Species Differentiation in Tilia

Transcription

Species Differentiation in Tilia: A Genetic Approach
Thesis
to obtain Master of Science Degree in Tropical and International
Forestry at the Faculty of Forest Sciences and Forest Ecology,
Georg-August-University Goettingen
By
Rajendra K.C.
Supervisors:
Prof. Dr. Reiner Finkeldey
Dr. Ludger Leinemann
Goettingen, Germany
January 2009
To,
My parents
Meena K.C. and
Chhatra Bahadur K.C.
iii
Acknowledgements
Acknowledgements
I am extremely grateful to Prof. Dr. Reiner Finkeldey who has scholarly guided this
research throughout. Besides his scientific guidance, his parental support and care
toward my family at the difficult situation will always remain into our heart and soul.
I am very thankful to my co-supervisor Dr. Ludger Leinemann who was always
supporting me with his scientific guidance. I was nurtured with his valuable
suggestions, feedbacks and comments throughout the work. I would like to share all
credits to my supervisors for the worthy findings and outputs in this study. However, I
will be solely responsible for any errors or omissions.
I express my sincere thanks to Prof. Dr. Martin Ziehe and Dr. Elizabeth M. Gillet for
their support in data analysis. I am thankful to Dr. Oliver Gailing for his support and
guidance in conducting this research. The technical supports provided by Christine
Radler, Olga Artes, Oleksandra Dolynska, August Capelle and Gerold Dinkel can never
be forgotten.
I would like to express my profound gratitude to Prof. Dr. Edzo Veldkamp, Prof. Dr.
Christoph Kleinn, Prof. Dr. Ralph Mitlöhner, Prof. Dr. Dirk Hoelscher and Dr. Uwe
Muus for their continuous support and kindness.
I am also very thankful to Dr. Stefan Fleck, Jasmine Weisse, Nicole Legner, Meik
Meißner and other colleagues for their support to conduct field works.
I would like to extend my sincere acknowledgement to Dr. Nicolas-George Eliades, Dr.
Aye Bekele Tayele, Nga Phi Nguyen, Marius Ekue, Oleksandra Kuchma and Amaryllis
Vidalis for their assistance in many aspects of the research. I am also very thankful to
Marita Schwahn for her tremendous support in providing favourable working
environment.
I am deeply indebted to our entire classmate (TIF 2006-2008) for their help, cooperation
and friendship throughout the study period and establishing effective network for our
future endeavours.
I am very thankful to Prof. Dr. I.C. Dutta and Dr. Ridish Kumar Pokhrel (IOF,
Pokhara); and Ram Prasad Paudel, Gopal Kumar Shrestha, Dr. Udaya Raj Sharma and
iv
Acknowledgements
late Dr. Damodar Prasad Prajuli of the Ministry of Forests and Soil Conservation for
their constant support and inspiration to continue my academic career.
Our friends from Goettingeli Nepalese Society (GöNeS) helped us in each and every
step of life. I am highly indebted to Netra Bahadur Bhandari, Lok Nath Paudel,
Baburam Rijal, Dr. Ajaya Jang Kunwar, Rosan Devkota, Jeetendra Mahat, Dev Raj
Gautam, Bharat Budthapa, Ajaya Pandey and Archana Guali for their great support. I
would like to provide my sincere gratitude to Uschi Demmer (Christophorus
Kindergarten) for her parental support to us.
I would like to thank Birgit Skailes and Stefan Heinemann (DAAD) for their instant
support. They were so kind to us, we never required asking twice for any kind of help
that we needed. I am deeply grateful to German Academic Exchange Service (DAAD)
for providing me DAAD scholarship to study M.Sc. in Tropical and International
Forestry (TIF). I am very thankful to Antze Henkelmann for her support throughout the
study period.
I am very grateful to Ministry of Forests and Soil Conservation and Department of
Forests, Nepal who supported me by granting study leaves.
I would like to offer all my indebtedness, gratitude and humbleness to my parents,
brothers and sisters. My parents-in-laws also deserve special thanks for their continuous
support and encouragement.
I am very thankful to my wife Aasha Khattri in flourishing the career and maintaining
my life. She never let me feel sad even in the most difficult situation in our family. Even
when our daughter Aarju was struggling for life, she tried to maintain entire
environment favourable for my study. My children Ravi Raman, Avi Raman and Aarju
have been providing millions of smiles in my life despite I could not provide them
enough time, sufficient care and better opportunities.
Rajendra K.C.
Göttingen, 26 January 2009
Table of Contents
v
Table of Contents
ACKNOWLEDGEMENTS ................................................................................................. III
TABLE OF CONTENTS .....................................................................................................V
ABBREVIATIONS .......................................................................................................... VII
LIST OF TABLES .......................................................................................................... VIII
LIST OF FIGURES ........................................................................................................... IX
APPENDIXES .................................................................................................................. X
1.
INTRODUCTION............................................................................................... 1
1.1
Introduction to the Species ............................................................................... 1
1.1.1 The Family Tiliaceae .................................................................................... 1
1.1.2 The Genus Tilia ............................................................................................ 1
1.2
Distribution ....................................................................................................... 2
1.3
Silivicultural Characteristics ............................................................................ 3
1.4
Morphology ...................................................................................................... 3
1.5
Phenology ......................................................................................................... 6
1.6
Use and Economic Importance of the Species ................................................. 6
2.
LITERATURE REVIEW .................................................................................... 8
2.1
Genetic Aspect of Tilia ..................................................................................... 8
2.2
Reproduction Biology in Tilia .......................................................................... 9
2.3
Prevalent Methods of Species Identification in Tilia ..................................... 10
2.3.1 Morphological Identification ...................................................................... 10
2.3.2 Genetic Identification ................................................................................. 13
2.3.2.1 Biochemical Marker ........................................................................... 13
2.3.2.2 Molecular (DNA) Marker.................................................................... 14
3.
OBJECTIVES AND RESEARCH HYPOTHESIS ................................................ 17
3.1
3.2
4.
Objectives ....................................................................................................... 17
Hypothesis ...................................................................................................... 17
MATERIALS AND METHODS......................................................................... 18
4.1
Materials ......................................................................................................... 18
4.1.1 Collection and Preparation of Samples ...................................................... 18
4.1.1.1 Study Area .......................................................................................... 18
4.1.1.2 Collection of Samples......................................................................... 19
4.2
Methods .......................................................................................................... 20
4.2.1 Study of Nuclear Information ..................................................................... 20
4.2.1.1 Preparation of Materials ..................................................................... 20
4.2.1.2 Preparation of Buffer Systems............................................................ 21
4.2.1.3 Preparation of Gels ............................................................................. 21
4.2.1.4 Loading the Samples .......................................................................... 22
4.2.1.5 Slicing the Gel .................................................................................... 23
4.2.1.6 Staining of the Enzymes ..................................................................... 23
4.2.1.7 Scoring the Zymogramme .................................................................. 24
4.2.1.8 Use of Enzyme Systems ..................................................................... 24
Table of Contents
vi
4.2.2 Study of cpDNA ......................................................................................... 26
4.2.2.1 Extraction of DNA ............................................................................. 27
4.2.2.2 Polymerase Chain Reaction ................................................................ 27
4.2.2.3 Agarose Gel Electrophoresis .............................................................. 28
4.2.2.4 Gene Scanning and Genotyping ......................................................... 30
4.3
Data Analysis .................................................................................................. 30
4.3.1 Analysis of Isozyme Data ........................................................................... 30
4.3.1.1 Species Assignment ............................................................................ 30
4.3.1.2 Determination of Genetic Pattern ....................................................... 34
4.3.2 Analysis of cpDNA Data ............................................................................ 35
4.3.3 Genetic Software for Data Analysis ........................................................... 36
5.
RESULTS........................................................................................................ 37
5.1
Interpretation of Isozyme Pattern ................................................................... 37
5.1.1 Menadione reductase .................................................................................. 37
5.1.2 Leucine Aminopeptidase ............................................................................ 38
5.1.3 Phosphoglucose isomerase ......................................................................... 38
5.1.4 Phosphoglucomutase .................................................................................. 40
5.1.5 Shikimate Dehydrogenase .......................................................................... 41
5.1.6 Alcohol Dehydrogenase ............................................................................. 41
5.1.7 Glutamate Oxalacetate Transaminase ........................................................ 42
5.2
Identification of Species ................................................................................. 43
5.2.1 Summer Linden (T. platyphyllos) ............................................................... 44
5.2.2 Hybrid Linden (T. × europaea) .................................................................. 45
5.2.3 Winter Linden (T. cordata) ........................................................................ 46
5.3
Genetic Patterns in Tilia spp........................................................................... 47
5.3.1 Species-wise Genetic Pattern in Tilia ......................................................... 49
5.3.1.1 Genetic Pattern of T. platyphyllos ...................................................... 49
5.3.1.2 Genetic Pattern of T.× europaea ........................................................ 49
5.3.1.3 Genetic Patterns of T. cordata ............................................................ 50
5.3.2 Genetic Pattern of Tilia spp. across Study Plots ......................................... 50
5.3.2.1 III-1 Plot ............................................................................................. 50
5.3.2.2 V1 Plot ................................................................................................ 51
5.3.2.3 V2 Plot ................................................................................................ 52
5.4
cpDNA Genetic Patterns ................................................................................ 53
5.4.1 Allelic Frequency ....................................................................................... 53
5.4.2 Haplotypes Frequency ................................................................................ 54
6.
DISCUSSION AND CONCLUSIONS.................................................................. 56
7.
SUMMARY ..................................................................................................... 58
8.
