Assessing Biological Impacts of Land Rec

Water Air Soil Pollut (2016) 227: 104
DOI 10.1007/s11270-016-2803-5

Assessing Biological Impacts of Land Reclamation in a Mining
Region in Canada: Effects of Dolomitic Lime Applications
on Forest Ecosystems and Microbial Phospholipid Fatty Acid
Signatures
K. K. Nkongolo & P. Michael & G. Theriault & R. Narendrula &
P. Castilloux & K. N. Kalubi & P. Beckett & G. Spiers
Received: 13 July 2015 / Accepted: 1 March 2016 / Published online: 10 March 2016
# Springer International Publishing Switzerland 2016

Abstract The ability of an ecosystem to withstand serious disturbances may depend in part on the diversity of
the system, with soil microbiological communities being coupled to their associated vegetation. The main
objectives of the present study were (1) to determine
the association between soil microbial diversity, abundance, and activities with diversity and sustainability of
remediated mining-damaged ecosystems in Northern
Ontario, and (2) to assess the effects of dolomitic lime
applications on aboveground and belowground biodiversity and community structures. Results revealed that
liming increases soil pH, cation exchange capacity
(CEC), forest tree species diversity and abundance,

and the overall ecosystem health even 25 to 35 years
after dolomite applications. The mean Shannon index

K. K. Nkongolo (*) : P. Beckett
Department of Biology, Laurentian University, Sudbury, ON P3E
2C6, Canada
e-mail: knkongolo@laurentian.ca
K. K. Nkongolo : P. Michael : G. Theriault :
R. Narendrula : K. N. Kalubi
Department of Biomolecular Sciences program, Laurentian
University, Sudbury, ON P3E 2C6, Canada

value was significantly higher in limed compared to
unlimed sites. Tree species richness was 4.0, 6.0, and
7.7 for unlimed, limed, and reference sites, respectively.
Overall, the mean health index revealed a significant
improvement in population health in limed sites (index
score = 7) compared to unlimed areas (index score = 4).
Soil microbial biomass and respiration were also increased by liming. Surprisingly, the main component
of soil microbial biomass in limed, unlimed, and reference sites within the vicinity of the restored lands was

bacteria (mostly Gram (−). Significant difference was
also observed between limed, unlimed, and reference
sites for arbuscular mycorrhizal fungi, Gram (−) bacteria, Gram (+) bacteria, anaerobe, and actinomycetes
abundance. The ratios between fungi and bacteria and
among other phospholipid fatty acid (PLFA) measures
were extremely low suggesting that the targeted region
is still under environmental stress. No apparent associations among soil microbial biomass, soil respiration,
and forest plant diversity or abundance were observed.
Soil pH levels and organic matter amounts and quality
appear to be the main factors affecting these parameters.
Molecular analysis of main tree species within the impacted region revealed no change in genetic variation
among plant populations on either limed or unlimed
lands contaminated with metals over two generations.

P. Castilloux
Collège Boréal, 21 Lasalle Blvd, Sudbury, ON P3A 6B1, Canada
G. Spiers
School of the Environment, Laurentian University, Sudbury, ON
P3E 2C6, Canada

Keywords Soil liming . Plant diversity and abundance .
ISSR analysis . Soil respiration . PLFA . Microbial
biomass

104 Page 2 of 13

1 Introduction
Soil biota is implicated in most of the key functions soil
provides in terms of ecosystem services, driving many
fundamental nutrient cycling processes, soil structural
dynamics, degradation of pollutants, and regulation of
plant communities. The protection of soil organism
biodiversity is a major way to maintain the proper
functioning of the soil. The living population inhabiting
soil includes macrofauna, mesofauna, microfauna, and
microflora (Cannon 1999; Nannipieri et al. 2003)
There is evidence that soil biotic communities are
coupled to their associated vegetation, with a mutual
dependence between aboveground and belowground
communities such that compromised soil communities

may curtail formation of particular plant assemblages
(Luo and Zhou 2006). Recent studies suggest that, by
characterizing diversity, one will be able to understand
and manipulate the working of ecosystems. The ability
of an ecosystem to withstand serious disturbances may
depend in part on the diversity of the system (Nannipieri
et al. 2003).
Fungi and bacteria play crucial roles in ecosystem
function. They constitute the dominant organism groups
involved in C/N cycling, primarily in the degradation of
organic matter. Critical stages in lignin decomposition
are almost exclusively carried out by fungi, with the
breakdown of complex biomolecules such as cellulose
and tannins in soils being due mostly to fungal enzymatic activity. Fungi account for up to 90 % of total
living biomass in forest soils, and soil fungal respiration
has been found to be two to four times greater than that
of bacteria (Anderson and Domsch 1978). The longterm stability of ecosystems is therefore dependent on
the continued contribution of fungal activities. Little is
known about the structure and composition of the microbial communities inhabiting Northern Ontario
(Canada) soils, and thus of the biological processes

shaping the assembly of those communities.
The Greater Sudbury region of Northern Ontario is
known globally for extensive nickel, copper, and other
metal deposits, with the aerosolic emissions from the
roasting and smelting of the ores rich in these elements
causing disastrous effects on the vegetation and overall
environment (Amiro and Courtin 1981; Gratton et al.
2000; Narendrula et al. 2012; Nkongolo et al. 2008;
Vandeligt et al. 2011). More than 80,000 ha became
semi-barren to completely barren, and several studies
implicated sulfur dioxide emissions and metal

Water Air Soil Pollut (2016) 227: 104

particulates in soils as being the main cause of these
effects (Wren et al. 2012). Dolomitic lime which contains calcium and magnesium carbonate was applied to
soils from 1978 to today as part of a Land Reclamation
(Regreening) Programme in the Greater Sudbury region
It is believed that at least some minimum number of
species are essential for ecosystem functioning under

steady conditions, with a large number of species probably being important for maintaining stable processes in
changing environments (Nannipieri et al. 2003). Threats
to soil systems such as erosion, decline of organic matter
content, and contamination will likely compromise or
destroy the soil biota and thus affect soil biodiversity.
However, even polluted or severely disturbed soils still
support relatively high levels of microbial diversity with
bacterial populations being more susceptible to certain
soluble contaminant metals than fungi (Khan 2000).
The main objectives of the present study were (1) to
determine the association between soil microbial diversity, abundance, and activities with diversity and sustainability of mining-damaged ecosystems in Northern
Ontario, and (2) to assess the effects of applications of
dolomitic liming materials on aboveground and belowground biodiversity and community structures.

