produce any amplification, two amplified poorly

and the remaining three primers produced scoreable bands. Species-specific markers were

identified when red and jack pines were compared.

Acknowledpents

1 would like to thank Dr. K. Nkongolo for helping me reaiize that hard work, dedication and

perseverance are essentiai to become a good scientist. Your guidance, patience and

encouragement will aiways be remembered. I am grateful to Dr. P. Beckett and Dr. V.

Clulow for their advice and cntical reviews and to Dr. G. Spiers for his insight on metal

analysis. Technicd assistance of Messrs. Luc Taillefer, Danny Gratton and Normand Graiton

tt-ith smple coIlections is gratefuiiy acknowIedged, Thanks to Dr. B. Bowin, Mr. John

Hechler and Ms. Seanna Hoendermis fiom the Ministry of Northem Development and

Mines for ail their analytical assistance. Thanks are also extended to Pierre Thibodeau, staff

and faculty of Laurentian University BioIogy Department, Mary Taylor tiom the Laurentian

University Audio Visual Centre, Petawawa Nationai Forest Institute and the Ontario Ministry

of Natural Resources in Angus, Ontario for ail their technicd support. Special thanks to the

Robert Spencer Foundation for financiai support.

Table of contents

Abstract .............................................................. iv

....................................................... Acknowledgments v

....................................................... Table of contents vi

... .......................................................... List of tables vin

.......................................................... List of figures ix

..................................................... General introduction 1

Chapter 1 : Literature review .............................................. - 3

Chapter 2: Metal accumulations in soi1 and jack pine needles ..................... 8

2.1. Introduction ............................................... - 8

2.2. Materials and Methods ....................................... - 9

2.2.1. SampIe collection ..................................... - 9 ..................................... 2.2.2. Tree core analysis - 9

2.2.3. Metai analysis ........................................ 12 2.2.4. Statistical analysis ..................................... 13

2.3. Results and Discussion ...................................... 14

2.3.1. Tree core dendrochronology ............................. 14 ........................... 2.3.2. Jack pine needle metal analysis 13

2.3.3. Total soi1 metal analysis ................................. 30 2.3.4.SoilpH ........................................... - 3 8

Chapter 3: RAPD characterization of jack pine populations fiom the Sudbury region and uncontaminated sites ........................................ 40

3.1. Introduction ........................................... - 4 0

3.2. Materials and Methods ................................... 32

...................... 3.2.1. Jack pine seedling germination -42 .................................. 3.2.2. DNA extraction - 4 2

...................... 3 .2.3. Amplification of RAPD markers 43

3.3. Resdts and Discussion ...................................... -45

................. 3.3.1. DNA concentration and purity values - 4 5 ......... . 3.3 .2 RAPD characterization of jack pine populations - 45

..................................................... Generalconclusions 56

............................................................ References 58

vii

List of tables

Table 1. Longitude and latitude coordinates of sampling sites .................. 10

Table 2. Age determination of jack pine populations fkom the Sudbury region ........................................ and uncontaminated sites 1 5

Table 3. Analyticai results of pine needle reference material (NIST 1 575) ......... 1 7

Table 4. Metal concentrations in jack pine needies fiom the Sudbury region ........................................ and uncontaminated sites. 18

Table 5. Metal range concentrations in jack pine needles from the Sudbury region ....................................... and uncontaminated sites. - 2 0

Table 6. Spearman correlation coeficients of total metal concentrations in jack pine needles. .............................................. -22

Table 7: Anaiyticai r e d t s of soi1 reference material (CCRMP-Till 1) ............. 3 1

Tabie 8: Total metal concentrations in soil fiom the Sudbury region and ........................................... uncontaminated sites - 3 2

Table 9: S p e m a n common correlation coefficients of total metal concentrations in soi1 samples ................................................ - 3 5

Table 10. Total metal concentrations in 0-5 cm soil profiles fiom the Sudbury region and uncontaminated sites. ....................................... - 3 6

Table 1 1 : DNA concentration and purity values from jack pine seedling sarnpies. .... 46

Table 12. RAPD markers fiom jack pine populations fiom Sudbury and .......................................... uncontaminated sites. - 4 7

Table 13. RAPD markers and species-specific bands for jack and red pine populations. ........................................... - 4 8

viii

List of figures

Figure 1 . Location of soil aud jack pine population sampling sites within the ............................................. Sudbury region 11

Figure 2 . Tree cores collected near Sudbury smelters revealing distinct sensitive ringpatterns .................................................. 16

Figure 3 . Lead concentrations (mg kg*') in jack pine needles collected fiom Sudbury ................................................ and control sites 23

Figure 4 . Copper concentrations (mg kg-') in jack pine needles collected fiom Sudbury ............................................... and control sites- 25

Figure 5 . Nickel concentrations (mg kg-') in jack pine needles collected nom Sudbury ............................................... and control sites- 26

Figure 6 . Cadmium concentrations (mg kg-') in jack pùie needles collected fiom Sudbury andcontrolsites- ............................................... 27

Figure 7 . Zinc concentrations (mg kg') in jack pine needles collected ftom Sudbury ................................................ and control sites 29

Figure 8 . pH values of soi1 depths fiom Sudbury and control sites . . . . . . . . . . . . . . . . 39

Figure 9 . RAPD markers fiom jack pine populations using primer P.2 . . . . . . . . . . . . 49

Figure 10 . RAPD markers fiom jack pine populations using primer P.8 ............ 50

Figure I 1 . RAPD markers in jack pine populations using primer P.9 . . . . . . . . . . . . . 51

General introduction

The Sudbury region is known for the rnining and smelting of high sulphide ores containing

nickel, copper, iron and precious metals. Early practice of open roast yard rnining, followed

by the construction of smelters, released large quantities of sulphur dioxide and metai

particulates to the atmosphere resulting in elevated metal content in soi1 and vegetation near

Sudbury smelters (Freedman and Hutchinson, 1980; Hutchinson and Whitby, 1977:

Hutchinson and Whitby, 1974). Years of intense Wigation of more than 100 million tonnes

of suIphur dioxide and tens of thousands of tomes of metal particulates created barren and

semi-barren lands near smelters (Amiro and Courtin, 198 1 ; Struik, 1973). Forest

comrnunities with a 15 km radius of smelters were nearly eliminated.

Plant species near Sudbuy smelters have been characterized mainly as birch and maple

transition communities (Amiro and Courtin, 198 1). Communities are composed essentially

of white birch (Berula papyrzyera), although red maple (Acer rubrurn), large tooth aspens

(Popttlus grandidentata), red oak (Quercus borealis), red pine (Pinus resinosa) and jack

pine (Pinus banksiana) do exist. The colonization of the barren landscapes by metal-tolerant

species like Deschampsia caespitosa and Agrostis gigantea have suggested possible genetic

base tolerance (Winterhalder, 1 995). Previous investigations of the Sudbury ecosystem have

provided information on metal levels in soils and their uptake and accumulation by plants,

but knowledge of genetic effects of plants growing in these contaminated areas is limited.

Recently, differences in the genetic structure of plants growing in contaminated areas have

been reported (Müller-Starck, 1989, 1985; Bergmann and Scholz, 1987; Mejnartowicz.

1983).

Although reduction in industrial emissions during the last 30 years has resulted in significant

improvements to the local ecosystem, continued investigation and monitoring of soil and

vegetation is essential. Random amplified polymorphic DNA (RAPD) analyses ofjack pine

trees were conducted to determine if genetic differences exist between populations growing

in Sudbury contaminated soil and uncontaminated sites. Determination of current levels of

metal content in soil and jack pine needies fiom these sites were also investigated.

Chapter 1: Literature review

The community of Sudbury located in Northern Ontario (46O30' N, 8 1°00' W, 259 m above

mean sea level) has a long history of mining. The discovery of a rich ore body in 1883 durhg

the construction of the transcontinental route of the Canadian Pacific Railway caused an

enormous mhing boom. The deposits, extending dong the 150 km rirn ofthe Sudbury Basin,

contained high sulphide ores of nickel, copper, iron and precious metds. Mthough fur

trading and lumbering existed within the region, Sudbury becarne known for its nch sulphide

ores. Several mining companies within the region soon established themselves, however, the

International Nickel Company (Inco Ltd.) and Falconbridge Lirnited became dominant in

Sudbury.

Early metal extraction fiom sulphide ores involved open roast yard rnining. Large quantities

of ore were piled on beds of corewood, ignited and aliowed to bwn for long periods.

Sulphides within the ore oxidized and were released as sulphur dioxide. Smelting then

followed to separate metals. Although dense sulphur dioxide fimes, emitted fiom roast beds,

mostly at ground level, killed plants and acidified soils, widespread contamination of

Sudbury area soils was caused mainly by smelter fiimes fiom stacks containing high levels

of sulphur dioxide, copper and nickel particles. R o m beds produced Iarge amounts of

sulphur dioxide but only localized metal particdate deposition, resulting in fewer permanent

effects to the surrounding soils (Winterhalder, 1 995).

Investigations over the last 40 years have provided critical information. The combinations

of roast beds and mining smelters interacted to produce dramatic changes to the Sudbury

landscape. Fumigation by area smelters caused the elimination of vegetation and ground

cover near smelters- Erosion caused rich nutrient soil horizons to be washed away

(Lautenbach, 1987). Acidification and metal toxicity of soils caused serious breakdowns in

plant-soi1 relationships (Winterhalder, 1984). Ecological degradation soon followed and

large areas of forest communities near smelters disappeared creating barren and semi-barren

landscapes (Stniik, 1973)-

Early investigations by Limon (1 958) and Gorham and Gordon (1960) revealed few plant

species near smelters. Sensitive species such as lichens and white pine were virtudly absent

(Leblanc and Rao, 1966; Gorham and Gordon, 1960). Amiro and Courtin (1 98 1 ) showed that

white birch was the dominant species within the serni-barren iandscapes. Severe surface soil

contaminations were also noted near smelters (Freedman and Hutchinson, 1 980; Hutchinson

and Whitby, 1977). High levels of copper, nickel and sulphur dioxide were found in airborne

pollutants, rainfall and snow sarnples (Freedman and Hutchinson, 1980; Hutchinson and

Whitby, 1 977; Hutchinson and Whitby, 1974). Extensive ecological investigation and long

term precipitation and air monitoring prograrns were then developed. Early reports indicated

that sulphur dioxide concentration had a direct effect on vegetation and soil, However.

permanent and widespread metal contamination in soil and vegetation was attributed to

smelter fumes emitted fiom stacks containing high metal content and suiphur dioxide

(Winterhdder, 1996; Freedman and Hutchinson, 1980; Hutchinson and Whitby, 1974).

Reports of soil and vegetation sampling indicated higher metal concentrations within

fumigation and pollution zones established by Struik (1 973) and Dreisinger and McGovem

(1 969). Soi1 profile analysis indicated higher metai content in surface soils (Hazlett er al.,

1 983 ; Freedman and Hutchinson, 1980; Rutherford, and Bray, 1 979; Hutchinson and Whitby,

1977). Plant metal accumulations were above nomal levels (Freedman and Hutchuison,

1980; Hutchinson and Wbitby, 1974), however, metal levels declined substantially in soil

and vegetation with increasing distance fiom smelters (Freedman and Hutchinson, 1980;

Whitby et al. 1976; Hutchinson and Whitby, 1974). Plant species richness aiso increased

m-i th distance (Hutchinson and Whitby, 1974).

The effects of mining activities have aiso k e n recognized in other parts of the country and

around the world. in Canada, both Trail (BC) and Wawa (ON) exhibited effects of smeiting

(Archibold, 1978; Gordon and Gorham, 1963). Internationally, the devastations caused by

the copper-nickel smelter of Monchegorsk in Russia have been compared to Sudbury

(Kryuchkov, 1993). Contaminations of local biotaand soils near mining complexes have also

been reported elsewhere (Jordan, 1975; Little and Martin, 1972)-

Much attention has focused on the environmental effects of mining pollution. Although

reports provide information of metai levels in soil and their uptake and accumulation by

plants, knowledge of genetic effects on plants growing in contaminated areas is limited.