REFERENCES................................................................................................. 61
Abbreviations
vii
Abbreviations
AAT
Asparatate Amino Transferase
ADH
AP
bp
CCMP
cm, mm, m
cpDNA
DNA
dNTP
EDTA
GOT
GSED
Alcohol Dehydrogenase
Aminopeptidase
Base Pair
Consensus Chloroplast Microsatellite Primer
Centimeter, Millimeter, Meter
Chloroplast Deoxyribo Nucleic Acid
Deoxyribo Nucleic Acid
Deoxynucleotide Tri Phosphate
Ethylene Diamine Tetra Acetic Acid (Na2 Salt)
Glutamate Oxaloacetate Transaminase
Genetic Structure from Electrophoresis Data
He
Expected Heterozygosity
HL
Ho
ml, l
MNR
NAD
NADH
NADP
PCR
PGI
PGM
PPL
RF
SGE
SKDH
SL
SSR
TAE
WL
Hybrid Linden (T.× europaea)
Observed Heterozygosity
Millilitre, Litre
Menadione Reductase
Nicotinamide Adenine Dinucleotide
Nicotinamide Adenine Dinucleotide Hydrate (Reduced Disodium salt)
Nicotinamide Adenine Dinucleotide Phosphate
Polymerase Chain Reaction
Phospho Glucose Isomerase
Phospoglucomutase
Proportion of Polymorphic Loci
Relative Frequency
Starch Gel Electrophoresis
Shikimate Dehydrogenase
Summer Linden (T. platyphyllos)
Simple Sequence Repeats
Tris Acetate Ethylenediaminetetraacetic Acid
Winter Linden (T. cordata)
viii
List of Tables
List of Tables
Table 1:
Morphological traits to distinguish species in Tilia ................................... 11
Table 2:
Differences in morphological traits in Tilia spp. ........................................ 12
Table 3:
Enzymes used for isozyme electrophoresis ............................................... 26
Table 4:
Expected size and position of cpDNA microsatellite primers .................... 26
Table 5:
List of species specific alleles in Tilia spp. ................................................ 31
Table 6:
Identification of hybrid genotypes based on Mnr-A and Lap-D loci ......... 31
Table 7:
The nomenclature of haplotypes produced by ccmp10 and ccmp3............ 35
Table 8:
Grouping of cpDNA haplotypes according to species ............................... 44
Table 9:
List of identified T. platyphyllos trees ........................................................ 44
Table 10:
List of identified T.× europaea trees ......................................................... 45
Table 11:
List of identified T. cordata trees ............................................................... 46
Table 12:
Distribution of haplotypes at study sites ................................................... 54
List of Figures
ix
List of Figures
Figure 1:
Photograph of a Linden tree ......................................................................... 2
Figure 2:
Photograph of various parts of Linden tree .................................................. 7
Figure 3:
Map of Hainich National Park .................................................................... 18
Figure 4:
Distribution of Tilia spp. inside research plots .......................................... 19
Figure 5:
Photographs showing steps of isozyme electrophoresis ............................. 25
Figure 6:
UV light photograph showing DNA amplification .................................... 27
Figure 7:
Variations detected after PCR followed by agarose gel electrophoresis with
tested all universal primers ......................................................................... 28
Figure 8:
Photograph after agarose gel electrophoresis of PCR products ................. 29
Figure 9:
Electropherograms showing size variation in chloroplast microsatellites
ccmp3 and ccmp10 ..................................................................................... 35
Figure 10: Photograph and schematic diagram of Mnr-A locus .................................. 37
Figure 11: Photograph and schematic diagram of Lap loci ......................................... 38
Figure 12: Photograph and schematic diagram of Pgi-loci .......................................... 39
Figure 13: Photograph and schematic diagram of Pgm loci ........................................ 40
Figure 14: Photograph and schematic diagram of Skdh loci ....................................... 41
Figure 15: Photograph and schematic diagram of Adh-A locus .................................. 42
Figure 16: Photograph and schematic diagram of Got loci.......................................... 42
Figure 17: Frequency of identified T. platyphyllos ..................................................... 43
Figure 18: Allelic patterns across populations recorded from isozyme study ............. 47
Figure 19: Allelic frequency of Tilia spp. in all isozyme gene loci ............................. 48
Figure 20: Allelic frequency of Tilia spp. at III-1 plot ................................................ 50
Figure 21: Allelic frequency of Tilia spp. at V1 plot ................................................... 51
Figure 22: Allelic frequency of Tilia spp. at V2 plot ................................................... 52
Figure 23: Relative frequency for alleles amplified with ccmp3 and ccmp10 ............ 53
Figure 24: Relative frequency of haplotypes in Tilia .................................................. 55
Figure 25: Allelic patterns across populations recorded with cpDNA study ............... 55
x
Appendixes
Appendixes
Appendix 1:
Recipe for preparation of homogenization buffer .................................. 69
Appendix 2:
Recipe for preparation of gel buffer ....................................................... 69
Appendix 3:
Recipe for preparation of electrode buffer ............................................. 70
Appendix 4:
Recipe for preparation of starch gel ...................................................... 70
Appendix 5:
Recipe for preparation of staining buffer .............................................. 71
Appendix 6:
Electricity applied for electrophoresis .................................................... 73
Appendix 7:
The protocol for running PCR for cpDNA ............................................ 73
Appendix 8:
Isozyme and cpDNA data for individual trees at III-1 .......................... 74
Appendix 9:
Isozyme and cpDNA data for individual trees at V1.............................. 75
Appendix 10: Isozyme and cpDNA data for individual trees at V2 ............................. 78
Appendix 11: Allelic structure of T. cordata ............................................................... 81
Appendix 12: Allelic structure of T.× europaea .......................................................... 82
Appendix 13: Allelic structure of T. platyphyllos ....................................................... 83
Appendix 14: Genotypic structure of T. cordata ......................................................... 84
Appendix 15: Genotypic structure of T.× europaea ...................................................... 85
Appendix 16: Genotypic structure of T. platyphyllos .................................................. 86
Appendix 17: Genetic parameters for T. platyphyllos .................................................. 87
Appendix 18: Pairwise population matrix of genetic distance for T. platyphyllos ...... 87
Appendix 19: Genetic parameters for T.× europaea ..................................................... 87
Appendix 20: Pairwise population matrix of genetic distance for T.× europaea .......... 88
Appendix 21: Genetic parameters for T. cordata .......................................................... 88
Appendix 22: Pairwise population matrix of genetic distance for T. cordata.............. 88
Appendix 23: Various genetic parameters for Tilia spp. at III-1 .................................. 89
Appendix 24: Pairwise population matrix of genetic distance at III-1 ......................... 89
Appendix 25: Various genetic parameters for Tilia spp. at V1 ..................................... 89
Appendix 26: Pairwise population matrix of genetic distance at V1 ............................ 90
Appendix 27: Various genetic parameters for Tilia spp. at V2 ..................................... 90
Appendix 28: Pairwise population matrix of genetic distance at V2 ............................ 90
Introduction
1.
Introduction
1.1
Introduction to the Species
1.1.1
The Family Tiliaceae
1
Tiliaceae is one out of nine families under the order Malvale. Watson and Dallwitz
(1992) have extensively described the characteristics of the family Tiliaceae which
includes trees, shrubs and in some cases herbs too.
This family has various leaves forms; mainly alternate, spiral or disctichous, petiolate,
non sheathing, stipulate and simple leaves. Plants under this family constitute mostly
hermaphrodites, or monoecious, or polygamomonoecious (Pigott, 1991; Watson and
Dallwitz, 1992). It might have either solitary flowers or aggregated in inflorescence
(Pigott, 1991). Flower contains a large number of androecium (15-100), free of one
another or coherent, and has stylate gynoecium (Pigott, 1991; Watson and Dallwitz,
1992). It produces fleshy or non fleshy; dehiscent or indehiscent or schizocarp fruits
(Watson and Dallwitz, 1992). It has endospermic seeds and its endosperm is rich in oil
content.
The family has around 450 species from 50 different genera (Watson and Dallwitz,
1992). The plants under this family have world wide distribution. These species are
primarily distributed from tropical, subtropical to the temperate regions (Pigott, 1991).
1.1.2
The Genus Tilia
Many of the species are found hybridizing in wild and in cultivation. Therefore, it is
difficult to identify the exact number of species under this genus, however it is
commonly agreed that the genus Tilia has around 35-50 species (Środon, 1991). The
genus includes the species, deciduous and tall trees, mostly native to northern
hemisphere. The species under this genus have been distributed to Europe, America and
Asia.
In this study, we will focus on Small Leaved Linden (Tilia cordata), Large Leaved
Linden (Tilia platyphyllos) and their putative hybrid (Tilia× europaea).
2
Introduction
The taxonomical classification of the Tilia spp. is as
follows:
Kingdom
: Plantae
Subkingdom
: Tracheobionta
Division
: Magnoliophyta
Class
: Magnoliopsida
Order
Family
Genus
: Malvales
: Tiliaceae
: Tilia
Species: T. cordata Mill.
T. platyphyllos Scop.
Figure 1. A Linden Tree
T.× europaea L. and many other species.
Local name: Linden tree (Deutsch), Lime tree (English)
T. cordata and T. platyphyllos and their hybrid (T.× europaea) are the large trees of
various forms. It can grow up to 40 meter high, with a fairly cylindrical trunk up to 1
meter diameter at breast height. It produces clear bole up to two thirds of its height. The
trees on woodlands grow taller than open areas however the branching in the trees at
open areas start nearer to ground than woodland.
1.2
Distribution
Tilia species are widely distributed in Europe. Pigott (1991) has extensively studied the
geographical distribution of Tilia species. Among many species of Tilia, T. cordata is
one of the most widely distributed trees of Summer green deciduous forests of the
temperate low land of Europe and a small part of Western Siberia (Pigott, 1991). Its
distribution is sub oceanic to sub continental. T. cordata reaches maximum elevation in
the Alps at 1450m whereas T. platyphyllos has a range in central and southern Europe,
and the most elevated stands have been reported also from the Alps at 1800m
(Boratynska and Dolatowski, 1991). The distribution range of T. platyphyllos is quite
limited in comparison to T. cordata (Jensen et al., 2008) however both of Tilia spp. are
distributed all over Europe, in different proportion (Green, 1955; Pigott, 1991; Środon,
1991; Chytry et al., 1997; Wicksell et al., 1999; Ücler and Mollamehmetoglu, 2001;
Introduction
3
Jensen et al., 2008). Due to its broader distribution in Europe, it can be referred as true
European broad leaved tree species.