2 Materials and Methods
2.1 Sampling
Nine forest populations in six areas were selected for
this study. Three areas were contaminated with metals
and each had limed and unlimed sites; three other areas
were metal-uncontaminated and used as comparison

sites. Contaminated sites were located within the
81,000 ha affected by smelter emissions and include
Wahnapitae Hydro Dam, Kingsway Road, and Kelly
Lake sites (Fig. 1). They were highly or moderately
disturbed based on their proximity to smelters as described in Nkongolo et al. (2013). Comparison sites
were at St. Charles (Hagar), Onaping Falls, and Capreol
at distances >40 km from Sudbury.
2.2 pH and Cation Exchange Capacity
Soil pH was measured in water and a neutral salt solution pH (0.1 M CaCl2) (Carter 1993). The exchangeable
cations (Al3+, Ca2+, Fe3+, K+, Mg2+, Mn2+, and Na+)
were quantified by ICP-MS analysis of ammonium

Water Air Soil Pollut (2016) 227: 104

Page 3 of 13 104

Capreol

On
naping

Falls
F

*
St.Charless
(Hagar)
Kingsw
way

*

*

Waahnapitae
Hyydro Dam

Kelly Lakee

Fig. 1 Location of red maple (Acer rubrum) sampling sites in
Northern Ontario within the Greater Sudbury region. Site 1: Kelly

Lake; Site 2: Kingsway; Site 3: Wahnapitae Hydro Dam; Site 4: St.

Charles Hagar (reference); Site 5: Capreol (reference); Site 6:
Onaping Falls (reference). *Represent smelter sites

acetate (pH 7) extracts of soil samples as developed by
Lavkulich 1981, with the total exchange capacity being
estimated as the sum of the exchangeable cations as
described in Hendershot et al. (2000).

richness, and species evenness were estimated. A health
index was also assigned to each population using a 1 to
10 scale with the grade 1 representing a population
without trees or herbaceous layer (all vegetation below
50 cm in height) and a grade of 10 representing a
population with maximum complexity with no apparent
disturbance.

2.3 Plant Species Diversity, Abundance, and Population
Health

Plant community structures were analyzed using
established ecological assessment procedures
(Theriault et al. 2013). In each population, three quadrats of 10-m diameter each were used to assess plant
species richness and abundance. Specifically, Shannon–
Wiener index, Simpson’s index of diversity, species

2.4 Molecular Analysis
Leaf samples from Betula papyrifera (the most dominant species) and Quercus rubra were collected from
both limed and unlimed sites in Northern Ontario. For
each species, 70 trees representing over 50 % of the

104 Page 4 of 13

targeted population were selected. Leaf samples were
wrapped in aluminum foil, immersed in liquid nitrogen,
and stored at −20 °C until DNA extraction.
2.4.1 DNA Extraction
Genomic DNA was extracted from fresh frozen leaf
material using the CTAB extraction protocol
(Nkongolo et al. 2005; Mehes et al. 2007). This protocol

is a modification of Doyle and Doyle (1987) procedure.
The modifications included the addition of 1 % polyvinyl pyrrolidone (PVP) and 0.2 % beta mercaptanol to
the cetyl trimethylammonium bromide (CTAB) buffer
solution, two additional chloroform spins prior to the
isopropanol spin, and no addition of RNAse. After
extraction, DNA was stored in a freezer at −20 °C.
2.4.2 ISSR Analysis
A total of 15 inter simple sequence repeat (ISSR)
primers were pre-screened for polymorphism and reproducibility. Of these, eight primers were identified. These
are 178 99A, 178 99B, UBC 841, UBC 825, ISSR 5, 8,
9, and 10. Five of these eight primers (178 98B, UBC
825, ISSR 5, 9, and 10) that produced strong bands were
selected for ISSR analysis.
PCR amplification was carried out as described by
Mehes et al. (2007) in a 25-μL total volume containing a
master mix of 11.4 μL distilled water, 2.5 μL MgSO4,
2.1 μL 10× buffer, 0.5 μL of dNTPs (equal parts dTTP,
dATP, dCTP, dGTP), 0.5 μL of ISSR primer, a Taqmix
of 3.475 μL distilled water, 0.4 μL 10× buffer and
0.125 μL Taqpolymerase (Applied Biosystems), and
4 μL standardized DNA. Each primer contained a negative control of master mix and Taq mix without any
DNA. All samples were covered with one drop of
mineral oil to prevent evaporation and amplified with
the Eppendorf Mastercycler gradient thermocycler. The
program was set to a hot start of 5 min at 95 °C followed
by 2 min at 85 °C to which the Taq mix was added. In
total, 42 cycles of 1.5 min at 95 °C, 2 min at 55 °C, and
1 min of 72 °C were performed. This was followed by a
final extension of 7 min at 72 °C after which samples
were removed from the thermocycler and placed in a
freezer set at −20 °C until further analysis.
Amplified DNA products were separated for analysis
on a 2 % agarose gel in 0.5× TBE with ethidium bromide. Then, 5 μL of 2× loading buffer was added to the
PCR products and 10 μL of this solution was loaded