Several authors have reported differences in the genetic structure of plants growing in

contarninated areas ( Müller-Starck, 1989,1985; Bergmann and Scholz, 1987; Mejnartowicz.

1 983). Enzymatic studies of Norway spruce revealed genetic differences between groups of

sensitive trees in polluted areas (Bergmann and Scholz, 1987; Scholz and Bergmann, 1 984).

Obsemations of higher heterozygosity in tolerant plants of European beech in Germany

(Müller-Starck, 1985), scots pine in Gemany and Great Britain (Geburek et al., 1987; Farrar

et al., 1977) and trembling aspen and red maple in the United States (Berrang et al., 1986)

have been reported. Mejnartowicz (1983) presented evidence of loss of genes and

heterozygosity in tolerant scots pines.

During the last 10 years, DNA technologies have been used to study plant population

structure and species differentiation. However, many of these new techniques are costiy and

laborious for large numbers of individuals (such as restriction fragment length

polyrnorphisms (RFLP)) or limited in their genomic distribution and level of diversity they

reveal (such as isozyrnes) (Sobral and Honeycutt, 1994). The newly developed polymerase

chôin reaction technique (PCR) is simple and cost efficient. It consists of 3 steps: thermal

denaturation of DNA, annealing of oligonucleotide prhers to template DNA and primer

extension by DNA. polyrnerase with nucleotide triphosphates (Saiki er al., 1985). The

discoveries of thermally stable DNA polymerases (Saiki er al., 1988) and the development

of automated thermal cyclers have allowed DNA amplification to become widely used by

scientists interested in comparing organisrns at the molecular level.

Various methods of PCR such as random amplified polymorphic DNA (RAPD) have

evolved recently. RAPD markers, obtained by PCR amplification of random DNA segments

fiom single arbitrary primers, are based on mismatches in primer binding sites andior

insertioddeletion events resulting in the presence or absence fiom a single locus (Welsh et

a' 1 990, Williams er al. 1990). Genetic maps of Arabidopsis sp. (Reiter et al., I 992),

sugarcanes (Al-Janabi et al., 1 993), soybeans (Williams et al., 1990) and pine species

(Carlson el al., 199 1 ) have been produced using RAPD methodology. RAPD has been

successfûl in plant systematics and population genetic studies (Nkongolo, 1998, Furman et

al. 1997 Gunter et al., 1996, Yeh, et al., 1995; Sweeney and Danneberger, 1995; Van

Coppenolle el al., 1993).

Chapter 2: Metal accumulations in soi1 and jack pine needles

2.1. introduction

Environmental conditions within the Sudbury area have improved considerably during the

last 30 years (Dudka et al., 1995; Gunderman and Hutchinson, 1993; Negusanti and

McIlveen, 1990). Improvements to smelting technology, greater emission dispersal by

smelting stacks, closure of the Coniston smelter and stricter governmental emission

guidelines have reduced both sulphur dioxide and metal particle emissions (Gunn et al. 1 996;

Winterhalder, 1996; Negusanti, 1995). Furthemore, reclamation efforts by the Municipality

of Sudbury and local mining companies have resulted in significant improvements of the

Sudbury landscape. Vascular and nonvascular plants such as conifers, birches and lichens

have re-invaded semi-barren landscapes (Gunn, 1996). However, recolonization of bmen

lands is Iimited to very few species, mostly metal-tolerant (Rauser and Winterhalder. 1985).

Lack of reestablishment and/or stunted growth exhibited by some species are presumably in

response to a combination of unfavorable microclimate conditions and continued soil toxicity

(Courtin, 1 994).

Continued investigation and monitoring of soil and vegetation within the Sudbury region are

essential to understand the recovery. Jack pine populations, consisting mainly of small

groups ranging fiom 30 to 50 individuals, were analyzed for metal content. Soi1 profile

analysis for metal content was also conducted- Sites were selected based on population size.

location and direction in relation to Sudbury smelters.

2.2, Materials and Methods

2.2.1. Sample collection

Samples of soil, jack pine needles and tree cores were collected fiom 8 sites within 15 km

of Inco Ltd- and Falconbrïdge Ltd. smelters during September and October 1995 (Table 1,

Figure 1). Two control sites, each located approximately 100 km northwest and northeast

from Sudbury, were aiso sampled (Table 1). Needles were rinsed with deionized distilled

water, oven dried for 16 h and homogenized. Soi1 sarnples, collected in intervals of 5 cm to

a 15 cm depth, were air dried, lightly ground with a ceramic mortar and pestle and sieved to

2 mm. Tree core samples, collected using an Uicrement borer, were air dried and mounted

on wooden backings. Samples were stored until analysis.

2.2.2 Tree core analysis

Annual rings were cross-dated using the narrow ring pattern method from living trees

(Yamaguchi, 199 1). Tree cores were sanded using grades of sandpaper, fiom 60 to 600 grit.

to clariQ ring structures. Rings were counted backwards microscopically in time from the

outermost ring, labeling each decade ring (e.g. 1990, 1980, etc.). Years of rings that were

noticeably narrower than adjacent rings were recorded and Iabeled as marker nngs. Cores

fiom living trees were aged and cross-dated quickly and efficiently by listing the narrow

rings patterns (marker nngs) present in each core and comparing to other cores for shared

narrow rings patierns.

Table 1 . Latitude and longitude coordinates of sampling sites

Sites Location Latitude Longitude

near Falconbridge smelter

near Falconbridge smelter

near Falconbridge smelter

near Inco smelter

near h c o smelter

near h c o smelter

Inco tailing

Faiconbridge property

Temagarni (control)

Low Water Lake (control)

Figure 1. Location of soi1 and jack pine population sampling sites within the Sudbury region.

2.2.3. Metal analysis

Metal analyses were performed as described by Marr (1979) and Carter (1993) with some

modifications. Samples (0.2 g) of homogenized soi1 in Teflon beakers were completely

digested to clear liquid with 15 ml of reagent T4A (400 ml HF, 40 ml HCIO, 40 ml HCI)

and placed on a hotplate at 120°C for 24 h. An additional 15 ml of reagent T4B (30 ml

HCIO,, 70 ml HCI, 380 ml distilled H,O) was added and let to stand for 24 h at 120°C- Eight

drops of HCl, 1 ml of HNO, and 4 drops of HF were added individually, swirled, cooled for

1 min and let to react for approximately 2-3 min. Fifieen millilitres of distilied H,O was then

added. beakers placed on hotplate for 30 min and volumes brought down to approximately

10 ml. SoIutions were diluted to a final volume of 100 ml with distilled H,O.

Samples (0.5 g) of needles were oven dried at 400 "C and digened using a modified HN0,-

30% H,O, - - procedure (Jones et al., 1 99 1 ). Five millilitres of concentrated HNO, were added

to needle samples in 50 ml centrifuge tubes and subjected to cold digestion for 16 h. Samples

were then heated to 1 10 O C for 1 h on digestion blocks, dlowed to cool to 70°C and 2 ml of

30% H201 was added and maintained at 70°C for 45 min. Samples were cooled and diluted

to a final volume of 10 ml with distilled H20. Al1 solutions were analyzed by ICP-MS for

total metal content.

Soi1 and needle sample digests were analyzed for metal concentrations of Cd, Co, Cu, Fe,

Mn, Ni, Pb and Zn at the Geoscience Laboratories of the Ministry of Northem Development

and Mines, Sudbury, Canada. Dataquality was assessed by digestion and analysis ofcertified

reference material for vegetation NIST 1575 (NBS 1575) and soil (Till 1, CANMET) and

analytical and method duplicates. The QNQC analyzed totaled 25%.

Soil pH was measured by adding 20 ml of distilled H,O to 10 g of air dried soil (c 2 mm)

material. Soil suspension was çtirred intermittently for 30 min and let to stand for 1 h

(Carter, 1993). Using a Fisher Scientific Accumet pH meter 910 rnodel, soil pH was

measured three times, 10 min apart, and averaged.

2.2.4. Statistical analysis

Statistical analyses were performed on soil and needle samples using SPSS-X and SPSS 7.5

for Windows (SPSS Inc., 1996)- For al1 statistical tests, differences were significant at

ps0.05. Kolmogorov-Smimov normality test was performed on soil and needle samples to

determine the degree of normality in data distribution. Both Bartiett-Box F and Cochrane's

C were used to evaiuate the degree of homogeneity of variance between data subsets.

Nonparametric analysis of variance Kniskal-Wallis followed by nonparametric Tukey-type

multiple cornparison (Zarr, 1996) were perfonned on soil and needle samples since data

transformations were unsuccessful to satis@ criteria of normality and homogcneity of

variance. Potential reiationships between metal content in needles were evaluated using the

Speamian rank correlation coefficients. individual soil depth correlation coefficients for

metal content were pooled when no significant differences were determined (Zarr, 1996).

Speaman common correlation coefficients were used to evduate any possible relationships.

2.3. Results and discussion

2.3.1. Tree core dendrochronology

Cores analyses revealed that mean age of populations ranged fiom 28.1 to 55.3 years for

sampling sites located near Sudbury smelters (Table 2). Ages fiom transpIanted trees on h c o

Ltd. tailing and Falconbndge Ltd. property were much younger. Control sites, Temagarni and

Low Water Lake, had mean ages of 37.9 and 81 -6 years respectively. Observation of cores

collected near smelters revealed distinct sensitive ring patterns prominent during the 1960's

and earl y 1 970's (Figure 2). Narrow rings for years 196 1 - 1962, 1 966- 1 967 and 1 97 1 were

observed on most of the sampling cores fiom Sudbury. Previous reports indicated tree ring

widths are influenced by age, geometric conshn t s and environmentai interactions (Cook,

1987). High levels of sulphur dioxide have been shown to cause reduced tree ring widths

(Thompson, 198 1 ; Kelly, 1980; Linzon, 197 1)- This was not investigated in the present study

but high levels of sulphur dioxide have been well docurnented in Sudbury during the 1 960's

and ear!y 1 970's (Potvin and Negusanti, 1995, Chan and Luis, 1 985).

2.3.2. Jack pine needle metal analysis

Reliability of the analytical digestion indicated that results were within acceptable ranges

for certified reference material NIST 1575 (Table 3). Only lead was below certified ranges.

Meta1 concentrations in jack pine needles are shown in table 4. High Ievels of cadmium,

copper, nickel and lead were found in needles fiom the Sudbury region. Concentrations

within the Sudbury area were between 3 and 10 times higher compared to control sites.

Table 2. Age determination of jack pine populations fiom the Sudbury region and uncontaminated sites

Sitea Range of age in years Mean age in years

10 85- 1 O 0 90.6 "Sites I,2,3 = sites located near Falconbridge Ltd. smelter; sites 4,5,6 = sites located near lnco Ltd. smelter; sites 7 = lnco Ltd. tailing; site 8 = site located on Falconbridge Ltd. property; site 9 = Temagarni (control site) and site 10 = Low Water Lake (control site).

Figure 2. Tree cores colIected near Sudbury smelters revealing distinct sensitive ring patterns. Figures A and B represent ring patterns fiom a tree cotlected £iom site 2 and 5 respectively.

Table 3. Analfical results of pine needle reference material (NIST 1575); concentrations are in m g kg", dry wt.

Elements Certified mead Reference rangea Study mean % Deviation

WIST, Washington. bElement should be interpreted with caution-

Table 4: Metai concentrations (*SEM) in jack pine needles (n=100) fiom the Sudbury region and uncontaminated sites; concentrations are in mg kg-', dry wt.