Linden tree has long traditional, cultural and historical importance in many countries in
Europe as it has widely distributed in the region. For example it is regarded as the
national tree of Czech Republic (www.chez.cz) and Slovakia.
1.3
Silivicultural Characteristics
Linden trees generally occur as an associate of other species therefore rarely constitute
pure continuous forest. Lindens are deciduous trees which shed leaves during winter as
an adaptation strategy to severe cold. T. cordata is exceptionally tolerant to very low
temperature. It has been recorded undamaged after exposure to an air temperature of 48°C (Pigott, 1991).
Seedlings and saplings are relatively shade tolerant therefore they can survive even in
the frosty regions. T. cordata is lesser tolerant to shade than T. platyphyllos (Pigott,
1991). T. platyphyllos is found at the intermediate stage whereas T. cordata is more
advance and found at the intermediate-climax stage of succession (Eriksson, 2001).
T. cordata, T. platyphyllos and their hybrids have remarkable capacity for vegetative
reproduction. They are good coppicers. Trees of all ages used to produce vigorous
shoots (coppices). Deer, sheep and cattle generally browse on their seedlings and
saplings (Pigott, 1991); therefore it is difficult to establish Linden trees in forest areas
with high game density.
1.4
Morphology
1.4.1
Crown
It has normally emergent and semi hemispherical crown spreading up to 5-12 m in
diameter. Old trees may form parabolic crown and reach up to 10-15 m in diameter.
Introduction
1.4.2
4
Branch
The lower branches in Linden mostly have horizontal and arching shape whereas
middle or second order branches usually have horizontal, ascending or vertical forms.
The upper layer branches are generally ascending and vertical.
1.4.3
Bark
Its bark is smooth, greyish with rhombus lenticels at the young age. The lenticels grow
into shallow fissures with increasing age and finally lie in deep fissures. The bark is still
thin but very firm and has scales on the ridges.
1.4.4
Leaves
Linden trees have simple, distichously arranged and palmately veined leaves. Leaves
are alternate in two opposite rows. It has a long and slender petiole with a stipule. The
petiole of T. cordata is slender and ranges from 0.8 to 1.0 mm in diameter and 3-6 cm in
length which is generally 0.5 to 2 times longer than lamina (Pigott, 1991).
The leaf is abruptly acuminate at the tip but its base is cordate or rarely truncate. It has
dentate margin on entire lamina except on the base. Leaves at the illuminated part of the
crown are flat and thicker than shadowed leaves. Leaves at the basal sprouts are
exceptionally larger and differ from those of crown in shape, marginal teeth and
distribution of hairs (Pigott, 1991).
Leaves of T. cordata are smaller than that of T. platyphyllos in length and width of the
leaf blade and apex. Hence T. cordata is also called as Small Leaved Linden and T.
platyphyllos as Large Leaved Linden.
1.4.5
Flower
All Linden flowers are hermaphrodite (Hildebrand, 1869; Pigott, 1991). They have
complete flowers with five valvate sepals, five pale yellowish petals, large numbers of
stamens with yellowish anthers and pistil with spherical ovary. The floral formula for
Tilia is K5 C5A∞G (5) (Fromm, 2001). The flower is dish shaped and 1.0 to 1.5 cm
large in diameter. The inflorescence of T. cordata is weakly erect while T. platyphyllos
Introduction
5
and their hybrids T. × europaea have pendulous inflorescence. There are five flowers in
average, ranging from 2-16, in an inflorescence.
1.4.6
Bract
They have distinctly visible bract with unique forms. Flowers are connected with
stalked bract. The bract is yellowish green and membranous in structure which is
oblong in shape and has 10-15 cm length with 1.5-2.5 cm width. It is important to
attract various pollinators. It acts as the wing for fruit assisting especially in dispersal of
seeds to longer distance.
1.4.7
Fruit and Seeds
Fruits are smaller nuts which are spheroid, ellipsoid or ovoid in shape. They are covered
with thin greyish wall with full of brownish and smaller hairs. Each fruit ranges from
five to eight mm in diameter with miniature acuminated tip.
A fruit normally contains one seed however in few cases, it may have two or three
seeds. Each seed has 2-3 mm diameter. Single seeded oven dry T. cordata fruit has
34.5±3.5 mg whereas a seed has 24.7 ±2.5 mg weight (Pigott, 1991). Seeds possess a
crustaceous seed coat, a fleshy and yellowish endosperm and a well-developed embryo.
Thick and relatively hard seed coat restricts its natural regeneration from seed. Seeds
are orthodox in storage behaviour hence properly dried seeds can be stored for longer
period in airtight container.
1.4.8
Root
Linden trees possess well developed and long lived root system (Rowe and Blazich,
2008). It has relatively longer tap root spread into soil with several axes. Some of the
roots grow horizontally and some descend obliquely till 1.5 to 2 m or deeper. It has
extensively branched roots with 90% covered with fine root systems. Mycorrhizas are
located within 20 cm of soil surface (Samoilova, 1968; Pigott, 1991).
Introduction
1.5
6
Phenology
Linden sheds leaves during winter. Leaf defoliation starts from October. As a result,
trees become completely leafless during whole winter. Leaf buds start to swell in April
and trees are again full with expanded leaves from middle of the May.
Linden starts flowering normally at the age of 25-30 years (Büsgen and Münch, 1929;
Pigott, 1991) in woodland, and several years younger in open areas. Linden trees bloom
generally in between June and July. T. cordata flowers 10-15 days later than T.
platyphyllos and T. × europaea (Pigott, 1991; Chalupa 2003). Fruits grow throughout
the month of August and mature at the end of September.
1.6
Use and Economic Importance of the Species

Tilia is very important for its aesthetic and cultural value as a part of urban
forestry and landscape management. Tilia and their hybrids are among the most
favourite avenue trees in Europe.

The inner bark or “bast” consists of long and tough fibres that once were used in
the production of cordage, mats, and clothing (Rowe and Blazich, 2008).

Tilia produces good lumber popularly known as white lumber or brass wood.
But it can not be ideally used as the construction materials since it is soft and
rots easily. As wood is soft, straight grained, even textured and easy to work, it
is famous for wood carving.

Due to good acoustic properties, the wood is widely used in manufacturing the
musical instruments such as electrical guitar, drum shells, piano keys and others.
It does not produce splinters hence it is considered as an ideal wood for
manufacturing handle for various tools and items (Rowe and Blazich, 2008).

Tilia flowers have pleasant fragrance and produce large quantities of nectar.
Therefore, it is highly favoured by bee keepers for the production of honey. T.
cordata is famous for many medicinal uses. The leaves and flowers can be used
as the medicine against flu and cough.
7
Introduction
b
a
c
d
e
f
g
a) A matured Linden tree
b) Alternate leaves foliage
c) A simple leaf
d) Rough bark on mature tree
e) Smooth bark on young tree
f) An inflorescence
g) A bract
& h) A fruit
h
Figure 2: Parts of Linden tree:
Literature Review
2.
Literature Review
2.1
Genetic Aspect of Tilia
8
A systematic description of genus Tilia is difficult in view of the polymorphism of
species and the presence of numerous hybrids. The most commonly given number of
species are 35-50 under this genus (Giertych, 1991).
All three studied Tilia spp. are polyploids. The occurrence of more than two sets of
homologous chromosomes in the nucleus is called as polyploidy. Polyploid types are
named according to number of chromosome sets in the nucleus. Stebbins (1950)
estimated that about one third of the angiosperms species are polyploids. Further, Grant
(1981) estimated about half of all angiosperms are polyploids. The occurrence of
polyploidy in animals is very rare events. The occurrence of polyploidy is a mechanism
of evolution and speciation in organism (Wright, 1976; Schultz, 1980).
Chromosomes of Tilia are very small, almost ovoid (Giertych, 1991), about 1µm long
and 0.5µm wide (Dermen, 1932; Pigott, 2002). Tilia has the very unusual high basic
chromosome number of 41 (Wright, 1976). Most of the Tilia species are diploid
(2n=84) and few of them are polyploids such as tetraploid, hexaploid and octoploid.
Pigott (2002) found out eight species as diploid (2n=84), five as tetraploid (2n=4×=164)
and one as octoploid (2n=8× =328) out of 14 analyzed Tilia species.
T. cordata and T. platyphyllos are the hexaploids that might have been evolved due to
inter species hybridization in the remote pasts. The genus Tilia has n=41 chromosomes,
whereas all its relatives have n=7 chromosomes (Wright, 1976). A hexaploid (6n=42)
Tilia was presumably produced from an n=7 ancestor and lost a chromosome to become
n=41 (Wright, 1976). Chimera may occur in nature and it used to be induced by low
temperature (Giertych, 1991).
Literature Review
2.2
9
Reproduction Biology in Tilia
Tilia species are hermaphrodite plants. The availability of all reproductive organs in a
flower increases the chances for selfing. Selfing occurs in both T. cordata and T.
platyphyllos (Giertych, 1991). Fromm (2001) estimated that 30.1% average selfing rate
in T. cordata.
Fromm (2001) made extensive study about the production of pollen in T. cordata. As
per his study, a Linden flower produces around 43,500 (±3,430) pollens. If the mean
number of flower is considered as five then there will have around 200,100 pollens in
an inflorescence. A ten year old branch usually has around 445 inflorescences so single
Linden branch may produce up to 89 million pollens. In total the production of pollen is
lower than other temperate tree species such as Carpinus betulus (>95 mill.), Quercus
petrea (>110 mill.) and Acer pseudoplatanus (>336 mill.) (Pohl, 1936; Fromm, 2001).
The pollen size of T. cordata is estimated as 30 µm (Fromm, 2001). The sink rate of
pollen flow is 3.2 cm/second (Knoll, 1932; Fromm, 2001). Average pollen transport
distance is around 79 m for T. cordata (Fromm, 2001). The stigma of Tilia is receptive
up to 2-5 days in general but up to 7 days in the cooler weather (Fromm, 2001).
Linden largely used to be pollinated by insects. A large number of insects used to visit
the flower. Both types of insects, diurnal and nocturnal visit Linden flowers because the
flowers used to open throughout the day and night. The insects are the most important
pollinators in Tilia species however the wind may also contribute in pollination.