Water Air Soil Pollut (2016) 227: 104

into the wells of the gel. The gel was run at 3.37 V/cm,
documented with the Bio-Rad ChemiDoc XRS system,
and analyzed with Image Lab Software. The ISSR bands
on each gel were scored as either present (1) or absent
(0). Popgene software version 1.32 (Yeh and Boyle
1997) was used to determine percentage of polymorphic
loci, observed and effective number of alleles, Nei’s
gene diversity, and Shannon’s information index. The
genetic distances were calculated using Jaccard’s similarity coefficients with Free Tree Program version 1.50.
A neighbor-joining dendogram was produced from the
similarity coefficients. The method starts with a starlike
tree with no hierarchical structure and in a stepwise
fashion finds the two operational taxonomic units that
minimize the total branch length at each cycle of clustering. The unrooted tree generated by the neighborjoining method is constructed under the principle of
minimum evolution (Saitou and Nei 1987).
2.5 Microbial Analysis
2.5.1 PLFA Analysis and Soil Respiration
Phospholipid fatty acid (PLFA) analysis was performed
at FAME Lab, Microbial ID. Inc, Newark, DE (USA),
as described in Buyer and Sasser (2012). The procedure
was originally developed by MIDI Inc. (Newark, DE)
for analysis of fatty acids from laboratory- grown pure
cultures. Extraction of fatty acids from the soil involves
saponification of the soil at 100 °C followed by acid
methylation at 80 °C for fatty acid methyl ester (FAME)
formation. This was followed by an alkaline wash and
extraction of FAME into hexane for analysis by gas
chromatography (GC) (Kaur et al. 2005).
Respiration for dried soil samples incubated for 24 h
at 50 % H2O (v/v) was assessed using the Solvita Soil
Test™ as described in Goupil and Nkongolo (2014). A
colorimetric CO2 probe inserted into the incubation jar
was read using a Digital Color Reader (DCR), with
interpretations of the DCR providing soil respiration
data estimates based on Solvita’s guidelines.
2.6 Statistical Analysis
Data for limed, unlimed, and reference sites were compared using Student’s t tests. One-way analysis of variance (ANOVA) tests were also conducted, and differences among means were determined using Tukey’s
multiple range test (P ≤ 0.05).

Water Air Soil Pollut (2016) 227: 104

Page 5 of 13 104
25

3.1 Soil Acidity and CEC
Soil pH and cation exchange capacity (CEC) estimates
were significantly higher in limed areas compared to
those in unlimed sites (Figs. 2 and 3). The mean pH
values were 6 and 4.1 for limed and unlimed sites,
respectively. CECs were 21 cmol/kg for limed sites
and 2.3 cmol/kg for unlimed.

3.2 Species Richness, Abundance, and Forest
Population Health
Tables 1 and 2 describe the proportions of different tree
species and cover found in limed, unlimed, and reference areas, with key differences in species distribution
being observed between limed and unlimed areas. In
assessing species composition, B. papyrifera was more
abundant in unlimed sites (68 %) than in limed (34 %).
Likewise, there was a decrease of Acer rubrum in limed
sites compared to unlimed areas. Salix spp. were more
prevalent in limed areas (30 %) compared to those in
unlimed sections (0 %). In reference sites, A. rubrum
was the most dominant (44 %) followed by
B. papyrifera (35 %; Table 1).
For the ground cover vegetation, Deschampsia spp.
cover was 36 and 19 % in limed and unlimed areas,
respectively, followed in relative abundance by
Vaccinium angustifolium with 26 % in the unlimed area
and 21 % in the limed areas. Polytrichum juniperum
mostly found in unlimed sites (21 %) was rare in limed
areas (3 %). The ground cover in reference sites, on the
7

*

6
5
pH

4
3
2
1
0
Limed

Unlimed

Fig. 2 Soil pH for limed and unlimed sites in the Greater Sudbury
Region. Means were significantly different based on the Student’s
t test (P ≤ 0.05). Bars represent standard error

Mean CaƟon Exchange (cmol/kg)

3 Results

*

20
15
10
5
0
Limed

Unlimed

Fig. 3 Mean estimated cation exchange capacity (CEC) for limed
and unlimed sites in the Greater Sudbury Region. Means were
significantly different based on the Student’s t test (P ≤ 0.05). Bars
represent standard error

other hand, was dominated by Pteridium aquilinum
(44 %; Table 2).
The mean Shannon index value was significantly
higher (1.06) in limed compared to that in unlimed sites
(0.76). Tree species richness was, on average, 4.0, 6.0,
and 7.7 for unlimed, limed, and reference sites, respectively (Table 3). Figures 4 and 5 depict differences in
tree species diversity and abundance between limed and
unlimed areas. The proportion of cover was higher in
unlimed sites compared to that in limed. Overall, the
mean health index revealed a significant improvement
in population health in limed sites compared to that in
unlimed areas. The average health index values were 4,
7, and 10 for unlimed, limed, and comparison sites,
respectively.
3.3 Molecular Analysis
Ten ISSR oligonucleotides primers (Table 4) were used
for the amplification of Q. rubra and B. papyrifera DNA
to determine the level of molecular variation since these
species represent over 50 % of all tree species in the
region. For each population in each species, the levels of
polymorphism for the two generations analyzed were
similar. Thus, the data for two generations for each
species at each site were compiled and used to compare
limed and unlimed populations, with no significant differences in levels of polymorphism loci being observed
between limed and unlimed sites. The genetic variation
was moderate in the two targeted species. Specifically,
the levels of genetic variation in all populations were
54 % in Q. rubra and 49 % in B. papyrifera populations.
The mean Shannon’s information index was 0.21 in

104 Page 6 of 13

Water Air Soil Pollut (2016) 227: 104

Table 1 Total number (#) and percentage (%) of tree species in limed, unlimed, and reference sites in Northern Ontario
Unlimed

Limed
#

Reference

Tree species

#

%

Abies balsamea

0

0.0

0

0.0

47

4.15

Acer rubrum

106

18.18

33

3.93

508

44.84

Betula papyrifera

399

68.44

288

34.29

406

35.83

Fraxinus americana

0

0.0

0

0.0

14

1.24

Ostrya virginiana

0

0.0

0

0.0

46

4.06

Picea glauca

0

0.0

0

0.0

16

1.41

Pinus banksiana

1

0.17

16

1.90

0

0.0

Pinus resinosa

7

1.20

46

5.48

2

0.18

Pinus strobus

0

0.0

0

0.0

7

0.62

Populus grandidentata

0

0.0

3

0.36

22

1.94

Populus tremuloides

0

0.0

114

13.57

44

3.88

Quercus rubra

70

12.01

86

10.24

14

1.24

Salix spp.