Elements

o . i ) ( 0 i ) (*0.05), (11 31, (* 1 51, (*0.25), ( 0 - 1 (*2-0), Means in columns with common subscripts are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric comp&son ( p a ~ . ~ 5 ) .

"Sites 1,2,3 = sites located around Falconbridge Ltd. smelter; sites 4,5,6 = sites located around Inco Ltd. smelter; site 7 = Inco Ltd- tailing, site 8 = site located on Falconbridge Ltd. property; site 9 = Temagarni and site 10 = Low Water Lake.

Reports of high concentrations of nickel aad copper have k e n observed in plant species

within the Sudbury region (Freedman and Hutchinson, 1980; Hutchinson and Whitby,

1974). However, since copper and nickel requirernents in plant tissue tend to be low, a

dramatic increase in either or both element content may be attributed to pollution (Adriano.

1986). Background levels of copper in gyrnnosperms have been quoted at 15 ppm (1 5 mg kg'

') by Bowens (1966) and 5 to 25 mg kg-' of copper are needed for normal physiological

processes in plants (Adriano, 1986) (Table 5). In this study, copper concentrations in jack

pine needles fiom Sudbury ranged fiom 10.3 to 28.6 mg kg-'. Although significantly different

in cornparison to control sites, values in general were within normal levels found in tissue

(Kabata-Pendias and Pendias, 1992; Jones et al., 1 99 1 ) and comparable, or lower than, those

previously detennined in gymnosperms and angiosperms species located near Sudbury

smelters (Negusanti and McIlveen, 1990; Freedman and Hutchinson. 1980; Hutchinson and

Wlitby. 1977). Only site 5, located north of the Copper Cliff smelter, exceeded Ontario

Ministq of Enviromnent and Energy (OMEE) guidelines of 20 mg kg-' of copper in

vegetative tissue (Table 4). Higher copper concentration results fiom the sites close

proximity to the smelter and the effects of prevailing winds fiom the south and south-west.

In contrast, nickel concentrations in jack pine needles tiom Sudbury are elevated.

Concentrations ranged fiom 28.9 to 50.8 mg kg-' exceeding both typicai background levels

and controI site values 7 to 10 fold. Normal concentration of nickel in plants seldom

exceeds 5 mg kg" (Kabata-Pendias and Pendias, L 992; Bowen, 1966). Concentrations

obsewed in Sudbury fa11 within excessive or toxic levels reported in vegetation and exceeded

Table 5. Meîai range concentrations in jack pine needles from the Sudbury region and uncontaminated sites

Eiements Sudbury Uncontaminated Background Excessive or OMEEc region regions levelsa toxic levelsb upper limits (mg kg-') (mg kg-') (mg kg-') 0% kg-') (mg kg-')

Cd 0.2-0.3 0.05-0.1 0-05-0.2 5-30 d a

Fe 145-342 107-222 w'a d a 500

Zn 1 O .4-20 43 -0-66.4 27- 1 50 100-400 d a

'Levels considered normai in vegetation (Kabata-Pendias and Pendias, 1992; Jones er al., 199 1 ; Bowens, 1966).

bLevels considered toxic or excessive in vegetation (Kabata-Pendias and Pendias, 1992). 'OMEE upper Iimits guidelines for individual etements (Negusanti and Mcllveen, 1990). n/a=not available.

the OMEE upper Limit guidelines of 30 mg kg-' of nickel in plant tissue for nearly al1 sites

(Table S)(Kabata-Pendias and Pendias, 1 992; Negusanti and McIlveen, 1 990).

Particulate emissions have decreased since the early 1970's (Negusanti and McIlveen, 1990).

Negusanti ( I 995) reported that nickel concentration decreased significantly in white birch

during the Iast 20 years. However, comparison of total nickel content in jack pine needles

obtained in this study to previous data h m red pine needle analyses (Beckett and

Negusanti. 1990; Freedman and Hutchinson, 1980) indicate continued elevated levels of

nickel in plant species. Interestingly, Beckett et al. (1995) found that metal content in pine

needles increased with age. This higher concentration indicates continued elevated nickel

deposition surrounding smelters and/or higher nickel availability for plant root uptake from

soi1 as a result of historical deposition.

Significant correlation between copper and nickel concentrations were also observed (r=0.82.

ps 0.05) indicating a uniform deposition pattern (Table 6). Emission studies conducted by

the OMEE revealed that copper and nickel are major elements emitted by Sudbury smelters

(Negusanti and McIlveen, 1990). Chan and Luis (1985) demonstrated that copper and nickel

showed the greatest impact of smelting within the Sudbury region, with lead and cadmium

input being slightly less significant. Cornparison of lead content in needles fiom Sudbury

showed significant differences compared to control sites (Figure 3). Elevated metal

content were between 3 and 10 times higher in Sudbury. However, normal concentration

of 30 mg kg-' of lead tissue was not exceeded at any sites (Table 4) (Negusanti and

Table 6. Spearman correlation coefficients of total metai concentrations in jack pine needles (n= 1 00)

Speannan correlation coefficients

Cd Co Cu Fe Mn Ni Pb Zn

Zn -0.31* -0.43* -0.63* -0.33* 0.10 -0.69* -0.704 1.00 'Correlation is significant at the 0.05 level.

Sudbury sites Contml sites

E3 Pb concentration

Figure 3. Lead concentrations (mg kg-') in jack pine needles (n=lOO) collected from Sudbury and control sites. Means (SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric comparison (pz0.05).

McIlveen, 1990). Iron, considered a major element emitted fiom Sudbury smelters, was

within normal levels of 500 mg kg" of iron in tissue. Concentrations in needles from

Sudbury were only significantly different fiom the Low Water Lake control site (Table 4).

Udike copper, nickel and iron, cadmium, cobalt and manganese are present in considerably

iower arnounts in smelter emissions (Negusanti and McIlveen, 1990). Significant differences

for cadmium in needles fiom Sudbury were found in cornparison to contro1 sites (Table 4).

Although slightly higher than normal levels found in vegetation, cadmium concentrations

were well below toxic levels (Kabata-Pendias and Pendias, 1992). In general, cobalt

concentrations in Sudbury jack pine needles were found only to be significantly different

than the control site of Low Water Lake (Table 4). The Temagarni control site showed

sirnilar concentrations to some sites within the Sudbury area Cobalt concentrations observed

\\ithin the Sudbury region were within normal levels found in tissue (Kabata-Pendias and

Pendias. 1 992). Significant positive correlations between copper and cobalt (r=0.77, p r0.05)

and nickel and cobalt (r=0.74, ps0-05) suggest sarne deposition pattern (Table 6). Unlike

cobalt. some sites located in Sudbury showed elevated levels of manganese in needles.

Values observed exceed typical values found in vegetation (Kabata-Pendias and Pendias.

1992; Bowen, 1979). Elevated levels of manganese were also found at the Temagarni site.

Spatial distribution for metd concentrations in needles indicated a variability arnong sites

in Sudbury. Higher copper and nickel levels were found primarily near Falconbridge Ltd.

Sudbury sites Control sites

El Cu concentration

Figure 4. Copper concentrations (mg kg") in jack pine needles (n=lOO) collected from Sudbury and control sites. Means (&SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric cornparison (pz0.05).

Sudbury sites Control sites

Iii3 Ni concentration

Figure 5. Nickel concentrations (mg kg") in jack pine needles (n=100) collected from Sudbury and control sites. Means (*SEM) with common notations are not sig nificantly different as indicated by Kruskal-Wallis test followed by nonparametric cornparison (p10.05).

Sudbury sites Control sites

El Cd concentration

Figure 6. Cadmium concentrations (mg kg") in jack pine needles (n=lOO) collected from Sudbury and control sites. Means (*SEM) with common notations are not significantly different as indicated by Kniskal-Wallis test followed by nonparametric cornparison (pz0.05).

smelter (site 1,2 and 8), near Inco Ltd. smelter (site 5) and Lnco Ltd. tailing (site 7)(Figure

4 and 5). Cadmium concentrations were consistent among dl sampling sites in Sudbury

(Figure 6). Cobalt and lead distribution patterns were similar to nickel and copper. High

metal deposition patterns north of the smelters seem to be influenced by the dominant

regional wind vector fiom the south and south-west.

In contrast to hi& nickel, copper, cadmium and fead fevels, a decreased concentration of zinc

was observed in jack pine needles fiom Sudbury. Zinc, an essential trace element necessary

for numerous enzyme activities, had a concentration range fiom 43 to 66 mg kg' fiom the

Low Water Lake and Temagarni sites respectively (Table 4). Significant differences were

found between sites fiom Sudbury and control sites (Figure 7). Jack pine needle

concentrations fiom Sudbury were 3 to 4 times lower than control sites and below normal

ranges found in vegetation (Kabata-Pendis and Pendias, 19%; Jones et al., 199 1 ).

Significant negative correlations between copper and zinc (F-0.63, psO.OS), nickel and

zinc (r-0.69, ps0.05), and Iead and zinc (r-0.70, psO.05) suggest a possible antagonistic

interaction (Table 6). Zinc foliar deficiency in birch leaves observed near a copper-nickel

smelter in Russia was also suggested as possible antagonistic interaction between copper and

zinc (Kozlov er al. 1 995). Antagonistic interactions between anthropogenic elements copper,

nickel and lead with zinc have k e n reported (Kabata-Pendias and Pendias, 1992; Adriano,

1986).

bcd -7

Sudbury rites Control sites

E l Zn concentration

Figure 7. Zinc concentrations (mg kg-') in jack pine needles (n=lOO) collected from Sudbury and control sites. Means (SEM) with cornmon notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric cornparison (p~0.05).

2.3.3. Total soil metal analysis

Reliability of the analytical method for total soil digest was acceptable for al1 metals (Table

7 ). Results of soil depth analysis indicated that most sites located near Sudbury smelters had

significantly higher metal concentrations in the first 5 cm of soil compared to the lower soil

depths (Table 8). In general, concentrations o f nickel, copper, lead, cobalt and cadmium were

2 to 10 times higher in the first 5 cm of soil. Sites located dong the dominant wind vector

had the highest surface metal concentrations exceeding background levels and ministry

guidelines. No significant differences in metal concentratims between soil depths were

found in control sites.

Previous soil sampling in Sudbury have concentrated near the closed Coniston smelter

(Rutherford and Bray, 1979; Hutchinson and Urhitby, 1977; Hutchinson and Whitby, 1974).

Results indicated that nickel and copper were the main contaminants of soil. Hi&

concentrations of both metals were found in surface horizons nearthe smelter, decreasing

with both depth and distance (Hutchinson and Whitby, 1977; Hutchinson and Whitby. 1974).

Metd analyses of copper and nickel in this study fiom sites located near Inco and

Falconbridge smelters were 3 to 8 times higher in the first 5 cm of soil compared to lower

soil depths,exceeded ministry guideiines of60 mg kg-' ofcopper or nickel in uncontaminated

soil. Concentrations were also higher in cornparison to the first 5 cm of soil surveys fiom the

Sudbury and Garson regions (Negusanti and Mcllveen, 1990). Extensive sampling of topsoil

(first 20 cm) by Dudka et al. (i935) indicated that only 25% and 38% of 73 locations in

Sudbury had concentration of copper and nickel be tow rninisûy guidelines. Strong positive

Table 7: Anaiytical results o f soi1 reference materiai (CCRMP-Till 1); concentrations are in mg kg-', dry wt unless othenvise noted

EIement Certified mean' Study means % R.S.Db % Deviation (n=2)

Cd d a 0.25 6.8 nia

Zn 98 89 5 9 'CANMET, Natural Resources Canada, Ottawa, bReiative standard deviation. n/a=not available.