During the flowering season of Linden, it is only tree species in its distribution range
that flowers. Linden flowers produce attractive fragrances due to the secretion of
chemicals such as Fernasols and others (Pigott, 1991). Fernasols act as the pheromone
for bee (Fromm, 2001). The large numbers of flowers, availability of lots of sugars such
as fructose, glucose and sucrose, and conspicuous pale-greenish bracts etc. are the main
reasons of attraction for large number of insects. Prominently bees, flies and moths are
the major pollinators for these species.
Seed sterility is a common phenomenon in Linden (Pigott, 1991) however T. cordata
yields much more viable seeds than T. platyphyllos (Giertych, 1991). Seed germination
is poorer and irregular in Tilia spp. due to their hard seed coat and immature embryo
Literature Review
10
(Ücler and Mollamehmetoglu, 2001). Natural regeneration of Tilia from seed is very
difficult hence the species frequently fail to regenerate. Kärkönen (2000) explains about
poor reproduction of T. cordata in Finland. Fromm (2001) mentioned about the
incapability of T. cordata to reproduce by apomixes.
The mating system varies from autogamous to xenogamous. (Giertych, 1991) has
mentioned about the existence of natural hybridization and introgression between T.
cordata and T. platyphyllos. If T. cordata (2n=6 ×=84) and T. platyphyllos (2n=6 ×=84)
are found sympatrically in a region, they used to take part in spontaneous hybridization
(Pigott, 1991; Fromm, 2001).
T. cordata fruits fall within a distance of 100 meters from parent trees (Pigott, 1991),
and the seed flow in T. platyphyllos is assumed to be equal due to the similar
morphological traits of seeds to that of T. cordata. The limited distance flow of seeds
creates a condition that the second generation offspring of Tilia species used to grow
relatively closer to its mother plant. This situation provides better opportunities for the
formation of family structures and back crossing between offspring and parents.
2.3
Prevalent Methods of Species Identification in Tilia
The species identification for Tilia spp. is possible through the study of morphological
characteristics or genetic traits. In following chapters we will summarize theoretical
concepts and prevalent practices of species identification for Tilia spp. based on
published literatures.
2.3.1
Morphological Identification
Pigott (1969); and Wicksell and Christensen (1999) studied extensively the hybrids and
hybridization of T. cordata and T. platyphyllos in England and in Denmark respectively
on the basis of its morphological characteristics. Both studies have mostly relied on the
morphological characteristics of leaves to distinguish between both species and their
hybrids.
Wicksell and Christensen (1999) looked at leaf characteristics as the major source of
species identification for T. cordata, T. platyphyllos and their hybrid (T.× europaea).
Size of lamina, leaf venation, width of apex, petiole size, serration of teeth, presence of
11
Literature Review
hairs on leaf and on petiole, its colour etc. were taken as the major basis of species
identification (see Table 1).
Table 1: Morphological traits to distinguish species in Tilia
Traits
T. cordata
T. platyphyllos
Length of leaf including basal lobe
45-106 mm
53-144
Length of leaf excluding basal lobe
40-91 mm
48-135 mm
Width of lamina
40-82 mm
40-113 mm
Width of apex
2-9
3-15 mm
Length of petiole
25-62 mm
22-62 mm
Number of teeth per cm on the broadest part of leaf
blade
3-7
3-5
Numbers of lateral veins of first order
4-6.5
6-10
Presence of hairs on upper and lower surface of leaf
Glabrous
Pubescent
Types of hairs
Stellate (forked)
Simple
mm
Reddish brown
Colour of hairs
Colour of abaxial surface of leaf blade
Glaucous
Lateral veins of 2nd and 3rd order on abaxial surface
Not raised
mm
White
Green
Raised
13. Presence of hairs on petiole
Glabrous
Pubescent
14. Presence of hairs on twig
Glabrous
Pubescent
15. Inflorescence
Obliquely erect
Pendulous
(Adapted from Wicksell & Christensen, 1999)
Anderson (1949) and Pigott (1969) suggested morphological traits for the analysis of
natural hybridisation and species identification (see Table 2).
Andrew (1971) mentioned that the pollen of T. cordata is characterized by smaller
average size, finer reticulation, a rounder outline than that of T. platyphyllos.
Chamber and Godwin (1971) studied the species identification based on the microscopy
of Tilia pollen. They studied pollen structure to distinguish T. platyphyllos and T.
cordata and their putative hybrid T. × europaea. They found differences in size and
shapes of pollen grain, pollen walls and in size and structure of funnels.
12
Literature Review
Table 2: Differences in morphological traits in Tilia spp.
Traits
T. cordata.
T. × europaea
T. platyphyllos
< 8 cm
8-10 cm
>10 cm
Leaves
Largest leaves on second
order shoots
Flat, tertiary veins not
raised on abaxial
Intermediate
surface
Rugose, tertiary vein
prominent on abaxial
surface
Hairs on adaxial surface
No hairs
Scattered hairs
Many hairs
Abaxial surface
Glaucous
Intermediate
Green
Axils of veins on abaxial
surface
Brown hair
Pale hairs
No tufts of hair
Veins of abaxial surface
Hairless
Scattered hairs
Very hairy
Hairs between veins of
abaxial surface
No hairs
Scattered hairs
Hairy
Diameter of petiole
<1.2 mm
1.2-1.5 mm
>1.5 mm
Hairs on petiole
No hairs
Few hairs
Many hairs
No hairs
Few hairs
Many hairs
Adaxial surface
Petiole
Twigs
Hairs on young twigs
(Adapted from Pigott, 1969)
The morphological traits as described by Pigott (1969); and Wicksell and Christensen
(1999) provide clear indication about the prevalence of many morphological traits
which are common and overlapping in both species. The size ranges of all
characteristics mentioned in Table 1 and 2 are continuous and overlapping. Further
morphological traits are highly influenced by environmental factors whereas genetic
markers are not influenced by the environmental interaction. These are the reasons why
we use genetic markers for species identification.
Therefore, identification of the species on the basis of genetic approaches has been
experimented. Isozyme and chloroplast SSR (cpDNA) markers were applied to identify
the species in Tilia.
Literature Review
2.3.2
13
Genetic Identification
Genetic identification of Tilia spp. can be done by using genetic markers. The genetic
trait or the phenotypic characters that can be unambiguously assigned to a set of
genotypes is called genetic marker. If the genetic trait is due to the one genotype, it is
called gene marker (Gillet, 1999).
Genetic markers have contributed tremendously to the development of the discipline of
genetics to the advanced stage. Genetic markers are applied to study the genetics of all
kinds of organisms. It can be used in wild and domesticated population of any animal or
plant population. Genetic markers have been used to identify the genetic variation,
mating systems, evaluating the impact of management, identifying the species,
development of plant phylogeny and so on.
There are several genetic markers frequently used in Forest Genetics according to the
needs and objectives of study. For the wider use and application, the genetic marker
should possess basic characteristics such as: inexpensive to develop and use, unaffected
by environmental variation, polymorphic and highly reproducible.
There are several genetic markers that have been developed and used in different
chronological period. Among them biochemical markers and molecular markers has
been tremendously effective and in application since their origin to date.
2.3.2.1 Biochemical Marker
Monoterpenes and isozyme are major biochemical markers. Due to many reasons
isozyme markers gradually replaced monoterpenes (White et al., 2007). Isozyme marker
will be described in detail as we applied it in the laboratory to distinguish species.
Literature Review
14
Isozyme Marker
Isozymes are special kinds of proteins that act as catalysts in chemical reaction in the
organisms. They act to enhance the rates of chemical reactions without themselves
being consumed.
Isozymes are the enzymes with similar or even identical functions (Finkeldey and
Hattemer, 2007). Markert and Moller coined the term „isozyme‟ to explain the different
molecular forms of an enzyme in a species that share a common catalytic activity
(Acquaah, 1992). The multiple molecular forms of isozymes are quite common in
organisms and these isozymes share a common catalytic activity irrespective of its
differential molecular forms. The isozymes are often tissue or cell specific. Each and
every isozyme has a particular role and function in the metabolic pathway.
Isozymes arise in nature due to genetic and epigenetic mechanisms. Mutations such as
chromosomal and genetic aberrations constitute genetic origin of isozymes whereas
physical and chemical alteration of polypeptides during translation is the source of
epigenetic isozymes in organisms.
Isozymes have been successfully applied in investigations of the genetics of a large
number of organisms since 1960s (Liengsiri et al., 1990; Weising et al., 2005).The
characteristics such as inexpensive operational costs, comparatively simpler and easier
laboratory work, availability of standard protocols for larger number of plant species,
codominant expression, relatively higher level of polymorphisms etc. have made
isozymes popular and extensively used marker in population genetics (Liengsiri et al.,
1990; Acquaah, 1992; Gillet, 1999; Bergmann and Leinemann, 2000; Finkeldey and
Hattemer, 2007; White et al., 2007). It has been generally used to explain pattern of
genetic variation within and among population, estimating mating systems and gene
flow (White et al., 2007).
2.3.2.2 Molecular (DNA) Marker
There are only a small number of different marker loci therefore genetic information
obtained from biochemical marker, such as Terpene or Allozyme, may not be sufficient
representation of genes throughout the genome (White et al., 2007). Thus, it may lead to
erroneous conclusion about their protection and management (Szmidt and Wang, 1991).
15
Literature Review
The limitation of biochemical marker insisted scientists to work for the development of
molecular or DNA based genetic marker.
Geneticist had developed this marker, so they applied first in the beginning of 1980s
(Botstein et al., 1980; White et al., 2007). Soon after the invention, molecular markers
were used in plant for genetic analysis (Szmidt and Wang, 1991). The invention of PCR
and other advanced technologies made the application of molecular marker easier and
popular. With the advancement of technology and knowledge, various molecular
markers and analyses have been developed and applied for many purposes. White et al.
(2007) mentioned two general types of DNA markers:
a) Based on DNA-DNA hybridization such as Restriction Fragment Length
Polymorphism (RFLP).
b) Based on amplification of DNA sequences using the PCR such as Random
Amplified
Polymorphic
DNA
(RAPD),
Amplified
Fragment
Length
Polymorphism (AFLP), Microsatellite or Simple Sequence Repeats (SSR) etc.