0

0.0

250

29.76

5

0.44

Thuja occidentalis

0

0.0

4

0.48

2

0.18

Total

583

840

%

#

%

1133

Unlimed and limed sites: Wahnapitae Hydro Dam, Kingsway, and Kelly Lake; reference sites: St. Charles, Onaping Falls, and Capreol

populations from both species. No significant difference
for the level of polymorphism was observed between
the two groups (Table 5).

3.4 PLFA Analysis and Soil Respiration
Results of phospholipid fatty acid analysis are described
in Tables 6 and 7. This analysis revealed higher microbial biomass in samples from limed areas compared to
that in unlimed samples. Specifically, significant difference was also observed among limed, unlimed, and
reference sites for arbuscular mycorrhizal fungi, Gram
(−) bacteria, Gram (+) bacteria, anaerobe, and actinomycetes abundance. Surprisingly, there were more bacteria than fungi in all the sites analyzed (Table 6). In fact,
total bacteria represent 68.4, 74.3, and 71.4 % of the
total microbial biomass for unlimed, limed, and reference sites, respectively. These values were only 9.8 %
(for unlimed), 6.7 % (for limed), and 9.4 % (for reference sites) for total fungi biomass. There were twice as
many Gram (−) bacteria than Gram (+) in all the sites.
The ratio between fungi and bacteria was very low for
all the groups, with a significant difference being observed for the fungi/bacteria ratio for limed and unlimed
sites (Table 7).

In addition, sensible differences between limed and
unlimed sites were observed for the total fungi and
eukaryotes (Table 6). Surprisingly, there were more
unsaturated PLFAs than saturated in limed and reference
sites compared to those in unlimed samples. Overall, the
ratios between unsaturated and saturated PLFA were
low.
Soil respiration rates were higher in limed sites compared to those in unlimed areas, with the samples having
been collected in the fall (Fig. 6). In general, respiration
rates for the reference sites were similar to those documented for the limed sites, with soil bacteria,
representing 70 % of the microbial communities, being
the main components involved in soil respiration in
these sites. No associations among soil respiration data,
total soil microbial biomass, and aboveground diversity
indices were observed (Table 8).

4 Discussion
4.1 Soil Acidity and Metal Content
The pH values in unlimed and comparison sites were
0.05). Unlimed and
limed sites: Wahnapitae Hydro Dam, Kingsway, and Kelly Lake; reference sites: St. Charles, Onaping Falls, and Capreol

104 Page 8 of 13
1200
1000
Total number of trees

Fig. 4 Total number of trees for
unlimed, limed, and control sites
from the Greater Sudbury Region.
Unlimed and limed sites:
Wahnapitae Hydro Dam,
Kingsway, and Kelly Lake;
reference sites: St. Charles,
Onaping Falls, and Capreol

Water Air Soil Pollut (2016) 227: 104

800
600
400
200
0
Unlimed sites

Reference sites

widely used for comparing diversity among various
habitats (Clarke and Warwick 2001; Ma 2005; Stirling
and Wilsey 2001; Wilsey and Stirling 2007). Shannon’s
index is an entropy measure and has mathematical properties useful for combining diversity measures, e.g.,
when calculating regional diversity. In the present study,
Shannon–Wiener and Simpson indices were used to
compare limed and unlimed species. The Shannon–
Wiener index is more sensitive to richness and less
sensitive to evenness compared to the Simpson diversity
index (Clarke and Warwick 2001; Ma 2005; Stirling and
Wilsey 2001; Wilsey and Stirling 2007). The two indices are more robust than species richness—the number
of species in the populations. Diversity and evenness
can be positively or negatively related, and evenness
and diversity indices are not consistently regulated by
richness (Ma 2005). In general, in both limed and
unlimed sites, the diversity indices were low but the
diversity and evenness in limed sites were higher than
in unlimed areas. As in previous analysis (Theriault

pH of 5 of the Sudbury Land Reclamation Program. The
CEC followed the same trend with values higher in
limed compared to those in unlimed sites.
Previous reports describing metal content of the same
sites revealed that the limed samples contained higher
levels of total and bioavailable Ca and Mg compared to
those in unlimed sites (Nkongolo et al. 2013; Theriault
et al. 2013). There were also higher levels of total and
bioavailable P in limed samples compared to those in
unlimed. On the other hand, higher contents of bioavailable Al, Co, Fe, Ni, Sr, and Zn were observed in unlimed
sites compared to those in limed areas (Nkongolo et al.
2013; Theriault et al. 2013), reflective perhaps of the
slightly increased solubility of metal-hosting minerals at
lower pH.
4.2 Ecological Analysis
Among the various indices used in ecological diversity
assessment, the Shannon–Wiener index is the most

Ground Cover
Shrub Species
Tree Species

20
18
16
Species Richness

Fig. 5 Species richness for
ground cover, shrub, and tree
species for unlimed and limed
sites from the Greater Sudbury
region

Limed sites

14
12
10
8
6
4
2
0
Unlimed

Limed

Kingsway

Unlimed
Limed
Wahnapitae
Hydro-Dam

Unlimed

Limed

Kelly Lake

Water Air Soil Pollut (2016) 227: 104

Page 9 of 13 104

Table 4 ISSR primers used for amplification of DNA from
Quercus rubra (red oak) and Betula papyrifera (white birch) tree
samples from limed, unlimed, and reference sites in Northern
Ontario
Primer
Nucleotide sequence (5′→3′)
identification

G+C
content
(%)