Table 8: Total mean metal concentrations (*SEM) in soi1 (n=9) from the Sudbury region aiid uncoiitaminated sites; concentrations in mg kg". dry wt. ---

Sampling sites"

Elcm- Dcpth I 2 3 4 5 6 7 8 9 10 ents (cm)

correlation between copper and nickel (r=0.82, p s 0.05) fUrther acknowledges wide spread

copper and nickel deposition near smelters (Table 9).

Spatial distribution indicated that metal concentrations were quite variable in Sudbury and

probably reflected contamination fiom smelters in the past. Significant differences between

soi1 depths for cadmium, cobalt, manganese and lead were found primarily at sites 2, 3

(near Falconbridge Ltd. smelter) and 5,6 (near Inco Ltd- smeiter). Furthemore, both copper

and nickel showed the highest concentrations in these sites. Al1 metal concentrations from

the upper soil profiles for these sarnpling sites exceeded OMEE guidelines and/or

background levels (Negusanti and McIlveen, 1990; Spiers et al., 1989; McKeague et al..

1979) (Table 10). Manganese concentration exceeded background levels o d y at site 5 (near

Inco Ltd. smelter). High metal levels in these sites are probably a result of metal deposition

fiom adjacent smelters? influence ofthe prevailing winds fiom the south and south-west and

sampling sites close proximity to the smelters.

In contrast, zinc and iron concentrations showed no significant differences between soil

depths for nearly al1 sampling sites. Zinc is ernitted only in small quantities fiom Sudbury

smelters (Negusanti and McIlveen, 1990; Chan and Luis, 1985) and is considered by some

a non pol lutant (Freedman and Hutchinson, 1 980; Hutchinson and Whitby, 1 977). Although

iron is considered a major element emitted fiorn smelters, concentrations observed were

comparable to previous studies (Hutchinson and Whitby, 1977; Hutchinson and Whitby.

1974). Most concentrations were within normal guidelines of 3.5 % Fe in soil established

Table 9. Spearman common correlation coefficients of total metal concentrations in soi1 samples (n=30)

- --

Spearman common correlation coefficients

Cd Co Cu Fe' Mn Ni Pb Zn

Cd

Co

Cu

Fe"

M n

Ni

Pb

Zn 'Correlation is significant at the 0.05 level. "iron concentration in mg kg" dry weight was used for soi1 correlation. n/a=not available.

Table 10. Total metal concentrations in 0-5 cm soil profiles fiom the Sudbury region and uncontaminated sites

Metals Sudbury Inco Falconbridge Uncontaminated Background OMEEb reg ion tailing P r O P e q regions levels' UPPer

(mg kg-9 (mg kg-') (mg 43-9 (mg kg-') (mg kg-') limits (mg kg'')

Cd 0.09-1 -21 0.26 0.27 O- 1 8-0.22 0.40- 1.1 d a

Zn 23-60 38 80 22-56 40- 125 nia "Normal levels in soil (Kabata-Pendias and Pendias, 1992; McKeague et al., 1979). b O ~ E ~ upper 1 imits guidel ines for individual elements (Negusanti and Mc1 lveen, 1 990). 'Fe concentrations are expressed in percentage (Fe %).

by the OMEE, aithough variability and naturaily high concentrations of uon in Sudbury area

soils have been shown in the past (Negusanti and McIlveen, 1990; Rutherford and Bray.

1979; Hutchinson and Whitby, 1977).

Metal soil analyses from inco tailing and Faiconbndge property sites indicated high

concentrations of cobalt, copper, lead and nickel. High levels of iron and manganese were

also observed at Faiconbridge Ltd.. Most soil metal concentrations exceeded both OMEE

guidelines and/or background levels. Since h c o tailing is a mixture of ore and sludge

containing iron-copper-nickel sulphides (Bouillon, 1995; Heale, 1993, high levels of these

metals were expected. High metal concentrations observed at the Falconbridge property site

were much lower than previous reports of soi1 metal analyses near the Coniston smelter

(Hutchinson and Whitby, 1977; Hutchinson, Whitby, 1974), probably a reflection of vastly

improved emission management at the new Falconbridge cornplex.

Except nickel and cadmium fiom the Inco tailing, soi1 profile analyses indicated no

significant differences in metal content between soil depths from sites inco tailing and

Falconbridge property (Table 8). Results contradict soil analyses Iiom sites near Sudbury

smelters which showed a decrease in metal content with depth. Lower soil depth analyses

from sites near Sudbury smelters revealed no significant differences for any metals

suggesting that most metal deposition is within the first 5 cm of soil, relatively immobile

within the pedon, and remains locaiized. Since Inco tailing is a combination of waste rock

and sludge mixed and pilled in large disposal areas and the site on Falconbridge property is

located next to the srnelter, consistent high levels of metals distributed throughout the soil

profile would be expected.

2.3.4 Soil pH

Soil pH values in Sudbury ranged fiom 3.8 to 4.6. Soil depth analyses indicated lower pH

values in the first 5 cm of soil increasing with depth. Although soil pH in the past have been

low (Negusanti and Mcnveen, 1990; Freedman and Hutchinson, I980), only site 2 and 3.

each located near Falconbrïdge Ltd. smelter, had a pH lower than 4.0. Lower than normal

pH values (pH Iower than 4.4) were found pnrnarily in the first 5 cm of soil within the

Sudbury region. In contrast, pH values increased with depth within normal ranges suggesting

sulfates are localized primarily withh the upper soil profiles. Significant differences in pH

values were found only at site 2, 3 and 8. (Figure 8). Control sites showed no significant

differences in pH values between soil depths. Values for Temagarni were within normal

range, while Low Water Lake had Iower pH values. inco tailing and Falconbndge property

had higher pH values because of the rehabilitation and liming of sites.

Sudbury rites Control sites

HO-5 cm depth 63 6-10 cm depth Ei11-15 cm depth

Figure 8. pH values of soi1 depths from Sudbury and uncontaminated sites. pH values w lh wmmon notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric cornparison (pr0.05).

Chapter 3: RAPD characterization of jack pine populations from the Sudbury region and uncontaminated sites

3.1. Introduction

Environmental studies of rnining pollution in Sudbury have focused in the past on the effects

of fùmigation and metal deposition. Reports provide information of landscape degradation,

so il toxicity , acidification and plant metal accumulation. It has been suggested recently that

anthropogenic stresses may affect the genetic structure of plant populations. Several authors

have reported differences in the genetic structure of plants growing in contaminated areas

(Müller-Starck, 1989,1985; Bergrnann and Scholz, 1987; Mejnartowicz, 1983). Different

responses to air pollution exposure have been observed for many forest trees species

indicating genetic differences among individuals (Bergmann and Scholz, 1989;

Mejnartowicz, 1983).

Metal analysis results presented in this study demonstrate that metal accumulation in soi1 and

plants growing in Sudbury is still an important problem. Resuits indicate that jack pine

populations from the Sudbury region continue to growth in elevated metal contaminated

soils. High metal concentrations are toxic to p lam and may impose selective pressure on

plant cornmunities. Since anthropogenic stresses have k e n implicated in large-scale forest

declines around the world, selection pressures may result in considerable changes to plant

community structure living in polluted environrnents. Lost of rare alleles, lower

heterozygosity and directional selection have been concerns of plant populations subjected

to air pollution (Bergmann and Scholz, 1989).

However, studies on the genetic structure of tree populations growing in environments

polluted by metal ernissions are lacking. Previous investigations of the Sudbury ecosystem

have provided information on metal levels in soi1 and their uptake and accumulation by

plants, but knowledge of genetic effects of plants growing in these contarninated areas is

lirnited. Studies of metal-tolerant gras species such as red top (Agrostis gigmtea), tickle

grass (Agrosris scabra) and tufied hair grass (Deschampsia cespifosa) colonizing the barren

and semi-barren landscapes of Sudbury have suggested enhanced metai tolerance

(Archambault and Winterhalder, 1995; Cox and Hutchinson, 1979; Hogan and Rauser,

1979). Plant colonization of metal contaminated soils around Sudbury area mines suggest

possible different genetic makeup (Winterhalder, 1 996). In the present study, jack pine

populations fiom Sudbury and non contarninated areas were analyzed by random arnplified

polymorphic DNA to determine any variation in ailelic frequency. The usefùiness of this

technique to differentiate pine species was also investigated.

3.2. Materiah and Methods

3.2.1. Jack pine seedling germination

Approximately 50 jack pine cones fiom the region of Sudbury were collected randomly fiom

jack pine trees at five sites (1,3,5,6,7) during September and October, 1995. Cones fiom

Temagarni (controi site) were also selected. Seeds were extracted, dewinged and bulked

together by the Ontario Ministry of Naturai Resource in Angus, Ontario as per normal

procedures for commercial lots. Seeds were placed in cIear polycarbonated "Petawawa

germination boxes" containing wet "Kimpak" celluIose paper and kept in a germinator at

25°C. Twelve day old seedlings were collected, roots and seed debris discarded, weighed,

fiozen in liquid nitrogen and stored at -80°C until DNA extractions.

3.2.2. DNA extraction

Jack pine DNA was extracted followïng a procedure described by Nkongolo er a[. (1 998).

Two grarns of eesh pine seedlings were finely ground in liquid nitrogen and transferred to

a 45 ml centri fùge tube. Twenty millilitres of 2X CTAB extraction buffer (2X buffer: 1.4 M

NaCl. 100 mM Tris-HC1 (pH 8.0), 20 m M EDTA, 2% hexadecyltrimethylarnmoniurn

bromide w/v) was added to the tube and the mixture was incubated at 60°C for 1 h.

Following incubation, 1 5 ml of chloroform:octanol(24: 1) was added, mixed and centrifùged

(1 3000 rpm, 15 min, 25°C). The aqueous phase was transferred to a fiesh centrifuge tube and

nucleic acids were precipitated by addition of equal volume of isopropanol. The nucleic acid

pellet was collected by centrifugation (6500 rpm, 3 min, 25°C) and resuspended in 10 ml of

TE buffer (10 m M Tns-HC1 (pH 8.0), ImM EDTA).

A second chloroform:octanol purification was perfomed followed by centrifugation ( 1 3 000

rpm, 5 min, 25°C) and aqueous phase was transferred to fresh tubes. Nucleic acids were

reprecipitated with 1 ml of 7.5 ammonium acetate, followed by 10 ml of cold ethanol

rnixing. The pellet was collected by centrifiigation (6 500 rpm, 3 min, 25°C)- drained, then

redissolved in 10 ml of 200 mM ammonium acetate, and a second ethanol precipitation was

performed. After the final centrifùgation (1 3 000 rpm, 2 min, 25°C) the pe!let was drained,

dried bnefly under vacuum and resuspended in 2 ml of TE buffer. DNA purity and

concentrations were estimated using a spectrophotometer.

3.2.3. Amplification of RAPD markers

Total DNA prepared from d l jack pine samples were used for PCR reactions. in a 25 pl

volume. approximately 200 ng plant DNA, 1.0 pM primers and 200 pM each of dATP.

dCTP, dGTP, dTTP were mixed with 10X PCR reaction buffer (Perkin Elmer) and

0.625unitd25 pl of AmpliTaqa DNA polymerase (Perkin Elmer). Samples were amplified

in a Perkin Elmer DNA thermal cycler model. After an initial denaturation step at 95°C for

5 min and a hot start step at 85°C for 2 min, 42 cycles consisting of 60 s denaturation at 95°C.

120 s annealing at 55°C and 60 s extension at 72°C were perfonned pnor to a final extension

of 5 min at 72°C. DNA amplifications were then cooled to 4°C. Analyses of RAPD

amplifications were preforrned on 1 .O% Gibco agarose gels in TAE buffer using a mixture

of 7 pl of PCR reaction and 5 pl of gel loading b a e r (Maniatis er al. 1989) and

electrophoresis was performed at 44 V for 2.5 h. Gels were stained in 0.5 p g h l ethidiurn

bromide solution and photographed in ultraviolet light. Eleven oligonucleotide primers

synthesized by the University of Calgary, University Core DNA described in table 12 were

used for the study. Primen were selected based on their ability to ampliS. spruce DNA

(Mcongolo, 1 998).