Microsatellite markers used in the study will be described in following chapter.
Microsatellites:
Microsatellites are also called as Simple Repetitive Sequences (SRS), Simple Sequence
Repeats (SSR) or Short Tandem Repeats (STR) (Weising et al., 2005). As the name
itself explains, SSR (or microsatellite) are sequences of tandem repeats. Hancock (1999)
defines microsatellite as sequence made up tandem repeats from one to six bases in
length which are arranged head to tail usually without interruption.
There are two widely used methods to categorise the microsatellite. The first one is
based on the number of nucleotide available in the motif and the second one is related to
the degree of perfectness of the array. As per the first method, the microsatellite is
prefixed with mononucleotide [motif with single nucleotide e.g. (A)n], dinucleotide
[motif with two nucleotides e.g. (CA)n], trinucleotide [motif with three nucleotides e.g.
(CGT)n] and quadri-nucleotide [motif with four nucleotides e.g. (CAGA) n] and so on.
On the basis of degree of perfectness of the array, it can be categorised into three types:
Literature Review
16
i) Perfect/pure: single uninterrupted array of particular motif such as ...(AG) n…
ii) Imperfect: not pure but interrupted by another bases within repeated motif such
as ...(TC) n A(TC) n …
iii) Compound: intermingled with perfect and imperfect arrays of several motifs
such as ...(AT)n(GT) n …
The microsatellites are most often found in non-coding regions (such as introns) and are
seldom found in coding regions (exons) of the genome (Hancock, 1999; Weising et al.,
2005). They occur in all eukaryotic genomes (Weising et al., 2005). Microsatellite
presents in both nuclear and organellor (chloroplast, mitochondria) genomes however it
occurs much lower frequencies in organellor than nuclear genomes (Weising et al.,
2005; White et al., 2007).
Microsatellite gene markers are highly polymorphic and codominant. The allele size can
be more correctly identified with the precision of 1 bp. It requires low quantities of
template DNA and is highly reproducible. These meritorious properties are the reasons
for the wide range of application of this gene marker (Pandey, 2005). But the
requirement of sophisticated equipments, technologies, chemicals and larger investment
limits its wider application.
Chloroplast Microsatellite:
Microsatellites are the regular constituents at chloroplast but their frequencies are
greater than mitochondria and much lower than nucleus. The chloroplast genome
undergo virtually no recombination therefore the cpDNA is uniparentally inherited
(Harris and Ingram, 1991; Röhr et al., 1998; Weising et al., 2005; Finkeldey and
Hattemer, 2007). In the case of angiosperm, it is inherited mostly from mother plant
whereas in the case of gymnosperm from pollen parent (Weising et al., 2005).
Therefore, it is transmitted only through seeds in angiosperm (Derero, 2007).
The cpDNA is highly conserved DNA (McCauley, 1995). Therefore, it displays low
intra specific variation (Weising and Gardner, 1999). These properties are very
important to separate unrelated species from the targeted one. Besides, chloroplast
microsatellite can be used in maternity or paternity inheritance, detection of
hybridization and introgression, analysis of phylogeny etc. (Weising et al., 2005).
Objectives and Research Hypothesis
3.
Objectives and Research Hypothesis
3.1
Objectives
17
T. cordata and T. platyphyllos have been growing sympatrically in the region for long
period. There are the admixture of T. cordata, T. platyphyllos and their putative hybrids
(T. × europaea). We are trying to identify the species of these individual Linden trees
on the basis of genetic markers. The main objectives of this study are:

To distinguish individual trees to T. cordata, T. platyphyllos and their hybrid on
the basis of already identified species specific genetic markers.

To compare species group concerning their cpDNA variation and differentiation.

To assess the genetic patterns in Tilia spp. (T. cordata, T. platyphyllos and T. ×
europaea).
3.2
Hypothesis
The main hypotheses of the research are as follows:
1. There is no evidence that species admixture of T. cordata, T. platyphyllos lead to
inter species hybrids in natural stands.
2. Effective gene flow occurs mostly in one direction in that the less frequent
species acts mainly as seed parents.
3. There is no evidence for species differentiation in cpDNA haplotypes.
Materials and Methods
4.
4.1
Materials
18
We analysed bud tissues of individual tree without prior knowledge about species
origin. Prior to genetic identification, we did not have any information regarding the
morphological traits of the selected trees.
4.1.1
Collection and Preparation of Samples
4.1.1.1 Study Area
The research sites are located at Hainich National Park in Western Thuringia, Germany.
The park covers an area of 75 square km, dominated by mixed deciduous forest
(www.nationalpark-hainich.de). Beech (Fagus sylvastica) is the dominant tree species,
grown in association with Ash (Fraxinus excelsior), Linden (T. cordata, T. platyphyllos
and T.× europaea), Hornbeam(Carpinus betulus), Oak (Quercus robur), Elm (Ulmus
glabra), Maple (Acer pseudoplatanus and A. platanoides) etc. Species composition of
the forests in Hainich has not largely disturbed by forestry operations since 1960s as it
was the part of military reservation area (Meisner, 2006). The park has the 7.5 °C and
700 mm mean annual temperature and rainfall respectively (Cesarz et al., 2007).
Figure 3: Map of Hainich National Park (Frech et al., 2003, referred in Meisner, 2006)
19
The samples were collected from three research plots namely III-1, V1 (new) and V2
established by Albrecht von Haller Institute of Plant Sciences, University of Goettingen.
The area of research plots are 50m wide and 50m long. The mean coordinate of research
sites are X=4394963 and Y=5661873 for III-1; X=4394956 and Y=5661852 for V1
(new) and X=4396845; and Y=5662679 for V2 research plots. These sites are around
367 m above sea level. The horizontal distance between III-1 and V1 is estimated 1.84
km whereas the distance between V1 (new) and V2 is merely 0.21 km. Linden trees are
concentrated around the middle part in III-1 research plot, and this plot is largely
dominated by F. sylvastica. The plots V1 and V2 have homogenous distribution of
Linden trees. We collected samples at these plots.
V1
III-1
Legend
Tilia
Fagus
Acer
Fraxinus
Carpinus
V2
Other research sub plots
Figure 4: Distribution of Tilia spp. inside research plots
(Source: https://ufgb989.uni-forst.gwdg.de/GK1086/)
4.1.1.2 Collection of Samples
Samples were collected in the second week of April, 2008. During this period, Tilia and
other associate species were completely defoliated. Twigs with 10-25 buds were
collected from the base of each tree for convenience. Collected twigs were labelled with
same number tagged on trees. In total samples from 332 trees were collected.
20
Twig samples were kept into climate chambers at 7°C. The bases of twigs were cut with
scissor to enhance vascular activities and the twigs with buds were dipped into water
bucket, which is kept inside refrigerator to use in longer period. For the DNA isolation
and subsequent tests, 4 to 5 buds were picked up, and put into 2ml Eppendorf tubes, and
these tubes were frozen into liquid Nitrogen (-176°C) for 10 seconds to prepare frozen
materials which were stored into refrigerator at -20°C.
4.2
Methods
Species assignment followed the methods suggested by Fromm (2001). He identified
the species specific markers and methods to distinguish T. cordata and T. platyphyllos
based on isozyme variation. WL and SL possess species specific alleles which permit a
differentiation of both species and identification of the hybrid (Fromm, 2001).
Following his methods, the identification of species was made. The Mnr-A and Lap-D
loci were mainly considered while differentiating the species. These gene loci show
species specific alleles/variants in Tilia.
Identified groups of the pure species T. cordata and T. platyphyllos as well as hybrids
were analysed with cpDNA markers to investigate differentiation at maternally
inherited genetic information. The combination of both isozyme gene loci and cpDNA
information allow us to estimate the direction of gene flow in mixed stand of both
species. Both isozyme and cpDNA study methods are discussed in following chapter.
4.2.1
Study of Nuclear Information
We applied the manual developed by Fromm (2001) for the isozyme electrophoresis.
The starch gel electrophoresis was undertaken for identifying the species variants. The
subsequent laboratory procedures for isozyme study are discussed in following chapters.
4.2.1.1 Preparation of Materials
The selection of plant materials and its phenological stage needs to be duly considered
for better results in isozyme study. Newly formed bud tissues and fresh leaves were
used as the plant material for isozyme electrophoresis. The newly grown leaves of T.
platyphyllos from the botanical garden of the Goettingen University were used as the
reference material at every electrophoresis run.
21
Around 1-2 cm newly burst leaves were minutely homogenized in the extraction buffer.
Green tissues were given priority to pale or brown. Soaking wicks with extracted
enzyme was better and more convenient from fresh and green materials. Getting better
extraction is the most important part of isozyme electrophoresis.
Different amounts, sizes of bud tissue or leaf tissue, with different amount of extraction
buffer, were tested in the beginning to optimize the material and protocol. Finally the
leaf tissues of 15-20 mm2 and ~10µg weight were macerated in 2-3 drops
(approximately 100µl) of extraction buffer. Special attention was paid to prohibit
contamination. The recipe of extraction buffer is given in the Appendix-1.
4.2.1.2 Preparation of Buffer Systems
The buffers impart electrical conductivity to the support media and enhance the
activities of protein molecules. The preparation of appropriate buffer systems is crucial
in promoting better electrophoresis to obtain better separation and resolution of bands.
The ionic strength of gel buffer should usually be lower than that of electrolyte
(Acquaah, 1992). The appropriate combination of gel and electrode buffers is the
important factor to affect the resolution of isozyme patterns. Electrode buffers were
regularly changed after two uses to maintain their ionic strength. The buffers could be
stored successfully for one to two weeks in the refrigerator (at ~ 7°C).
The homogenization (extraction) buffer, electrode buffers and gel buffers were prepared
as per the recipe from Fromm (2001) with little modification. The compositions of
different buffer systems are given in Appendixes 1-3.