ISSR 5

ACG ACG ACG ACG AC

64.29

ISSR 9

GAT CGA TCG ATC GC

57.14

ISSR 10
17899A

CTT CTT CTT CTT CTT CCT CCT 51.43
CCT CCT CCT CCT CT
CAC ACA CAC ACA AG
50.00

17898B

CAC ACA CAC ACA GT

50.00

UBC 825

ACA CAC ACA CAC ACA CT

47.06

UBC 827

ACA CAC ACA CAC ACA CG

52.94

UBC 841

GAA GGA GAG AGA GAG AYC

50.00

density. This adjustment is more appropriate for
assessing site recovery following damages caused by
air pollutants. The scale of 1 to 10 was easy to apply
since both extremes that include severely damaged sites
(with no trees and limited or no vegetation) and highly
dense forests (used as reference sites) were present in the
targeted region. When changes of forest ecosystem are
induced by human, they may lead to a global loss of
resilience and the increase of vulnerability of ecosystems to future disturbance (Aubin 2012; Laliberte et al.
2010).
4.3 Molecular Analysis

et al. 2013), the distributions of individual trees in the
populations were uneven on all the sites. The reference
sites also showed a moderate level of diversity that was
nevertheless significantly higher than in limed and
unlimed sites. The most significant differences
between limed, unlimed, and reference sites were
observed in forest population health. There was a
significant improvement of population growth quality
in limed compared to that in unlimed sites. But, there is
still a gap between limed areas and comparison sites
based on forest population health.
It should be pointed out that the population assessment used in the present study is based on the Aubin
(2012) and Drenovsky et al. (2012) approach with modifications. The current rating is based on tree canopy and
vegetation damages, together with the overall forest
Table 5 Levels of polymorphism and Shannon’s information
index in Quercus rubra (red oak) and Betula papyrifera (white
birch) populations from the Greater Sudbury region based on ISSR
data
P (%)

I

Limed

52.72

0.20

Unlimed

55.32

0.21

Limed

49.92

0.21

Unlimed

47.82

0.21

Quercus rubra

Betula papyrifera

Genetic diversity descriptive statistics. P: percentage of polymorphic loci; I: Shannon’s information index

Most of the forest ecosystems within the Greater Sudbury area of Northern Ontario have been improved
considerably during the last 30 years (Dudka et al.
1996; Gratton et al. 2000; Nkongolo et al. 2013) through
tree planting which followed significant reductions in
smelter emissions. To assess forest population sustainability, biodiversity and genetic diversity are the
key parameters considered for ecosystem stability
(Mehes-Smith et al. 2009; Rajora and Mosseler
2001a, b). Losses of rare alleles, lower heterozygoty,
and directional selection have been concerns noted
for plant populations growing in stressed environment (Slatkin 1985).
Several molecular markers have been used to determine genetic variability in different taxa. The intersimple sequence repeat (ISSR) system is a very useful
and efficient tool used in assessing genetic variation of
plant populations, combining features of SSRs and amplified fragment length polymorphism (AFLP) to the
universality of random polymorphic DNA (RAPD)
(Pradeep Reddy et al. 2002). There are a number of
advantages associated with the ISSR multi-locus technique. First, ISSRs are universal in the sense that microsatellite repeats are found in every eukaryotic genome
studied to date. Second, unlike their RAPD counterparts, ISSRs have high reproducibility. This reproducibility is most likely due to the longer lengths of the
primers which permit the use of higher annealing temperatures which, in turn, reduce non-specific binding
and results in higher stringency (Bornet and Branchard
2004; Qian et al. 2001). Furthermore, contrary to
microsatellites, amplification does not require prior
knowledge of the DNA sequence. ISSRs are quick and
easy to handle and reveal multi-locus, highly polymorphic patterns. Finally, because each band corresponds to

104 Page 10 of 13

Water Air Soil Pollut (2016) 227: 104

Table 6 Total microbial biomass, AM fungi, fungi, bacteria, eukaryote, anaerobe, and actinomycetes in soil samples from the Greater
Sudbury Region using phospholipid fatty acid (PLFA) analysis
Sites

Total microbial biomass AM fungi Fungi

Gram negative Gram positive Eukaryote Anaerobe Actinomycetes

Unlimed sites

145.72a
±33.73

6.10a
±2.12

11.81a
±6.13

67.10a
±40.06

Limed sites

339.63b
±83.25

14.91b
±2.57

Reference sites 431.81b
±80.36

19.35b
±2.57

37.29a
±16.51

5.06a
±2.64

1.64a
±0.68

16.72a
±6.95

26.72ab 174.61b
±14.17 ±41.66

76.06b
±15.45

7.39a
±2.90

4.61b
±0.70

35.33b
±6.62

42.49b
±13.25

95.32b
±20.23

16.11b
±0.60

5.19b
±2.15

40.59b
±9.95

212.76b
±32.21

Means in columns with a common subscript are not significantly different based on Tukey multiple comparison test (P > 0.05). Unlimed and
limed sites: Dam, Kingsway, and Kelly Lake; reference sites: Onaping Falls, Capreol, and Hagar. Data in ng/g

a DNA sequence delimited by two inverted
microsatellites, the amplified products, usually 200–
2000 bp long, are detectable by both agarose and polyacrylamide gel electrophoresis (Pradeep Reddy et al.
2002).
In the present study, molecular analysis of white
birch (B. papyrifera) and red oak (Q. rubra) that represent over 50 % of all the tree populations in the targeted
areas was completed. Molecular analysis showed that
the average level of polymorphic loci was 54 and 49 %
for Q. rubra and B. papyrifera, respectively. These
values indicate a moderate level of genetic variability,
with no significant differences in polymorphism being
observed among populations from limed and unlimed
areas. This observation suggests that, although the level
of tree population diversity and abundance has increased
in limed areas, the genetic variability within each species remains unchanged over two generations. There
were also no differences between metal-contaminated
and metal-uncontaminated populations at the reference
sites for genetic variability, a result consistent with previous data on conifer species (Dobrzeniecka et al. 2011;