3.3. Results and Discussion

3.3.1. DNA concentration and purity values

Genomic DNA concentrations of twelve day old jack pine seedlings are s h o w in table 1 1.

DNA concentrations ranged fiom 93 to 333 &nl with an average of 205 ~ g / m l . Mean

DNA purity values varied fiom 1.92 to 2.00 within acceptable ranges of 1.8 to 2.0.

3.3.2. RAPD characterizrition of jack pine popuiations

Six of the eleven primers screened (55%) did not produce any amplifications, two primers

(18%) amplified poorly and three primers (27%) consistently produced sharp and

reproducible RAPD bands (Table 12). A total of 18 W D markers ranging from 220 to

137 1 base pairs (bp) were produced by the 3 pnmers Cigure 9, 10 and 1 1). The level of

polymorphisms within and among different populations was low, only 4 loci (22 %) were

poIyrnorphic.

M D analysis using primer P-2 show no variation in patterns among al1 populations (Figure

9). Four RAPD bands ranging fiom 745 to 1265 bp were produced. Jack pine populations

fiom Northern Ontario and New Brunswick show sirnilar RAPD profiles. However, species-

specific RAPD markers were identified when jack and red pine populations were compared

(Table 1 3). Of the 5 bands produced, 4 fragments were present in both pine species while one

band (962 bp) was specific to red pine.

Polyrnorphic bands were also observed when pnmers P-8 and P-9 were used. Six to eight

Table 1 1 : DNA concentration and pwity values from jack pine seedling samples; ranges within parenthesis

Study sitesa Mean DNA concentration Mean DNA purïty Olg/ml)

1 195 (146 -278) 2.00 (1 -99 - 2-06)

9 157 (93 - 214) 1.93 (1 -88 - 1.99) 'Sites 1 and 3= sites located near Falconbridge Ltd. smelter, sites 5 and 6= sites located near Inco

Ltd. smelter; site 7 = Inco Ltd, taiIing, site 9 = Temagami (control site).

Table 1 2. Number of fragments, polymorphic RAPD markers and their size using jack pine DNA fiom Sudbury and uncontaminated populations

-- -

Primer Nucleotide Number of Fragment size Polymorphic identification sequence fragments range bands

(5' to 3') (range) @PI ACGACGTAGG

CCGCGGTTCC

CCGGCTGGAA

GAGGGCGTGA

GCTCCCCCAC

CGATGGCTTT

TAGCCCGCTT

GTAGACGAGC

GTGCGTCCTC

GTTCTCGTGT

AACACACGAG

Table 13. Mean number of RAPD and species-specifk markers for jack and red pine populations

Primer Nucleotide Mean bands amplified Number of species- identification sequence specific bands

(5' to 3') jack pine red pine jack pine red pine

ACGACGTAGG

CCGCGGTTCC

CCGGCTGGAA

GAGGGCGTGA

GCTCCCCCAC

GTAGACGAGC

GTGCGTCCTC

GTTCTCGTGT

AACACACGAG

Figure 9. RAPD markers fiom jack pine popdations using primer P-2. Numbers represent individuals from different sites. Species-specific marker is shown with an arrow. (Kb=l kb ladder; site 1 (near Falconbridge smelter) =1 to 3; site 3 (near Falconbridge smelter) = 4 and 5; site 5 (near Inco smelter) = 6 to 7; site 6 (near inco smelter) =8 and 9; site 7 (Inco tailing) =10 to 12; site 9 (Temagrni) = 13 to 15; New Bninswick=16 to 17; red pine= 18 and 19, blank=20).

Kb 1 2 3 4 5 6 7 8 9 10 Kbll 12 13 14 15 16 17 181920

Figure 10. RAPD markers fiom jack pine popdations using primer P-8. Numbers represent individuals from different sites. Species-specific markers are shown with an arrow. (Kb=l kb [adder; site 1 (near Falconbridge smelter) =1 to 3; site 5 (near Lnco smelter) = 4 and 5; site 7 (hco tailing) = 6 and 7; site 6 (near Inco smelter) =8 to 10; site 7 (Temagami) = 1 1,12,15; site 3 (near Fdconbridge smelter) = 1 3 and 14; New Brunswick=l6 to 17; red pine= 18 and 19; blank=20)-

Figure 1 1 . RAPD markers fÏom jack pine populations using primer P-9. Nunibers represent individuals from different sites. Species-specific markers are s h o w with an arrow. ( K k l kb ladder; site 1 (near Falconbi-idge smelter) =1 to 3; site 3 (near Faiconbridge smelter) = 4 and 5; site 5 (near Inco smelter) = 6 and 7; site 6 (near Inco smelter) =8 and 9; site 7 (hco tailing) =10 to 12; site 7 (Temagarni) = 13 to 15; New Bninswick=l6 to 17; red pine= 18 and 19; blank=20).

RAPD markers ranging fiom 220- 1 12 1 bp were produced with primer P-8 (Table 1 2). Two

trees, each found at site 1, located near the Garson-Nickel City durnp, had a missing band

of 344 bp. Two other trees, located near Skead (site 3) had an additional band of 1 121 bp.

Primer P-9 produced 4 to 6 RAPD hgments ranging fiom 396 to 1371 bp. Faint bands (71 7

and 1 195 bp) in some individuals were interpreted to be an artifact of weak amplification

rather than a polymorphism at that locus (Figure 1 1 ) . Close examination of the samples

showed extremely faint fragments withïn the background (Figure 1 1). However, tree samples

9 and 16 had either missing or additional bands. The two primers, P-8 and P-9 produced

species-specific markers when jack and red pines were compared (Figure 10 and Figure 1 1 ).

In general, amplification of control samples containing no genomic DNA produced no bands.

In isolated cases, such samples did generate some amplified products. However, none of

these bands corresponded to any jack or red pine RAPD markers. This phenornenon has been

attnbuted to primer rnultimer formation and would likely disappear when template DNA is

added to control samples (Yu et al., 1993).

The characterization of jack pine populations in Sudbury indicate low levels of genetic

diversity arnong populations. This corroborates observations made by Mosseler ef al. (1 992)

studying red pine populations in Newfoundland. These authors demonstrated by RAPD

analysis that red pine exhibited low levels of genetic variability. This contrasts results fiom

several allozyme studies which reported existence of genetic variability in ponderosa pine

(Pinus pondesora), Iodgepole pine (Pinus coniorta var Iarifolia) and jack pine (Pinus

banksiana) populations (Danick and Yeh, 1982; OYMal1ey et a', 1979; Yeh and Layton,

1979). Sirnikir analyses reveaied that the levels of genetic variability were lower in jack and

red pines compared to other pine species (Mosseler et al., 199 1 ; Danick and Yeh, 1983).

Although both isozymes and RAPD allow the anaiysis of genetic variability in plant species.

fundamentai differences exist between both methods. Isozyrne analysis reflect alterations

in the DNA sequence through changes in amino acid composition. (Hamrick, 1989). Changes

in amino acid composition will often alter protein charges thereby producing a change in

electrophoretic mobility. These differences in electrophoretic mobility of enzymes provide

an estremely useful method of evaluating levels of variation between individuais and

popuIations on the basis of gene loci coding for specific enzymes (Weeden and Wendel.

1 989). Although isozyme markers have been extremely informative in popuiation genetic

studies, the limited nurnber of isozymes (approximately 30) reflect only a small number of

the genome.

RAPD is a DNA based molecular marker. Genomic DNA is amplified using randomly

constructed oligonucleotides as primers. Unlike isoqmes, RAPD is relatively easy to apply

and the nurnber of loci that can be exarnined is essentiaily unlimited. Since the primers

consist of random sequences and do not discriminate between coding and noncoding regions.

the technique samples the genome more randomly than conventional methods (Lynch and

Milligan, 1994). RAPD results in the amplification of specific portions oftemplate DNA that

binds DNA primers while isozymes involve the differentiation of amino acid sequences

caused by alteration in DNA.

One of the difficulties in studying jack pine populations in Sudbury is the existence of the

small population size (approxùnately 30 to 50 trees). Most of the forest ecosystem within the

Sudbury region have been destroyed by Iogging, erosion and air pollution. Natural ranges of

forest trees are nearly non existent within 1 5 km fiom smelters. Very Little genetic differences

were observed even between populations with 30 year diRerences. Little genetic differences

within different populations studied were expected, but the lack of variation among trees

fiom contarninated and non-contaminated sites was surpnsing. Even jack pine populations

fiom New Brunswick showed similar RAPD patterns in cornparison to those fiom Sudbury.

Further studies using microsatellites (SS) and amplified fragment length polymorphisms

(AFLP) are warranted to confïnn these observations.

Plausible explanation for low genetic variability is a possible bottleneck due to the last

glaciation. The entire area of the present-day distribution ofjack pine is thought to have been

covered by ice during the last glacial stages. Geological and paleobotanical evidences fiom

fossil pollen depositions indicate that jack pine survived glaciation in an extensive refbgiurn

centered on the Appalachian Highlands of eastern North America (Yeatman, 1967). Upon

recession of the Wisconsin icecap, jack pine migration northward is though to have happened

rapidl y (Daubenmire, 1 978).

Evolution of jack pine in North America could have possibly followed the same pattern of

red pine which despite increases in population numbers and mutations, have not produced

much detectable genetic variation (Mosseler et al-, 1992). Fowler and Moms (1977)

attributed lack of genetic diversity in red pine to the occurrence of a genetic bottleneck

during the Holocene giaciation in which red pine migrated southward and survived in an

isolated glacial refugium. Self -p lba t ion may also have contributed to a loss of genetic

variation through inbreeding in smdl populations (Nei et al. 1975). The lack of continuous

forest within the Sudbury ecosystem could possibIy continue to contribute to low levels of

genetic variability between jack pine populations and other pine species since gene flow is

limited to small nurnber of individuds within the same populations.

Despite some limitations, RAPD is an effective tool in studying identity and relatedness

among plant species. Both jack and red pine species were easily differentiated based on their

RAPD fragment profiles. By s c o ~ g the presence and absence of RAPD markers. phenetic

relationships among pine species can easily be accomplished. This could shed light on

problems such as evolution divergences between cIosely related species and possibly help

in the understanding of evolution of species within the genus Pinus. RAPD c m also be useful

in the analysis of gene uitrogession between species. A classical example would be the

characterization of natural hybrids fiom jack and lodgepole pine in Western Canada where

the ranges of the two species overlap.

General conclusions

Aunospheric deposition of metals due to mining has ken, and still is an important

contributor to elevated rnetal levels in soil within the Sudbury region. Although reduction

of atmospheric metal deposition in the last 30 years has shown a decline in metal particdates

in soils fiom the Sudbury area (Ducika et al. 1995; Gundermann and Hutchinson, 1993).

element concentrations observed in this study continue to exceed OMEE upper limit

guidelines for uncontaminated soils. The range of values for these metals indicate a strong

impact of metal deposition fiom Sudbury smelters. Spatial distribution of metal

concentrations indicate that study sites located within dominant wind vector had higher

concentrations of metal contaminants compared to other sites. Sites near Falconbridge and

inco Ltd, smelters show metal increases of 3 to 8 times higher in the first 5 cm of soil. No

significant differences were observed within soil depths for control sites.

With the exception of nickel, reports of metal content above upper limits of normal

concentration for individual elements in needles seem to have decreased relative to recent

historical data. Reduction in particdate emissions have probably contributed to a decrease

in metal deposition on vegetation near smelters. Wind direction seems to have a profound

effect on location of metal depositions within the Sudbury region. Declining industrial

emissions should continue to diminish the anthropogenic effects on the local environment.