4.2.1.3 Preparation of Gels
We used hydrolysed potato starch from GERBU Company to prepare gel. Starch gel
imposes additional restrictions to the migrating molecules due to their molecular sieving
properties besides only due to pH. In addition to this advantage, the starch gel technique
is simpler and less expensive than other electrophoresis systems therefore it is the most
practiced media for isozyme electrophoresis.
22
Three different kinds of running systems such as Ashton and Braden (also called Trisborate) pH 8.1, Histidine-citrate pH 6.2 and Tris-citrate pH 7.4 were used for starch gel
electrophoresis. The recipe for the preparation of gel is given in Appendix- 4.
4.2.1.4 Loading the Samples
The gel was cut lengthwise around 2 cm away from the edge to load the wicks. The cut
divided the gel into two strips of different size; the narrow strip and wide strip. The
narrow strip was placed towards cathodal side and wider strip into anodal side to
provide longer space to hold isozyme variants. The cut itself performed as the origin of
isozyme separation.
The cut space was widened around 1 cm to load the wicks. The filter paper wicks of 5
mm width and 6 mm length were used to absorb the enzymes from crushed bud tissues.
Wicks were loaded vertically to the anodal strip of the gel. They were placed at the
regular interval of 1-1.5 mm. In between the group of ten wicks, around 3-4 mm wider
interval was maintained for the easy and undoubted identification of samples during
zymogramme scoring.
A wick soaked with the homogenate from known T. platyphyllos was also loaded at the
end of all samples for comparison. A wick saturated with Bromophenol blue
(C19H10Br4O5S) marker was loaded at the end to monitor the electrophoresis progress.
Altogether 32 wicks, 30 from samples, 1 each from control species and marker were
loaded to the gel of 13 cm×24 cm×0.8-1.0 cm size. After loading all wicks, the cathodal
strip was firmly pushed back against the anodal strip to eliminate any gap in between.
For the better results, there should be good contact between anodal and cathodal strips
of the gel.
Then the glass plate with loaded gel was placed on the cooler plate in the
electrophoresis box. The high temperature generated during electrophoresis might
degenerate enzymes, therefore it is required to cool down the plate. The temperature
was maintained at 7°C - 8°C during electrophoresis.
The gel was covered around 2 cm from each edge with electrode towel, one end dipping
in the electrode buffer and other end covering the gel. The towel piece saturated with
23
electrode buffer was placed firmly to ensure good contact with gel surface. Then, the
gel with towel was pressed with glass plate.
The electrophoresis was completed in four hours. The electricity was provided as
mentioned in the Appendix 6.
4.2.1.5 Slicing the Gel
The plates with gel were taken out after completion of the electrophoresis. The cathodal
gel was taken away and disposed. All the edges of anodal strip without any
electrophoretic variants were cut and removed. The upper right corner of gel was
diagonally cut to identify the numbering of samples afterwards.
The gels were sliced with metal filament. At first, the gel was sliced around 1 mm thin
and discarded. The gel was later sliced into two from Tris-citrate and Ashton and
Braden systems and three from Histidine-Citrate system. The thickness of slices was
maintained around 3 mm for all gels.
4.2.1.6 Staining of the Enzymes
The gel slice is dipped into staining solution to view the position of enzyme. The
staining solution contains optimum quantity of the enzyme substrate, appropriate
cofactors and a dye. The visible bands on the gel are the products of the enzymatic
reaction associated with the dye. The proper staining is crucial for rightly scoring the
zymogramme.
The charged enzymes used to migrate from the origin through the gel during
electrophoresis. Isozymes migrate to different positions on the gel depending on the
electrical charge, temperature and size of isozymes. Isozymes, in general, have a
different charge and/or size due to its different amino acids composition. The genetic
differences are revealed as mobility difference on the gel (White et al., 2007).
Gel slices were first dipped into pre-buffer for few minutes before pouring staining
solution. Flasks with well stirred staining solution were heated in microwave oven for
30 seconds. The warm staining solution was poured over slices in staining tray. Before
pouring the staining solution, pre-buffer was put back for later use.
24
The time and temperature play vital roles for enzymatic reaction to the dye. If the gel
slice is left for longer than optimum time, there can be the formation of additional
zones. That is not due to the true loci. Some enzyme system reacts easily in the room
temperature whereas some needs incubation at microwave oven. The trays with slice
and staining solution were incubated into microwave oven at 37°C temperature for 1-2
hours. The staining tray with ADH enzyme was covered with glass plate inside
incubator since it contains ethanol. Different staining solutions have different recipe to
prepare. The recipe for each staining solution is given in the Appendix-5.
4.2.1.7 Scoring the Zymogramme
The process of gathering information is termed as the scoring (Acquaah 1992). The
scoring of the bands in zymogramme is the final and critical step in isozyme
electrophoresis. The study and interpretation of the zymogramme needs to be made very
carefully, since many internal and external factors might influence correct reading of the
bands. The separation and resolution of the electrophoretic variant influences the study
of zymogramme.
The gel slices were scored immediately after completion of staining. The gel was not
drained before scoring. The fastest migrated zone is named as „A‟ zone and fastest
moved allele in a locus is named as „1‟. Similarly the slower migrated enzymes or zones
or alleles were named in ascending alphabets and numbers. The systems and
observation made by Fromm (2001) was implemented in scoring the zymogramme.
4.2.1.8 Use of Enzyme Systems
Seven different enzymes under three different running systems were applied for the
study. The Ashton and Braden, Tris-Citrate and Histidine-Citrate running systems were
successfully applied. Only the GOT electrophorants could not be studied due to the lack
of sharp and clear bands. The description of enzymes and their running systems is
briefly mentioned in Table 3.
25
b
a
c
d
e
f
g
h
Figure 5: Photographs showing the steps
of isozyme electrophoresis:
a) Samples of Tilia spp. in a bucket
b) Extraction of isozyme
c) Preparation of starch gels
d) Loading samples into the gel
e) Putting gel into electrophoresis box
f) Running isozyme electrophoresis
g) Slicing the gel h) Staining gel slices
26
Table 3: Enzymes used for isozyme electrophoresis
Enzyme and Nomenclature
Gene
Locus
Structure
Number
of Alleles
Running
Systems
Glutamate oxalacetate
transminase (GOT) / Aspartate
amino transferase (AAT)
E.C.2.6.1.1
Got-A
or
Aat-A
Dimer
2
Ashton and
Braden
Phosphoglucose-isomerase (PGI)
E.C.5.3.1.9
Pgi-B
Pgi-C
Dimer
4
2
Ashton and
Braden
Alcoholdehydrogenase (ADH)
E.C.1.1.1.1
Adh-A
Dimer
3
Tris-citrate
Phosphoglumutase (PGM)
E.C.2.7.5.1
Pgm-A
Pgm-D
Monomer
3
4
Tris-citrate
Leucine Aminopeptidase (LAP)
E.C.3.4.11.1
Lap-B
Lap-D
Monomer
4
Histidinecitrate
Menadione reduktase (MNR)
E.C.1.6.99.2
Mnr-A
Tetramer
4
Histidinecitrate
Shikimatedehydrogenase (SKDH)
E.C.1.1.1.25
Skdh-B
Monomer
6
Histidinecitrate
(Adapted from Fromm, 2001)
4.2.2
Study of cpDNA
Two universal chloroplast primers namely ccmp3 and ccmp10 were applied to study
maternally inherited chloroplast DNA. For this, the expected fragment size and
annealing temperature of the selected ccmp primers were taken from Weising and
Gardner (1999) for general idea about the sizes of amplified fragments after gene
scanning. The expected sizes of two tobacco cpDNA microsatellite are given in Table 4.
Table 4: Size and position of cpDNA microsatellites for ccmp3 and ccmp10
Primer
Primer sequence alignment
Tm
Expected size
ccmp3
5‟-CAG ACC AAA AGC TGA CAT AG-3‟
5‟-GTT TCA TTC GGC TCC TTT AT-3‟
51.3°c
112 bp
ccmp10
5‟-TTT TTT TTT AGT GAA CGT GTC A-3‟
5‟-TTC GTC GDC GTA GTA AAT AG-3‟
53.7°c
103 bp
(Adapted from Weising & Gardner, 1999)
27
4.2.2.1 Extraction of DNA
The DNA was isolated from Tilia buds following the protocol „Purification of total DNA
from fresh plant tissue of DNeasy Plant minikit (Qiagen, 2006). These buds were stored
in a refrigerator at the temperature -20°c before the extraction. All the necessary
chemicals and accessories to isolate DNA were provided by Qiagen.
Initially few samples composed of all three Tilia spp. were chosen to study the variation.
For these 15 samples from all three possible species and one T. platyphyllos sample from
New Botanical Garden of the Goettingen University were isolated in the beginning. Later
the DNA was isolated from 123 samples out of 332.
Figure 6: UV light photograph showing DNA amplification isolated from Tilia spp. on
1% agarose gel.
4.2.2.2 Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) allows the short fragment of DNA to be selectively
multiplied for further analysis (Finkeldey and Hattemer, 2007) hence it makes possible to
obtain result even if small amount of DNA is available.
The DNA was initially amplified for the test with eight out of ten universal cpDNA
primers. Eight tested fluorescence labelled consensus chloroplast primers were ccmp2,
ccmp3, ccmp4, ccmp6, ccmp7, ccmp8, ccmp9, ccmp10. Six out of eight primers showed
the amplification products. Only the primers ccmp3 and ccmp10 showed fragment length
polymorphism. PCR amplification was carried out in Peltier Thermal Cycler (PTC-0200
version 4.0, MJ Research). To run the PCR, the methodology mentioned by Demesure et
al. (1995) was followed with small modification in annealing temperature and
denaturation time.
28
The 15 µl PCR mixture containing 7.5 µl of Hot Star Master Mix from Qiagen, 2 µl of
10ng DNA template, 2 µl each of 5 pM forward and 5 pM reverse primers, 1.5 µl of
HPCL H2O per sample were used for PCR.
Figure 7: The variation detected after PCR followed by agarose gel
electrophoresis with all tested universal primers in Tilia spp.