Narendrula et al. 2013; Vandeligt et al. 2011). This result
suggests that the level of metals in plant tissue is too low
to play an important role in genotypic selection and
changes of the allelic frequencies.
4.4 Microbial Ecology
Microorganisms respond differently to prevailing environmental conditions, with different forest soil and vegetation properties influencing the composition of the soil
microbial community in a specific way (Hackl et al.
2005). In the present study, PLFA was used to analyze
the composition of the microbial communities of limed,
unlimed, and reference lands. The resulting data revealed that liming conditions increase forest diversity,
complexity, and health, with the PFLA data also
highlighting increases of total microbial biomass,
arbuscular mycorrhizal (AM) fungi, total fungi, Gram
(−) and Gram (+) bacteria, eukaryotes, anaerobe, and
actinomycetes in limed sites compared to unlimed areas.
Forest soil PLFA patterns have been found to change in
response to liming, application of wood ash, and fire

Table 7 Phospholipid fatty acid (PLFA) ratios analyzed in soil samples from limed, unlimed, and reference sites in Northern Ontario
Sites

Fungi/bacteria

Predator/prey

Gram positive/
Gram negative

Saturated/
unsaturated

Mono/poly

16w/16 cyclo

18w/19 cyclo

Unlimed sites

0.18a
±0.06

0.04a
±0.00

0.74a
±0.13

1.17a
±0.10

3.82a
±1.02

2.43a
±0.38

0.80a
±0.25

Limed sites

0.14b
±0.03

0.02a
±0.00

0.55a
±0.03

0.77b
±0.06

6.44a
±1.48

2.64a
±0.58

2.30a
±0.71

Reference sites

0.16a
±0.01

0.05a
±0.01

0.48a
±0.03

0.74b
±0.07

4.00a
±0.56

3.00a
±0.27

1.12a
±0.25

Means in columns with a common subscript are not significantly different based on Tukey multiple comparison test (P > 0.05). Unlimed and
limed sites: Dam, Kingsway, and Kelly Lake; reference sites: Onaping Falls, Capreol, and St. Charles (Hagar)

Water Air Soil Pollut (2016) 227: 104

Soil RespiraƟon CO2-C(mg/kg
(ppm))

80

Page 11 of 13 104

a

4.5 Relation Between Microbial Biomass, Activities,
and Aboveground Diversity

a

70

b

60
50
Limed

40

Unlimed

30

Control

20
10
0
Limed

Unlimed

Control

Fig. 6 Fall soil respiration rates for unlimed, limed, and reference
sites in the Greater Sudbury Region. Means with a common letter
are not significantly different based on Tukey multiple comparison
test (P > 0.05)

(Baath et al. 1995; Frostegard et al. 1993; Hackl et al.
2005). Soil pH appears to be responsible for the differentiation in soil microbial communities rather than total
metal contamination. Frostegard et al. (1996) found an
increase in fungal biomarkers with different levels of
soluble metal contamination (except Cu) in laboratory
experiments, an aspect not observed in the present study
because the level of bioavailable metals in soil solutions
was too low to generate any biomarkers. Soil chemical
composition and physical structure play also a critical
role in PFLA profile, with the low pH reference sites and
the less acidic limed sites showing a similar PLFA
profile. The high level of total and AM fungi in
reference and limed sites compared to that in unlimed
areas could be also the result of the higher organic
matter content observed in these sites as demonstrated
by Baath et al. (1995) and Bardgett et al. (2001). Surprisingly, the overall microbial communities in all the
sites were dominated by bacteria, mostly Gram (−),
indicating that the entire targeted region is still under
severe environmental stress.

The present study revealed no correlation between total
microbial biomass in the soil and forest complexity and
diversity. These two components are, however, functionally linked since microbes facilitate mineralization
of organic matter for both plant nutrient supply and the
provision of an appropriate physical environment for
microbial growth. The lack of association between soil
microbial biomass, soil respiration, and plant population
diversity could be, in part, attributed to the severe level
of land damage and the soil acidity level. The consequence of limited soil biodiversity is not well understood. From a functional perspective, soil species richness might be of little consequence, but the functional
repertoire of the soil biota is critical. There are several
microbial species involved in organic matter decomposition. But, processes such as nitrification (the oxidation
of ammonium) are carried out by a narrower range of
bacteria and there is less redundancy in this group
except for highly specific symbiotic associations. Reduced biodiversity will have differing consequences in
relation to different soil activities, and some specific soil
processes are impaired below a certain level of soil
biodiversity. A metagenomic analysis of soil sample is
in progress to establish the types of fungi and bacteria
present in each site and the level of soil microbial
diversity.

5 Conclusions
The ability of an ecosystem to withstand extreme disturbance may depend in part on the biotic and functional
diversity of the system. The latter is a function of species

Table 8 Correlation between microbial biomass, soil respiration, and ecological parameters for the unlimed, limed, and reference sites from
Northern Ontario
Variables

Total microbial
biomass

Total microbial biomass

0

Soil
respiration

Total number
of trees

Tree species
richness

Soil respiration

0.32

Total number of trees

0.13

−0.09

0

Tree species richness

0.39

−0.24

0.78*

0

Ground cover species richness

0.19

−0.24

0.3

0.1

*Significant correlation coefficients (P ≤ 0.05)

Ground cover
species richness

0

0

104 Page 12 of 13

richness or species abundance and species evenness or
species equitability. In the present study, these parameters were measured to assess the sustainability of damaged land following restoration mainly through the application of dolomitic limestone. Results from different
analyses revealed that liming increases forest trees diversity and abundance and the overall ecosystem health
even 25 to 35 years after dolomitic applications. Soil
microbial biomass and respiration were also increased in
soils following lime application. The main component
of soil microbiome in limed, unlimed, and comparison
sites within the vicinity of the reclaimed lands was Gram
(−) bacteria. The ratios between fungi and bacteria and
among other PLFA measures were extremely low suggesting that the targeted region is still under environmental stress. No associations among soil microbial
biomass, soil respiration, and forest plant diversity or
abundance were observed. Soil pH and organic matter
appear to be the main factors affecting these parameters.
However, molecular analysis of main tree species within
the targeted region revealed no change in genetic variation among plant populations from limed and unlimed
lands contaminated with metals over two generations.
Acknowledgments We would like to thank the Natural Sciences
and Engineering Research Council of Canada (NSERC), Vale
(Sudbury), and Sudbury Integrated Nickel Operations - Glencore
Company (formerly Xstrata Limited) for their financial support.