Genetic characterization of jack pine populations growing in Sudbury show linle variation

in RAPD profiles. RAPD patterns of jack pine trees fkom the Sudbury area closely resemble

those of control populations fiom Northern Ontario and New Brunswick. niese results

corroborate observations made by Mosseler et al. (1 992) studying red pine populations in

Newfoundland. However, results contrast allozyme studies sho wing genetic variabili ty in

pine species (Danick and Yeh, 1982; O'Malley et al., 1979; Yeh and Layton, 1979). The low

genetic variability in Sudbury is probably not related to pollution but possibly a result of a

bottIeneck occurrence during the last glaciation. Other possibilities include the different

abiiities of the RAPD primers to detect variation or to the limited nwnber of primers used

to characterize pine populations. Further in depth analysis of Sudbury populations is needed

to possibly identiQ variability. The potential of RAPD as a phylogenetic tool for the genus

Pinus was demonstrated. Jack and red pine species were easily differentiated based on their

RAPD profiles.

References

Adriano, D.C. (1 986). Trace EIements in rhe Terrestrial Environment. Springer-Verlag. New

York, 553 pp.

AI-Janabi, S.M., Honeycutt, R.J., McCIelland, M., and B.W.S. Sobral. (1993). A genetic

linkage rnap ofSucchortrm spontaneum (L) "SES 208". Genetics, 1 34: 1 249- 1 260.

Amiro, B.D. and G.M. Courtin. (1981). Patterns of vegetation in the vicinity of an

industriaiIy disturbed ecosystem, Sudbury, Ontario. Can. J. Bot. 59(9): 1623-

1639.

Archambault, DL-P. and K. Winterhalder. (1995). Metai tolerance in Agrostisscabra fiom

the Sudbury, Ontario, area. Can. J. Bot. 73:766-775.

Archibold, O.W. (1 978). Vegetation recovery following pollution control at Trail, British

Columbia, C m J. Bot. 56: 1625-1 637.

Beckett, P.J. and J.J. Negusanti. (1990). Using land reclamation practices to improve tree

condition in the Sudbury smelting area. Ontario, Canada. In Proceedings of the

1 990 Mining and Reclamarion Conference and Exhibition, 23-26 April 1 990,

Charleston. Vol 1. W.V. Edited by J. Skousen* J. Scencindiver, and D. Samuel. ,

West Virginia University PubIication Sewices, West Virginia. pp. 307-320.

Beckett, P.J., Negusanti, J., Peters, T., Vining, J., Miller, J. and W. Lautenbach. (1995). The

Sudbury regional land reclamation program - tree and shrub enhancement. In

Proceedings ofSudbury '95 -Minhg and the Environment, 28 May - I June

1995, Sudbury, Ont. Edited b y T.P. 1-Iynes and M.C.Blanchette. C ANMET,

Ottawa; Ont. pp. 1 103- 1 1 12.

Berrang, P., Kamosky, D.F., Mickler, R.A. and J.P. Bennett. (1986). Natural selection for

ozone tolerance in Populus iremuloides. Cam J, For. Res. 16: 12 14- 12 1 6.

Bergmann, F. and F. Scholz. (1987). The impact of air pollution on the genetic structure of

Norway Spruce. Silvae Genet. 36:80-83.

Bergrnann, F. and F. Scholz. (1989). Selection effects of air pollution in Norway spruce

(Picea abies) populations. In Genetics Effects of Air Pollutunts in Foresr Tree

Popufarions. Edited by F. Scholz, H.R. Gregorius and f)-Rudin. Springer-Verf ag.

Berlin. pp. 143-160.

Bouillon, D.F. (1995). Developments in emission control technologies/strategies: a case

study. In Environmenral restoration and recovery of an industrial region. Edited

b y J. Gunn. Springer-Verlag. New York. pp. 275- 285.

Bowen. H. J.M. ( 1 979). Environmental Chemistry of the Elements. Academic Press Inc.. New

York. 333 pp.

Bowen, H.J.M. ( 1 966). Trace EZements in Biochemistry. Academic Press Inc., New York,

241 pp.

Carlson, J.E. Tulsierarn, L.K. Glaubitz, J.C., Luk, V., KaufCeldt, C. and R. Rutledge. (1 99 1 ).

Segregation of random amplified polymorphic DNA markers in F 1 progeny of

conifers. Theor. Appl. Genet. 83: 194-200.

Carter, M. ( 1 993). Soil samples und methods ofanafysis. Lewis Publishers, Florida. 823 pp.

Chan, W.H. and M.A. Luis. (1985). Post-superstack Sudbury smelter emissions and their

fate in the atmosphere: an overview of the Sudbury environment study results.

Wuter Air Soil Pollut. 62:43-58.

Cook, E.R. (1987). The decomposition of tree-ring senes for environmental studies. Tree-

Ring Bull. 47:3-9.

Courtin, G.M. (1994). The last 150 years: a history of environmental degradation in

Sudbury. Sci. Total Environ. l48:W- 102.

Cox. R.M., and T.C. Hutchinson. (1980). Multiple metal tolerances in the gras

Deschampsia cespifosa (L.) Beauv. from the Sudbury smelting area New Phytol.

84:63 1 -647.

Danick, B.P. and F.C. Yeh. ( 1 983). Ailozyme variability and evolution of lodgepole pine

(Pinus conforta var lahyolia) and jack pine (P. banhiana) in Alberta. Can. J.

Genet. Cytol. 2557-64.

Daubenmire, R. ( 1 978). Plant geography. Academic Press. New York. pp. 3 3 8.

Dreisinger, B.R. and P.C. McGovem. (1 969). Sulphur dioxide levels and resultant injury to

vegetation in the Sudbury area during the 1968 season. Ontario Department of

Mines, Sudbury, Ontario

Dudka. S. Ponce-Hemandez, R. and TC. Hutchinson. ( 1 995) Current levels of total element

concentrations in the s d a c e layen of Sudbury's soils. Sci Total. Environ. 162:

161-171.

Farrar, J.F., Relton. I. and A.J. Rutter. (1977). Sulphur dioxide and the scarcity of Pinus

sylvesrris in the industrial pennines. Environ. Pollut. 1 4:63 -68.

Fowler, D.P. and R.W. Morris. (1 977). Genetic divenity in red pine: evidence for low genic

heterozygosity. Can. J . For. Res. 7:343-347.

Freedman, B. and TC. Hutchinson. (1980). Pollutants inputs fiom the atmosphere and

accumulations in soils and vegetations near a nickel-copper smelter at Sudbury,

Ontario, Canada. Can J. Bot. 58: 108-13 1.

Furman, B.J., GrattapagIia, D., Dvorak, W.S. and D.M. O'Malley. (1997)- Analysis of

genetic relationships of Central American and Mexican pines using RAPD

markers that distinguish species. Mol- Ecol. 6:321-33 1 .

Geburek, T-, Scholz, F., Knabe, W. and A. Vomweg. (1987). Genetic studies by isozynes

gene loci on tolerance and sensitivity in an air polluted Pinus syIvestris fieid trial.

Silvae Genet. 36:49-53.

Gordon. A.G. and E. Gorham. (1960). Some effects of smelters pollution north-east of

Falconbridge, Ontario. Cam J. Bot. 38:307-3 12.

Gordon, A.G. and E. Gorham. (1963). Ecological aspects of air pollution fiom an iron-

sintering plant at Wavva, Ontario. Can. J . Bot. 4 1 : 1063-1 078.

Gundermann, D.G. and T.C. Hutchinson. (1993). Changes in soi1 chemistry 20 years after

the closure of a nickel-copper smelter near Sudbury, Ontario, Canada. In Heary

rnefals in the environrnenr. Vol. 2. Edited by R.J. Allan and J.O. Nnagu. CEP

Consultants, Edinburgh, U.K. pp. 559-562.

Gunn, J- (1 996). Restoring the Smelter-Damaged Landscape Near Sudbury, Canada. Resror.

Manage. Notes. 14(2): 129- 136.

Gunter, L.E., Tuskan, G.A. and S.D. Wullschleger. (1996). Diversity arnong Populations of

Swithgrass Based on RAPD Markers. Crop. Sei. 36: 1 0 1 7- 1 022.

Harnrick, J.L. (1 989). Isozyrnes and the Analysis of Genetic Structure in Plant Populations.

In Isozyrnes in Plant Biology. Edited by D.E. Soltis and P.S. Soltis. Dioscorides

Press, Portland, Oregon. pp. 87- 1 05.

Hogan, G.D. and W.E. Rauser. (1979). Tolerance and toxicity of cobalt, copper, nickel and

zinc in clones of Agrostis gigantea. New Phytol. 83 :665-6 70.

Hazlett. P.W., Rutherford, J.K. and G.W. v d o o n . (1983). Metal contarninants in surface

soils and vegetation as a result of nickeVcopper smelting at Coniston, Ontario.

Canada. Reclam. Reveg. Res. 2: 123- 127.

Heale. EL. (1995). Integrated Management and Progressive Rehabilitation of Industrial

Lands. In Environmental restoration and recovery of an industrial region.

Edited by J . Gunn. Springer-Verlag. New York. pp 287-298.

Hutchinson. T.C. and L-M. Whitby- (1974). Heavy-metal pollution in the Sudbury mining

and smelting region of Canada. 1. Soil and vegetation contamination by nickel.

copper, and other metals. Environ. Conserv. 1 (2), 123- 132.

Hutchinson, T.C. and L.M. Whitby. (1977). The effects of acid raiddl and heavy metal

particulates on a boreal forest ecosystem near the Sudbury smelting region of

Canada. Water, Air, Soil Pollut. 7 : 42 1 -43 8.

Jones, J.B., WoIf, B., and KA. Mills. (1991). Plant Analysis Handbook: a pracrical

sampling. preparation and interpretation guide. Micro-Macro Publishing. Inc..

Georgia? 2 1 3 pp.

Jordon, M.J. (1 975). Effects of zinc smelter emission and fire on a chestnut-oak woodland.

Ecology, 56:78-9 1 .

Kabata-Pendias, A. and H. Pendias. (1 992). Trace Elemenfs in Soils and Plants. 2nd edition.

CRC press, Florida, 365 pp.

Kamosky, D.F. and P.C. Berrang. (1 989). Variation in and naturai selection for air pollution

tolerances in trees. in Genetics Effecfs of Air Pollutants in Forest Tree

Popufations. Edited by F . Scholt, H.R, Gregorius and D.Rudin. S pringer-Verlag,

Berlin, pp.29-37.

Kef I ÿ ' T. ( 1 980). The effect of a continuous sprïngtime fumigation with sulfur dioxidc and

carbon dioxide uptake and structure of the annual ring in spruce. Can. J. For.

Res. 10: 1-6.

Kozlov, M.V., Haukioja, E., Bakhtiarov, A.V. and D.N. Stroganov. (1995). Heavy metals

in birch leaves around nickel-copper smelter at Monchegorsk, Northwestern

Russia. Environ. Pollut. 9O(j):S9 1-299.

Kryuchkov, V.V. (1993). Degradation of ecosystems around the Severonikel smelter

cornplex. In Aerial PoZIution in the Kola Peninsula. Edited by M . V. Kozlov. E.

Haukioja and V.T. Yarmishko. Proc. International Workshop. April 1 4- 16.1 992.

St. Petersburg. pp. 35-36.

Lautenbach, W. E. ( 1 987). The greening of Sudbury. J. Soi1 Wafer Conserv. 7-8228-23 1 .

Leblanc, F. and D.N. Rao. (1966). Réaction de quelques lichens et mousses épiphytiques à

l'anhydride su1 fûreux dans la région de Sudbury, Ontario. Bryologist, 69: 3 3 8-

346.