The PCR was carried out with an initial activation (95°C for 15‟); thirty five cycles of
denaturation (94°C for 1‟), annealing (50°C for 1‟), and elongation (72°C for 1‟); and
final extension of (72°C for 10‟) and infinite time at 16°C before taking the PCR product
out from thermocycler. Primer with Hex (Green) fluorescent dye was used in PCR
process.
After completion of the process, the PCR product was tested in 1.5 % agarose gel
electrophoresis. The purpose of this test is to select better primers that show successful
amplification and to identify the strength of PCR products for gene scanning.
4.2.2.3 Agarose Gel Electrophoresis
The 1% and 1.5% of agarose (w/v) was used for the electrophoresis to check the quantity
and quality of DNA and PCR products respectively. 4.5 grams of agarose was kept into
300 ml of 1×TAE buffer in a Flask. The gel was cooked in microwave oven until the
agarose powder get complete dissolution. The dissolution of agarose powder can be
observed simply with unaided eyes. If there is any particle seen, it should be further
heated for complete dissolution.
29
Ethidium bromide was added as the staining solution into Flask. 9 µl of ethidium
bromide was put into the Flask. Since the ethidium bromide is very poisonous, it was
handled always inside the defume hoods wearing the protective gloves and glass.
The gel was poured into the plexiglass plate according to the requirement of the size of
gel. The masking tape was fixed tightly around the plate to make outer boundary and
stop leakage. Plastic combs were fixed into the plate before pouring the gel solution. The
DNA or PCR products were loaded later into these slots for electrophoresis.
The gel was cooled in room temperature for half an hour. The properly cooled agarose
gel was put into the running buffer (1x TAE) in the electrophoresis chamber and plastic
combs were removed. The samples (DNA / PCR products) were pipetted carefully into
the slots. The standard control was pipetted at the end of slots. Immediate after
completion of loading, the electricity was provided. The current and voltage provided
has strong influence on the speed of electrophoresis and its resolution. Electricity with
100 Volt potential was constantly applied for half an hour. After completion of
electrophoresis the gel was photographed with UV lights.
Figure 8: Photograph after agarose gel electrophoresis of PCR products
4.2.2.4
30
Gene Scanning and Genotyping
For analysis on capillary sequencer, the PCR products were diluted as per the strength of
electrophorised bands. On the basis of strength of PCR product, we decided the
appropriate dilution ration. The gene scanner has very sensitive capillaries. If very
concentrated PCR product is used, it may damage the capillaries and if the PCR product
is too dilute, it will not give clear and interpretable result. For the gene scanning, we
multiplexed both primers (ccmp3 and ccmp10) at the same tubes to be analysed.
As per the strength of electrophorized bands, we prepared the mixture with 1:100 rations.
1 µl of ccmp3 and 1 µl of ccmp10 primer were mixed with 98 µl of HPCL water. The
known internal size standard GS 500 Rox (fluorescent dye) from Applied Biosystems
was kept to the sample to compare the results afterward. Finally we loaded 14 µl [2 µl of
diluted PCR products and 12 µl Standard] per sample to the ABI 3100 Genetic Analyser.
Before loading probes to Gene Scanner, the assay was loaded into the PCR machine for
short denaturation at 90°C for 2 minutes. The assay was cooled down by dipping into the
ice for 5 minutes before loading it into the ABI Genetic Analyser. We used the ABI
Genetic Analyser 3100 with internal size standard fluorescent dye ROX (Gene Scan 500
ROX) from Applied Biosystems.
The individual alleles were analysed using Genescan version 3.7 (Applied Biosystems)
and genotyped using Genotyper 3.7 software.
4.3
Data Analysis
4.3.1 Analysis of Isozyme Data
4.3.1.1 Species Assignment
Species assignment followed the methods suggested by Fromm (2001). He identified the
species specific markers on the basis of genetic information. Following these methods,
the identification of species has been done.
Fromm (2001) mentioned about the presence of species specific alleles to recognize
particular species in Tilia (see Table 5). Different species specific alleles / variants have
been distinguished while scoring the zymogramme. On the basis of presence or absence
of these particular variants/alleles, we distinguished the species and grouped them into T.
31
cordata or T. platyphyllos or their hybrids (T.× europaea). Species specific alleles or
variants are mentioned in Table 5.
Table 5: List of species specific alleles / variants in Tilia
Locus
Pgi-B
Pgi-C
Pgm-A
Pgm-D
Mnr-A
Skdh-B
Lap-D
Adh-A
Species specific alleles /
variants
SL
WL
B4
×
×
×
×
×
D1
×
A1, A2
A3, A4
B5, B6
×
D3, D4
D1, D2
×
A1, A2, A3
Remarks
B1, B2, B3 may occur in both species
C1, C2 may occur in both species
A1, A2 may occur in both species
D2, D3 may occur in both species
B1, B2, B3, B4 may occur in both species
A1, A2, A3 may occur in both species
The presence of alleles or variants from both species of Tilia in an individual tree was
undertaken as the hybrid. A tree is assigned F1 generation hybrid if all discriminating loci
show heterozygote genotypes combined with alleles from both species and a tree is
assigned advanced generation hybrid if one locus indicates as hybrid and other locus as
any of pure species (Table 6). The Mnr-A and Lap-D loci were highly considered while
assigning the species as they were discriminating and discrete among all analysed gene
loci.
Table 6: The identification of hybrid generation based on Mnr-A and Lap-D loci
Species
T. cordata
Mnr-A
AxAy
Lap-D
DvDw
T. platyphyllos
AvAw
DxDy
1st generation hybrid
AvAx
DvDx
Advanced generation hybrid
All other cases
Remarks
Where,
X (y)= 3 or 4
V (w) = 1 or 2
32
4.3.1.2 Determination of Genetic Pattern
4.3.1.2.1 Genetic Structure
 Allelic Structure
Allelic structure can be explained as all frequency distributions of alleles of the
population at the respective marker gene locus. It can be calculated as either relative or
absolute frequency. The relative frequency of the alleles can be calculated as
pi =ni / 2N…………………..……....in the case of diploid species.
pi =ni / ∑ni
………………………….in the case of polyploid species.
Where,
pi= Relative frequency of Allele Ai
N= number of studied genotype
ni= Absolute frequency of Allele Ai n= number of alleles
 Genotypic Structure
Genotype is the collective genetic information within an organism at the studied gene
locus. Finkeldey and Hattemer (2007) describe genotypic structure as the frequency
vector of all (relative) frequencies of genotypes.
Pij=Nij / N
Where,
Pij= Relative frequency of genotype AiAj
Nij=Absolute frequency of genotype AiAj
N= Number of studied genotypes.
4.3.1.2.2 Proportion of Polymorphic Loci (PPL)
Proportion of Polymorphic Loci takes account only the occurrence of different genetic
types, but not their frequencies. It is calculated by dividing the number of polymorphic
gene loci by the number of all investigated loci including monomorphic loci.
PPL= PL /(PL+ML)
Where,
PPL = Proportion of Polymorphic Loci
PL = Polymorphic Loci
&
ML = Monomorphic Loci
33
4.3.1.2.3 Average Number of Alleles per Locus (A / L)
It is calculated by dividing of total number of alleles counted by the number of loci
observed.
Average Number of Alleles= ∑ ni / L
Where,
ni= Number of alleles at locus i
L= Total number of all observed loci
4.3.1.2.4 Genetic Diversity
 Effective Number of Allele (Ne)
The effective number of alleles explains about the distribution pattern of allele
frequencies. This is also related to the concept of expected heterozygosity. It reaches
maximum when the He is the maximum or when the distribution of all alleles are equal
in the population. Gregorius (1978) defined Ne as the allelic diversity (v) of the
population at the given gene locus (Finkeldey and Hattemer, 2007). This can be
calculated as
1 ≤ v = 1/∑ pi2 ≤ n
i
Where,
pi = Frequency of alleles in a locus
 Observed Heterozygosity (Ho)
The observed heterozygosity can be explained as the proportion of all heterozygote
genotypes present in the studied population.
n n
Ho = ∑ ∑ Pij = 1-∑Pii
i=1
j=1
Where,
Pij= Relative frequency of genotype AiAj (heterozygote)
Pii= Relative frequency of genotype AiAi (homozygote)
34
 Expected Heterozygosity (He)
Expected heterozygosity can be defined as the probability that may have „heterozygote
combination‟ from two alleles randomly drawn from individuals in a population. It is
most important and widely used parameters in comparing the genetic variation among
population (Finkeldey and Hattemer, 2007). It is also called as the gene diversity.
The term He can be explained in terms of the allelic structure only, hence creates
confusion since the „heterozygosity‟ refers to the genotypic structure of the organism.
He can be obtained by deducting the sum of the frequencies of expected homozygosity
from 1, as per the following formula:
He=1- ∑pi2
i
Where,
He = Expected heterozygosity
pi = Frequency of alleles in a locus
 Fixation Index
Fixation index is a measure of population differential based on genetic polymorphism.
Fixation index provides the idea about genetic make up of actual population calculated
from estimated genotypic frequencies (White et al., 2007).
The fixation index ranges from -1 to 1.The F=0 implies about the complete absence of
inbreeding and the perfect matches of genotypic frequencies with Hardy-Weinberg
principle. Whereas F=1 explains about the sole prevalence of inbreeding.
The formula to calculate fixation index is as follows
F= He-Ho
/ He
Where,
Both He (Expected heterozygosity) and Ho (Observed heterozygosity) are the
means calculated from all analysed gene loci.
35
4.3.2 Analysis of cpDNA Data
We have studied the cpDNA variation from 123 samples. The cpDNA provided genetic
information regarding the seed parents only. The ccmp3 and ccmp10 markers showed
clear results at expected fragment size. At the Figure 9, the first graph shows the
fragments size 112bp by ccmp10 and 128bp by ccmp3. In the second graph, the ccmp10
and ccmp3 produced 113bp and 128bp fragment size of DNA respectively.
Figure 9: Electropherograms showing size variation in chloroplast microsatellite
ccmp3 and ccmp10 as visualised by Genescan 3.7 and Genotyper 3.7
The linked fragment size loci obtained from the amplification with ccmp3 and ccmp10
were grouped into Haplotypes (Table 7).