References
Amiro, B. D., & Courtin, G. M. (1981). Patterns of vegetation in
the vicinity of an industrially disturbed ecosystem Sudbury
Ontario Canada. Canadian Journal of Botany, 59(9), 1623–
1639.
Anderson, J. P. E., & Domsch, K. H. (1978). A physiological
method for the quantitative measurement of microbial biomass in soils. Soil Biology and Biochemistry, 10(3), 215–221.
Aubin, I. (2012). From seed size to ecosystem health: the plant trait
approach. Frontline Express, 57(57).
Baath, E., Frostegard, A., Pennanen, T., & Fritze, H. (1995).
Microbial community structure and pH response in relation
to soil organic matter quality in wood-ash fertilized, clear-cut
or burned coniferous forest soils. Soil Biology and
Biochemistry, 27(2), 229–240.
Bardgett, R., Jones, A., Jones, D., Kemmitt, S., Cook, R., &
Hobbs, P. (2001). Soil microbial community patterns related
to the history and intensity of grazing in sub-montane ecosystems. Soil Biology and Biochemistry, 33(12–13), 1653–
1664.
Bornet, B., & Branchard, M. (2004). Use of ISSR fingerprints to
detect microsatellites and genetic diversity in several related

Water Air Soil Pollut (2016) 227: 104
Brassica taxa and Arabidopsis thaliana. Hereditas, 140(3),
245–248.
Buyer, J. S., & Sasser, M. (2012). High throughput phospholipid
fatty acid analysis of soils. Applied Soil Ecology, 61, 127–
130.
Cannon, P. F. (1999). Options and constraints in rapid diversity
analysis of fungi in natural ecosystems. Fungal Diversity, 2,
1–15.
Carter, M. R. (1993). Soil sampling and methods of analysis. Boca
Raton: Lewis.
Clarke, R. K., & Warwick, R. (2001). A further biodiversity index
applicable to species lists: variation in taxonomic distinctness. Marine Ecology Progress Series, 216, 265–278.
Dobrzeniecka, S., Nkongolo, K. K., Michael, P., Mehes-Smith,
M., & Beckett, P. (2011). Genetic analysis of black spruce
(Picea Mariana) populations from dry and wet areas of a
metal-contaminated region in Ontario (Canada). Water, Air,
and Soil Pollution, 215(1–4), 117–125.
Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation
procedure for small quantities of fresh leaf tissue.
Phytochemical Bulletin, 19, 11–15.
Drenovsky, R. E., Grewell, B. J., D'Antonio, C. M., Funk, J. L.,
James, J. J., Molinari, N., Parker, I. M., & Richards, C. L.
(2012). A functional trait perspective on plant invasion.
Annals of Botany, 110(1), 141–153.
Dudka, S., Ponce-Hernandez, R., Tate, G., & Hutchinson, T. C.
(1996). Forms of Cu, Ni and Zn in soil of Sudbury, Ontario
and the metal concentrations in plants. Water, Air, and Soil
Pollution, 90(3–4), 531–542.
Frostegard, A., Baath, E., & Tunlid, A. (1993). Shifts in the
structure of soil microbial communities in limed forests as
revealed by phospholipid fatty acid analysis. Soil Biology
and Biochemistry, 28(1), 55–63.
Frostegard, A., Tunlid, A., & Baath, E. (1996). Changes in microbial community structure during long-term incubation in two
soils experimentally contaminated with metals. Soil Biology
and Biochemistry, 25(6), 723–730.
Goupil, K., & Nkongolo, K. (2014). Assessing soil respiration as
an indicator of soil microbial activity in reclaimed metal
contaminated lands. American Journal of Environmental
Sciences, 10, 403–411.
Gratton, W., Nkongolo, K., & Spiers, G. (2000). Heavy metal
accumulation in soil and jack pine (Pinus banksiana) needles
in Sudbury, Ontario, Canada. Bulletin of Environmental
Contamination and Toxicology, 64(4), 550–557.
Hackl, E., Pfeffer, M., Donat, C., Bachmann, G., & ZechmeisterBoltenstern, S. (2005). Composition of the microbial communities in the mineral soil under different types of natural
forest. Soil Biology and Biochemistry, 37(4), 661–671.
Hendershot, W. H., Lalande, H., & Duquette, M. (2000). Ion
exchange and exchangeable cations. In M. R. Carter & E.
G. Gregorich (Eds.), Soil sampling and methods of analysis
(2nd ed).
Kaur, A., Chaudhary, A., Kaur, A., Choudhary, R., & Kaushik, R.
(2005). Phospholipid fatty acid—a bioindicator of environment monitoring and assessment in soil ecosystem. Current
Science (Bangalore), 89(7), 1103–1112.
Khan, M. (2000). Effect of metals contamination on soil microbial
diversity, enzymatic activity, organic matter decomposition
and nitrogen mineralization (a review). Pakistan Journal of
Biological Sciences, 3(11), 1950–1956.