Linzon, S.N. (1958). The influence of smelter fumes on the growth of white pine in the

Sudbury region. Department of Mines and Ontario Department of Lands and

Forests. Toronto. Ontario. Technical Report-

Limon, S.N. (1 97 1). Econornics effects of sulfur dioxide on forest growth. J. Air. Poilu<.

Conirol- Assoc. 2 1 :8 1-86.

Little. P. and M.H. Martin. (1 972). A survey of zinc, lead, and cadmium in soi1 and natural

vegetation around a smelter cornplex- Environ Pullut. 8: 149- 1 57.

Lynch. M. and B.G. Milligan. (1 994). Analysis of population genetic structure with RAPD

markers. Mol. Ecol. 3 :9 1-99.

Maniatis, T., Fritsch. E.F. and J. Sambrook. (1989). Molecufur Cloning: A laborutory

manual, 2"6 edition. Vol 1, II, m. Cold Spring Harbor Laboratory. Long Island.

New York.

Marr. I.L. (1979). A Handbuok of Decomposition Methodr in Anafytical Chemisrry.

International Textbook Company, London, 444 pp.

McKeague. I.A. Desjardins, J.G. and M.S. Wolynetz. (1 979). Minor elements in Canadians

Soils. Land Resource Research institute. Contribution No. LRRI 27- Research

Branch, Agriculture Canada. Ottawa. Ontario. 75 pp.

Mejnartowicz, L. (1983). Changes in genetic structure of Scoth pine (Pinus sylvestris L.)

population af5ected by industrial emission of fluoride and suiphur dioxide. Genet.

Polonica. 24:4 1-50.

Mosseler. A., Egger, K.N. and G.A. Hughes. (1992). Low levels of genetic diversity in red

pine confirmed by random amplified polymorphic DNA markers. Cam J. For.

Res. 22:1332-1337.

Mosseler, A., Innes, D.J. and B.A. Roberts. (1 99 1). Lack of allozymic variation in disjunct

Newfoundland populations of red pine (Pinus resinosa). Con. J. of For. Res.

2 1 525-528.

Moss, E.H. (1 949). Natural pine hybnds in Alberta. Can. J. Res. 27:2 18-229.

Müller-Starck, G. (1985). Genetic differences between 'tolerant' and sensitive beeches

(Fagus sylvatica L.) in an environmentally stressed adult forest stand. Silvea

Genet. 34% 1-246.

Müller-Starck, G. ( 1 989). Genetic implications ofenvironmental stress in adult forest stands

of Fagus sylvatica L. In Genetics Effecrs of Air Pollutants in Foresi Tree

Popularions. Edited by FScholt. H.R. Gregorius and D-Rudin. Sprïnger-Verlag.

Berlin. pp. 127- 142.

Nei, M., Maniyama, T., and R. Chakraborty. (1 975). The bottleneck effect and population

variability- Evolution, 29: 1 - 10.

Negusanti. J.J. (1995). Terrestrial metal trends in the Sudbury area. In Proceedings of

Sudbury '95 - Mining and the Environment. 28 May - 1 June 1995, Sudbury.

Ontario. Edited by T.P.Hynes and M-C-Blanchette. CANMET. Ottawa, Ontario.

pp. 1 143-1 150.

Negusanti, J. J. and W.D. McIlveen. (1 990). Studies of terrestrial environment in the Sudbury

area 1978-1 98 7. Northeastem Region, Ontario Ministry of the Environment.

Queen's Printer for Ontano, Toronto. Ontario.

Nkongolo, K.K., Kiimaszewska. K and W.S.Gratton. ( 1 998). DNA Yields and Optimization

of RAPD Patterns using Spruce Embryogenic Lines, Seedlings, and Needles.

Planr Mol. Biol. Rep. (In press).

Nkongolo, K.K. (1998). RAPD variations among pure and hybnd populations of Picea

mariana, P. rubenr, and P. g h c a and cytogenetic stability of Picea spp. hybrids:

identification of species-specific RAPD markers. Plant Sys~ Evol. (In press).

O'Malley, D.M., AUendorf, F.W. and G.M. Blake. ( 1 979). Inheritance of isozyme variation

and heterozygosity in Pinus ponderosa. Biochem. Genet. 17:233-250.

Potvin, R.R. and J.J. Negusanti. (1995). Declining induda1 emissions, improvhg air

quality, and reduced damage to vegetation. In Environmental restoration and

recovery of an industrial region. Edited by J. Gunn. Springer-Verlag, New York.

pp. 51-61.

Rauser, W.E. and K. Winterhalder. (1 985). Evaluation of copper, nickeI and zinc tolerances

in four g r a s species. Can. J , Bot. 63 :58-63.

Reiter. R.S., Williams, J.G.K., Feldmann, KA., Rafalski, J.A., Tingey, S.V. and P.A.

Scolink. (1 992). Global and local genome mapping of Arabidopsis thaliana by

using recombinant inbred lines and random amplified polymorphic DNAs. Proc.

Natl. Acad Sci USA. 89: 1477-1481.

Rutherford, G.K. and CR- Bray. (1979). Extent and distribution of soi1 heavy metai

contamination near a nickel smelter at Coniston, Ontario. J. Environ. Qual.

8(2):2 19-222.

Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Hom, G.T., Erlich, H.A. and N. Amheim.

(1 985). Enzyrnatic amplification of P-globin genomic sequences and restriction

site analysis for diagnosis of sickle ce11 anemia. Science, 230: 1350.

Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Hom, G.T., Mullis, K.B.

and H.A. Erlich. (1988) Primer-directed enzymatic amplification of DNA with

a thermostable DNA polymerase. Science, 239:487.

Scholz. F . and F. Bergmann. (1984). Selection pressure by air pollution as studies by

isozyme-gene-systems in Norway spnice exposed to sulphur dioxide. Silvae

Genet. 33:23 8-24 1.

Sobral. B. W.S. and RJ. Honeycutt. (1994). Genetics, Plants, and the Polymerase Chain

Reaction. In Polymerase Chain Reaction. Edited by K.B .Mullis, F. Ferre and

R.A.Gibbs. Birkhauser, Boston. pp- 304-3 19.

Spiers, G. A., Dudas, M.J. and L. W. Turchenek. (1 989). The chernical and mineralogical

composition of soi1 parent materials in Northeast Alberta. Can. J. Soii. Sci.

69:2 1-73 7.

SPSS Inc. (1996). SPSS 7.5 for Windows. Chicago.

Stmik, H. (1973). Photo interpretative study to assess and evaluate the vegetational and

physical state of the Sudbury area subject to industrial emissions. In Sudbz~ry

Environmental Enhancement Programme Summary Report. 1969-1 9 73. Ontario

Department of Lands and Forests, Sudbury, Ontario. pp. 6- 1 1.

Sweeney, P.M. and T.K. Danneberger. (1995) RAPD Characterization of Pou annua L-

Populations ui Golf Course Greens and Fairways. Crop Sci. 35: 1676- 1680.

Thompson, M.A. (1981). Tree rings and air pollution: a case study of Pinus monophylla

growing in east-central Nevada. Environ. Pollur. 26:25 1-266.

Treshow, M and F.K. Anderson. (1 989). Plant Stressfiorn Air Pollution. Wiley, New York.

283 pp.

Van Coppenolle, B., Watanabe, L, Van Hove, C., Second, G., Huang, N., and S.R.

McCouch. (1 993). Genetic diversity and phylogeny analysis of Azolla based on

D N A amplification by arbitrary primers. Genome, 36:686-693.

Weeden, N.F. and J.F. Wendel. (1989)- Genetics of Plant Isozymes. In IsoLymes in Plant

Bioloay. Edited b y D.E. Soltis and P.S. Sohis. Dioscorides Press, Portland.

Oregon- pp, 46-72.

Welsh. J. Chôda, K. Dald, SS. Ralph, D., Chang, R. and M. McClelland (1992). Arbitrary

pnmed f CR fmgerprinting of RNA. Nucl. Acids Res. 20:4965-4970.

Welsh, J. and M. McClelland. (1990). Fingerprinting genomes using PCR with arbitrary

primers. NucZ. Acids Res. 1 8:72 13-72 1 8.

Williams, J.G. Kubelik, A-R-, Livak, K.J., Rafdski, J.A. and S.V. Tïngey. (1990). DNA

polyrnorphisms amplified by arbitrary primers are useful as genetic markers.

Nucl. Acids Res. 18:6531-6535.

Winterhalder, K. (1 984). Environmental degradation and rehabilitation in the Sudbury area.

Laurenf ian Univ. Rev. 1 6(2): 1 5-47.

Winterhalder, K. (1 995). Early History of Human Activities. In Environmenial resrorarion

and recovery of an industrial region. Edited by J . GUM. Springer-Verlag.

New York. pp 17-3 1.

Winterhalder, K. (1996). Environmental degradation and rehabilitation of the landscape

around Sudbury, a major mining and smelting area. Environ. Rev. 4: 1 85-224.

Whitby, L.M., Stokes, P.M., Hutchinson, T.C. and G. Myslik, (1976). Ecological

Consequence of Acidic and Heavy-Metai Discharges fiom the Sudbury Smelters.

Can. Mineral. 14:47-57.

Yamaguchi. D.K. (1991). A simple method for cross-dating increment cores fiom living

trees. Can- J. For. Res. 21 :414-416.

Yeatrnan. C. W. (1 967). Biogeography of Jack pine. Can. J. of Bot. 67:220 1 -22 1 1.

Yeh, F.C.. Chong, D.K.X. and R.C. Yang. (1995). RAPD Variation Within and Among

Natural Populations of Trembling Aspen (Populrcs Tremuloides Michx.) fiom

Alberta. Heredis, 86:454-460.

Yeh. F.C and C. Layton. (1979). The organization of genetic variability in central and

marginal populations of lodgepole pine Pinus contorta spp. ZafroZia. Can. J.

Genet. Cytol. 2 1 :220 1-22 1 1.

Yu, Kang Fu, Deyneze, Allan Ven and K.P. Pauls. (1 993). Randorn Amplified Polymorphic

DNA (RAPD) Analysis. In Methods in Plant Mo[ecuZar BioZogy and

BiotechnoZogy. Edited by Bernard R. Glick and John E. Thompson. CRC Press

Inc., p. 287-301,

Zarr, J. ( 1 996). Biostatisfical Analysis. Third Edition, Prentice Hall, New Jersey. 662 pp.

Appendices

Appendix 1 : Age determination of jack pine populations -- -

Age determination of tree core samples c r o s y i d

A-5

-approximated age. da= not available. n= narrow ring, vn= very narrow ring.

Age of core

Tree

A- 10

Marker rings shared by most cores: 88,82,80,77,71 vn, 69,66vn, 62.60 Sensitive ring pattern shared by most cores: 77-78,73-74,64-65

93,92,88,84,82,80,77n, Mn, 71vn, 70,69,66vn, 61,62,60vn

Marker rings

Trees identified A represents trees from site 1 (near Falconbridge srnefter). Core samples were collected in October 1995.

93.9 1,88,86,85,83, 82,8 1,78,76vn, 7 1 vn, 69vn, 68,66vn, 63,62,60,59,57vn

1957

Last ring year

1954

Sensitive ring pattern

77-78, 64-65, 62-63

38

74-75, 70-7 1 , 64-65, 6 1 -62

41

Appendix 1 : continue

Age determination of tree core samples

8-8 1 92-90, 88, 82,79,77, 7 1,70,67vn, 65,6 ln, 1 1943 170-71.61-62 60,57,54,5 1 n, 49 1 52

using c r o s s - d a t i n n d

Core samples were collected in October 1995.

Age of core

B-10

n= narrow ring, vn= very narrow ring.

Sensitive ring pattern

Marker rings shared by most cores: 92,88,82,71,67 (vn or n), 6 ln, 57 Sensitive ring pattern shared by rnost cores: 70-7 1.66-67,6 1-62

Last ring Y-

Tree

Trees identified B represents trees fiom site 2 (near Falconbridge smeher).