Water Air Soil Pollut (2016) 227: 104
Laliberte, E., Wells, J. A., DeClerck, F., Metcalfe, D. J., Catterall,
C. P., Queiroz, C., Aubin, I., Bonser, S. P., Ding, Y.,
Fraterrigo, J. M., McNamara, S., Morgan, J. W., Sanchez
Merlos, D., Vesk, P. A., & Mayfield, M. M. (2010). Land-use
intensification reduces functional redundancy and response
diversity in plant communities. Ecology Letters, 13(1), 76–
86.
Lavkulich, L. J. (1981). Methods manual. Vancouver, BC:
Pedology Laboratory, Department of Soil Science,
University of British Columbia.
Luo, Y., & Zhou, X. (2006). Soil respiration and the environment.
Burlington: Academic Press.
Ma, M. (2005). Species richness vs evenness: independent relationship and different responses to edaphic factors. Oikos,
111(1), 192–198.
McKeague, J. A., Desjardins, J. G., & Wolynetz, M. S. (1979).
Minor elements in Canadian soils. Agriculture Canada,
Research Branch Land Resource Research Institute
Contribution No LRRI 27.
Mehes, M. S., Nkongolo, K. K., & Michael, P. (2007). Genetic
analysis of Pinus strobus and Pinus monticola populations
from Canada using ISSR and RAPD markers: development
of genome-specific SCAR markers. Plant Systematics and
Evolution, 267(1–4), 47–63.
Mehes-Smith, M., Nkongolo, K., & Kim, N. S. (2009). A comparative cytogenetic analysis of five pine species from North
America, Pinus banksiana, P. contorta, P. monticola,
P. resinosa, and P. strobus. Plant Systematics and
Evolution, 292, 153–164.
Nannipieri, P., Ascher, J., CECcherini, M. T., Landi, L.,
Pietramellara, G., & Renella, G. (2003). Microbial diversity
and soil functions. European Journal of Soil Science, 54(4),
655–670.
Narendrula, R., Nkongolo, K., & Beckett, P. (2012). Comparative
soil metal analyses in Sudbury (Ontario, Canada) and
Lubumbashi (Katanga, DR-Congo). Bulletin of
Environmental Contamination and Toxicology, 88(2), 187–
192.
Narendrula, R., Nkongolo, K. K., Beckett, P., & Spiers, G. (2013).
Total and bioavailable metals in two contrasting mining
regions (Sudbury in Canada and Lubumbashi in DRCongo): relation to genetic variation in plant populations.
Chemistry and Ecology, 29(2), 111–127.
Nkongolo, K. K., Michael, P., & Demers, T. (2005). Application of
ISSR, RAPD, and cytological markers to the certification of
Picea mariana, P. glauca, and P. engelmannii trees, and their
putative hybrids. Genome, 48(2), 302–311.
Nkongolo, K. K., Vaillancourt, A., Dobrzeniecka, S., Mehes, M.,
& Beckett, P. (2008). Metal content in soil and black spruce
(Picea mariana) trees in the Sudbury region (Ontario,
Canada): low concentration of arsenic, cadmium, and nickel
detected near smelter sources. Bulletin of Environmental
Contamination and Toxicology, 80(2), 107–111.

Page 13 of 13 104
Nkongolo, K. K., Spiers, G., Beckett, P., Narendrula, R., Theriault,
G., Tran, A., & Kalubi, K. N. (2013). Long-term effects of
liming on soil chemistry in stable and eroded upland areas in
a mining region. Water, Air, and Soil Pollution, 224(7), 1618.
Pradeep Reddy, M., Sarla, N., & Siddiq, E. A. (2002). Inter simple
sequence repeat (ISSR) polymorphism and its application in
plant breeding. Euphytica, 128(1), 9–17.
Qian, W., Ge, S., & Hong, D. Y. (2001). Genetic variation within
and among populations of a wild rice Oryza granulata from
China detected by RAPD and ISSR markers. Theoretical and
Applied Genetics, 102(2–3), 440–449.
Rajora, O. P., & Mosseler, A. (2001a). In G. MÃller-Starck & R.
Schubert (Eds.), Molecular markers in sustainable management, conservation, and restoration of forest genetic resources (pp. 187–202). Netherlands: Springer.
Rajora, O., & Mosseler, A. (2001b). Challenges and opportunities
for conservation of forest genetic resources. Euphytica,
118(2), 197–212.
Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new
method for reconstrucing phylogenetic trees. Molecular
Biology and Evolution, 4, 406–425.
Slatkin, M. (1985). Rare alleles as indicators of gene flow.
Evolution, 39, 53–65.
Stirling, G., & Wilsey, B. (2001). Empirical relationships between
species richness, evenness, and proportional diversity.
American Naturalist, 158(3), 286–299.
Theriault, G., Nkongolo, K. K., Narendrula, R., & Beckett, P.
(2013). Molecular and ecological characterisation of plant
populations from limed and metal-contaminated sites in
Northern Ontario (Canada): ISSR analysis of white birch
(Betula papyrifera) populations. Chemistry and Ecology,
29(7), 573–585.
Tran, A., Nkongolo, K. K., Mehes-Smith, M., Narendrula, R.,
Spiers, G., & Beckett, P. (2014). Heavy metal analysis in
red oak (Quercus rubra) populations from a mining region in
northern Ontario (Canada): effect of soil liming and analysis
of genetic variation. American Journal of Environmental
Sciences, 10, 363–373.
Vandeligt, K. K., Nkongolo, K. K., Mehes, M., & Beckett, P.
(2011). Genetic analysis of Pinus banksiana and Pinus
resinosa populations from stressed sites contaminated with
metals in Northern Ontario (Canada). Chemistry and
Ecology, 27(4), 369–380.
Wilsey, B., & Stirling, G. (2007). Species richness and evenness
respond in a different manner to propagule density in developing prairie microcosm communities. Plant Ecology,
190(2), 259–273.
Wren, C., Watson, G., & Butler, M. (2012). Risk assessment and
environmental: a case study in Sudbury, Ontario, Canada
(1st ed.). The Netherlands: Maralte B. V. Leiden.
Yeh, F. C., & Boyle, T. (1997). Population genetic analysis of codominant and dominant markers and quantitative traits.
Belgian Journal of Botany, 129, 157.