92,9 1, 89, 85,82,80vn, 78, 77,74,7 1,68, 67-6 1,60,58

Marker rings

1953 6 1-62 42

Appendix 1 : continue

Age determination of tree core samples

Marker rings shared by most cores: 90,87,82n, 71 n, 67,62 (vn or n) Sensitive ring pattern shared by most cores: 70-7 1,66-67,6 1-62

7n, 64vn,63vn, 62n, 6 ln

C-5

C-6

C-7

C-8

C-9

C-10

Trees identified C represents trees fiom site 5 (near inco srnelter). Core samples were collected in October 1995. 'approximated age. n= narrow ring, vn= very narrow ring.

68n ,67,64,62,6 1,60,57

91.87, 86,82,79,74, 7Sn, 67n, 63,62n, 6 1 n, 57, 56n, 54vn,

93,9 In, 90,87,83n, 82n,79,77,76,74,71, 65.63

94,90n, 89n, 87,83, 82,72,71,67n, 65, 6Zvn, 60,58,57vn, 5611,

93n, 9 1,87,82n, 78,76,74,7 1 n. 70,67,62, 57,56n,54n

90,89,87vn, 85,84vn, 82vn,77,75n, 72, 69,67,64,62,58,56,55

91,88,87,83,81,78,75,71,68,67,64,62, 61

1952

1961'

1953

1950

1951

1957

76-77, 66-67, 61-62

?a-7 1

70-7 1, 67-68, 6 1-62

7 1-72, 6 1-62. 56-57

70-7 1, 66-67, 6 1-62

7 1-72, 67-68, 6 1-62

43

34'

42

45

44

3 8

Appendix 1 : continue

Age determination of tree core samples usin~ross-da!

Tree Marker rings

ig method

Last ring Sensitive ring Year pattern

Age of core

D- 1 9 1,86n, 8Svn, 8211-8 1 vn, 79,78vn, 74n, 7 ln, 68n, 66n, 64,60,57,53,5 1 vn, 4 8 ~ 1 , 45,44,43

0-7 1 9 ln, 89,88,86,84,8 1,78,76,74,73,70, 68n,66n

D-IO 1 92n,91n,90un,88.84,81,80n,77.75, 72.70

Core samples were collected in October 1995. n= narrow ring, vn= very nmow ring.

Marker rings shared by most cores: 9 1 (vn or n), 84 (vn or n), 78,76vn, 68,66,48vn Sensitive ring pattern shared by most cores: 70-71.66-67,60-6 1

Appendix 1 : continue

Age determination of tree core samples

94,93,92,88,85,82n, 8On,77,67n, 65n,60 1957 66-6 7 1 38 Trees identified E represents trees fiom site 3 (near Falconbridge smelter).

- -

E-3

4

E-5

E-6

Core samples were collected in October 1995. n= narrow ring, vn= very narrow ring.

-

1955

1965

- - - ---

94,93,9 1,85, 82n780,78n, 77, 69,67,62 6 1 n, 56n

93,9 ln, 90n, 86,82n,77n,76 72,7 1.67n

Marker rings shared by most cores: 82n,80,77,67(vn or n) Sensitive ring panem shared by most cores: 7 1-72,6647

93-9 ln, 87,85,82n, 80n

93n. 89n, 88n, 86,82,77n, 74n, 69n, 68n,67vn, 6 1,60vn, 57n, 5 In, 42n,38,35vn, 3 1.28n

78-79, 66-67,

6 1 -6243

77-78, 7 1 -72

40

30

1978

1920

- 61-62-63,49-

50

17

75

Appendix 1 : continue

Age detemination of tree core samples -

u-oss-dath]

Marker rings

metbod

Last rimg Sensitive ring Age of Ymr pattern core

1932 66-67 63

Tree

93n, 89,87,83vn, 8 1 vn, 79,75,71,69,68vn, 67vn, 63vn,58,56,52,49,44,38vn, 3 4 , 3 2

66-67, 63-64, 54-55

er). Trees iden ied F represents tr& fiorn site 6 (near Inco sm~ Core samples were collected in October 1995. 'approximated age. n= narrow ring, vn= very n m w ring. Marker rings shared by most cores: 88,82,79,77,68,48,44 Sensitive ring pattern shared by most cores: 66-67,63-64

Appendix 1 : continue

Age determination of tree core samples bod 1

u s cross-dating m4 !tl

m

. I

1 997 Trees identified T represents trees fiom Low Water Lake area (control site). Core sarn ples were colIected in October 1995. n= narrow ring, vn= very nanow ring.

Tree Age of core

Marker rings Lln M g 1 Sensitive ring Year pattern

94,92n,86n,83,80n, 78vn. 75th 74n, 66n,65vnT 64 vn, 60vn,58vn, 54n,48vn, 44,40vn 3 9 ~ 3 6 , 28,24, 12n, 8n

- -

T- 7 94,92n, 89,84,83,78,72n, 67n, 63n, 56,55,54, 50,48,47,44, 40,36,34,29,28,26,25,24, Zln, 18, 14, 13vn

T-8 92n,86,85,78,77,74n, 67,65,64m, 63vn,57, 56,54n,53n, 46n,44vn, 40,36n, 33,3 1 vn, 2811, 26,22, 12vn

94,92,90,88n, 86, 84,82,81,79,77,76,74,72, 70, 65,64, don, 58,57,56n, 54,53,51,49,45, 44,42,4Ivn,38,36,31vn,28,27,25n, 17, 13, IO, 894, 1

---

93,91,88,86,84,81,77,76,75,74vn, 72vn, 67vn,65vn, 64,63 vn, 61vn,60vn, 57vn,S6vn, 53, 50n,43n, 38n,31,30, 28,25, 19, 18, 12, 11,

Marker rings shared by most cores: 92n, 86n, 78(vn or n), 64(vn or n), 63n, S4(vn or n), 44vn,40 (vn or n), 13

Sensitive ring pattem shared by most cores: 65-75,3545

Appendix 1 : continue

Age determination of tree core samples using cross-dating method

I 1 1 I Tree 1 Marker rings 1 Last ring 1 Sensitive ring ( Ageof

1 1 ~ e a r 1 pattern 1 core

1 46vn, 44 1 1 1 Trees identified R represents trees fiom Ternagarni area (control site). Core sarnples were collected in October 1995.- 'approxirnated age. n= narrow ring, vn= very narrow ring.

Marker rings shared by most cores: 88n, 82(vn or n ), 66(n or vn), 63,58,47 Sensitive ring pattern shared by most cores: no distinct pattern

Appendix 1 : continue

Age detemination of tree core samples

-

pattern

- --

X-IO 93n, 90,87, 85 1 1984 5ed X represents nees fiom site 7 (Inco tailing). Trees ideni

Core samples were collected in October 1995. 'approsimated age. n= narrow ring, vn= very narrow ring.

Marker rings shared by most cores: 93n,89,87n, 8511 Sensitive ring pattern shared by most cores: no distinct pattern

Appendix 1 : continue

Age determination of tree core samples

Y -9 9 I vn, 89n,86,85 1982 - Y- 1 0 9 1 vn, 89vn,86,85,84 1980 -

Trees identified Y represents trees fiom site 8 (on FaItonbndge property). Core sarnpIes were collected in October 1995. 'approximated age. n= narrow ring, vn= very narrow ring.

- -

Age of core

Marker rings shared by most cores: 9 I(vn or n), 89(vn or n), 85 Sensitive ring pattern shared by most cores: no distinct pattern

f3 Ni concentration Cu concentration

concentration

Sudbury sites Contml sites

Appendix 2. Nickel, copper and zinc concentrations (mg kg-') in jack pine needles (n=lOO) from pooled sampling sites from Sudbury and control sites. Means (&SEM) with wmmon notations are not significantly different as indicated by Kruskal-Wallis test followed by nonpararnetric cornparison (~10.05).

Eà 0-5 cm soif depth Q 6-1 O cm soi1 depth a11 -1 5 cm soi1 depth

Sudbury sites Control sites

Appendix 3. Copper concentrations (mg kg-') in pooled soi1 samples (n=30) from Sudbury region and control sites. Means (SEM) with common notations are not significantly different as indicated by Kniskal-Wallis test followed by nonparametric cornparison (pz0.05).

l30-5 cm soi1 depth H6-10 cm soit depth 9 1 1-1 5 cm soi1 depth

Sudbury sites Contml sites

Appendix 4. Nickel concentrations (mg kg") in pooled soi1 samples (n=30) from Sudbury and control sites. Means (SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametnc cornparison (pz0.05).

OO-5 cm soi! depth H6-10 cm soi1 depth Ei 4 1-1 5 cm soi1 depth

Sudbury sites Control sites

Appendix 5. Cadmium concentrations (mg kg-') in pooled soi1 samples (n=30) from Sudbury and control sites. Means (SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by non parametric cornparison (pz0 -05).

Sudbury s b Control sites

no-5 cm soi1 depth 6-10 cm soi1 depth

El 11-15 cm soi1 depth

Appendix 6. Lead concentrations (mg kg-') in pooled soi1 samples (n=30) from Sudbury and control sites. Means (SEM) with common notations are not significantly different as indicated by Kruskal-Wallis test followed by nonparametric comparison (pz0.05).

Sudbury sites Control sites

El O S cm soi1 depth 0 6-10 cm soi1 depth IB 11-1 5 cm soi1 depth

Appendix 7. Zinc concentrations (mg kg-') in pooled soi1 samples (n=30) from Sudbury and control sites. Means (*SEM) with cornmon notations are not significantly different as indicated by Kniskal-Wallis test followed by nonparametric cornparison (pz0.05).

Appendix 8: DNA concentrations (pg ml-') of jack pine samples

SampIes Site Site Site Site Site Site Site Site Site Site 1 2 3 4 5 6 7 8 9 10

Sites 1,2,3 = sites located near Falconbridge Ltd. smelter; sites 4,5,6 = sites located near Inco Ltd. smelter, sites 7= Inco Ltd. tailing; site 8 = site located on Falconbridge Ltd. property: site 9 = Temagami (control site) and site 10 = Low Water Lake (control site).

Appendix 9: DNA degradation tests of jack pine sarnples. (Kb= 1 kb ladder, site 1= 1 to 7, site 3= 8 to 14, site 5= 15 to 21 and site 6=22 to 28).

Appendix 10: Random amplified polymorphic DNA protocol

Components Volume per reaction Final concentration

Sterile deionized distilled H20: 10 X PCR buffer iI: dATP (IOmM): dCTP (1 OmM): dTTP (1 O m M ) : dGTP ( I OmM): Primer: Template DNA: AmpliTaq@ DNA Polymerase: 25 m M MgCI, Solution:

1X 10 rnM Tris-HCI, pH 8.3,50 mM KCl

200 pM 200 pM 200 pM 200 pM 1-0 gM 200 ng

0.625 units/25 pl 4.0 mM

Final volume: 25 DI

Appendix 1 1 : Formulas and products.

Concentration of DNA (pg ml-') = (Absorbance, - Absorbance 320) X 50 ~ ( g ml-' X dilution

DNA Purity = Absorbame!,, / Absorbance,,,

Products: a) Chloroform Molecular Biology Grade (Fisher Scientific) b) l -0ctanol (Sigma) c) Isopropanol anhydrous (Sigma) d) EDTA (United State Biochemicais) e) Hexadecyl trimethyl ammonium bromide (Sigma) f) Sodium Chloride (Fisher Scientific) g) Agarose Ultra Pure (Gibco BRL) h) 1M Tris/HCI pH 8.0 (United State Biochemicals) i) Ammonium Acetate (Sigma) j) 50X TE (United State Biochernicais) k) Polaroid 667 B and W instant film 1) 1 Kb DNA Ladder (Gibco BRL)