Table 7: The nomenclature of haplotypes produced through the combination of
ccmp10 and ccmp3 primers
ccmp3
ccmp10
103 bp
106 bp
112 bp
113 bp
127 bp
1
2
-
-
128 bp
-
-
3
4
129 bp
5
-
-
-
36
These haplotypes were compared and grouped against the particular species of Tilia
which was distinguished with the nuclear information obtained from isozyme study. We
attempted to find out the similarities and differences in distribution of specific
haplotypes to particular species groups. The grouping of haplotypes into groups of
species made on the basis of nuclear information provided the information regarding the
direction of gene flow during hybridisation.
4.3.3 Genetic Software for Data Analysis
The GSED (Genetic Structure from Electrophoresis Data) version 2.1 software was
used for the analysis of isozyme data. This software was developed at Institute of Forest
Genetics and Forest Tree Breeding of Goettingen University by Gillet E. M (19902008). Genetic Analysis in Excel (GenAlEx) version 6.1 of Peakall and Smouse (2006)
was also used while analysing isozyme data. Genescan 3.7 and Genotyper 3.7 (Applied
Biosystems) were used to produce chromatograms and analyse the study of cpSSR
allelic fragments.
37
Results
5.
Results
5.1 Interpretation of Isozyme Pattern
5.1.1 Menadione reductase
MNR is the tetrameric enzyme. Being a member of tetrameric enzyme system, it shows
low level of polymorphism than monomeric and dimeric enzyme. It produces only one
zone of activity. After proper staining, we can view clearly four alleles at this locus.
This enzyme system is very important for identification of species in Tilia. It produces
the species specific alleles in T. cordata and T. platyphyllos.
Figure 10: Photograph and schematic diagram of Mnr-A in Tilia
Alleles A3 and A4 are specific to Winter Linden (T. cordata) whereas A1 and A2 are
specific to Summer Linden (T. platyphyllos). Mnr-A locus showing one allele from WL
and another from SL, indicates that the particular tree is the Hybrid between these two
species. Genotypes A1A1, A1A2, A2A2 were specific to SL trees and A3A3, A3A4, A4A4
were WL specific whereas A1A3, A1A4, A2A3 and A2A4 were recorded only in hybrid
Linden (T.× europaea).
38
Results
5.1.2 Leucine Aminopeptidase
The sub cellular region of this monomeric enzyme is cytosol and this is used to be
activated by heavy metals. While staining, we used both leucine and alanine as per the
quantity mentioned in recipe at Appendix 5(F).
This enzyme system produced 4 different zones of activities however zone A and B
were observed blurred and zone C showed low variation. The D zone produces species
specific alleles, so that the Lap-D zone is very crucial while identifying the WL, SL and
their hybrids. D1 and D2 alleles are specific to T. cordata whereas D3 and D4 are specific
to T. platyphyllos. The heterozygous genotype containing either D1 or D2 with any of D3
or D4 alleles are considered as their putative hybrids (T.× europaea). Genotypes D1D1,
D1D2, D2D2 were found in T. cordata whereas D3D3, D3D4, D4D4 were found in T.
platyphyllos trees. Genotypes D1D3, D1D4, D2D3 and D2D4 were observed only in
Hybrids.
Figure 11: Photograph and schematic diagram of LAP loci in Tilia
5.1.3 Phosphoglucose isomerase
The phosphoglucose isomerage is the dimeric enzyme, which is also known as
glucosephosphate isomerase or phosphohexose isomerase. It functions in glycolysis
(Freeling, 1983; Acquaah, 1992). The sub-cellular location of PGI is cytoplasm and
plastids (Gottlieb and Weeden, 1981; Acquaah, 1992). After electrophoresis PGI
frequently shows polymorphic variation in plants (Lack et al., 1986).
39
Results
PGI showed three different zones of activities. All the zones showed the variation
however due to blurred phenotypes and also unavailability of nomenclature keys, Zone
A could not be analysed.
5.1.3.1 Pgi-B
Overall four alleles were observed at this locus. The alleles B1, B2 and B3 were common
in T. cordata and T. platyphyllos. SL showed completely different zymogramme from
T. cordata at this locus. These different patterns are due to the presence of variant B4,
but its confirmation of specific allele to T. platyphyllos must be tested by analysing the
larger number of T. platyphyllos samples.
5.1.3.2 Pgi-C
Two alleles were observed at C-zone. This Pgi-C locus produced minimum variation
however the zymogramme pattern of T. cordata and T. platyphyllos was different at this
locus too. This locus mostly used to be considered as the complementary to identify the
species in combination with other gene loci.
C1 allele for T. cordata and T. platyphyllos were phenotypically different hence
visualising it as different two alleles or variants. C1 allele of T. cordata was observed
faster and farther than that of T. platyphyllos. So, many times SL variant is named as C2.
Figure 12: Photograph and schematic diagram of PGI loci in Tilia
40
Results
5.1.4 Phosphoglucomutase
The sub cellular location of the phosphoglucomutase is chloroplast and cytosol
(Gottlieb and Weeden, 1981; Acquaah, 1992). It is a monomer enzyme. Being the
monomeric enzyme it is more polymorphic than dimeric and tetrameric enzymes. There
were four zones of enzymatic activities however only A and D zones were analysed as it
showed variation between T. cordata and T. platyphyllos electrophorants.
5.1.4.1 Pgm-A
The zymogramme clearly showed three different alleles/variants at this locus. They
were A2, A3 and A4. Variant A4 was observed only in the case of T. platyphyllos,
however to infer it as the SL specific variant was insufficient. For this, we need to have
more genetic analysis for larger number of progenies.
5.1.4.1 Pgm-D
Altogether four alleles were observed at this locus. The locus Pgm-D showed the
specific variant. The variant D1 was available only to T. platyphyllos whereas alleles D2,
D3 and D4 were present in both T. cordata and T. platyphyllos and their putative
hybrids. The rightly confirmation of the species based on the observation of D2, D3 and
D4 alleles at this locus may lead to wrong identification because these are the common
alleles found in both species.
Figure 13: Photograph and schematic diagram of PGM loci in Tilia
41
Results
5.1.5 Shikimate Dehydrogenase
SKDH is the monomeric enzyme which shows high level of polymorphism. It works in
the formation of aromatic amino acids in plants (Zubay, 1983; Acquaah, 1992). The Sub
cellular location of this enzyme is in cytosol and plastids (Gottlieb and Weeden, 1981;
Acquaah, 1992).
SKDH enzyme produced the highest number of alleles in our study. Fromm and
Hattemer (2003) mentioned that it produces 7 alleles however we obtained only six
alleles. Alleles B1, B2, B3 to B4 are common for both T. cordata and T. platyphyllos
whereas variants B5 and B6 are very specific to T. platyphyllos. It is hard to identify
correctly the species in Tilia on the basis of this locus alone. Many times the species
identified as Winter Linden, was confirmed as Hybrid or Summer Linden while
analysing other gene loci from other enzyme systems.
Figure14: Photograph and schematic diagram of SKDH loci in Tilia
5.1.6 Alcohol Dehydrogenase
Alcohol dehydrogenase is an enzyme discovered in the mid 1960s in Drosophila
melanogoster (Sofer and Martin, 1987). It is a dimer enzyme responsible for catalysing
oxidation of primary and secondary alcohols (Acquaah, 1992). It is found in cytosol or
intracellular fluids of the cells.
At this locus, we did not find any species specific alleles. Therefore, this enzyme system
was not considered at all while identifying the species in Tilia. This is not highly
42
Results
polymorphic loci. In SL and hybrids, they were found monomorphic. It was due to
incapability to capture polymorphism because of the small number of SL and hybrid
samples. Three different alleles namely A1, A2 and A3 were identified while scoring
zymogramme. There were larger numbers of homozygote than heterozygote at this
locus.
Figure 15: Photograph and schematic diagram of Adh-A in Tilia
5.1.7 Glutamate Oxalacetate Transaminase
Aspartate Aminotransferase is also called Glutamate Oxalacetate Transaminase which is
found in plastid, cytosol and mitochondria (Gorman et al., 1982; Acquaah, 1992) and
functions in transportation of amino group (Acquaah, 1992).
We found polymorphism in GOT however it was not scoreable. Consequently, it was
not included in the genetic study of Tilia however it was tested throughout the research.
Figure 16: Photograph of Got loci in Tilia
43
Results
5.2
Identification of Species
On the basis of nuclear information obtained from isozyme electrophoresis, we are able
to distinguish species for the individual trees of Tilia. Out of 332 selected trees, we are
able to identify the species for 294 trees. Other trees could not be identified due to the
lack of good quality materials. Among 8 different gene loci Mnr-A and Lap-D were
mainly considered for the identification of T. cordata, T. platyphyllos and their hybrids
since these loci show species specific alleles. The presence of WL specific allele with
the SL specific allele in a genotype is distinguished as T.× europaea.
Altogether 255 trees were identified as T. cordata, 12 trees as T. platyphyllos and 27
trees as T.× europaea. Among 27 identified Linden hybrids, at least 16 were identified
as the advanced generation hybrids.
Frequency of identified Tilia spp.
120
Frequency
100
80
T. cordata
60
T.x europaea
40
T. platyphyllos
20
0
III-1
V1
V2
Research plots
Figure 17: The frequency of identified Tilia spp. in research plots
The nuclear and chloroplast data were compared. Data acquired from the cpSSR were
compared against the data obtained from isozyme electrophoresis. Individual Tilia trees
identified as particular species from isozyme study, were grouped with cpSSR data and
matches were found with very few exception.
Haplotypes 1 and 2 were correctly identified as the haplotypes that confirm the
involvement of T. platyphyllos alleles. Therefore the trees that demonstrate these
haplotypes should be either pure SL tree or the hybrids. Similarly the haplotypes 3, 4
and 5 showed the contribution from WL. Therefore, these haplotypes are either purely
Winter Linden tree or hybrid Linden. Three trees identified as T. platyphyllos based on
44
Results
isozyme patterns also showed these haplotypes which can be explained as the
externalities or back crossing.
Table 8: The grouping of cpDNA haplotypes according to the identified species
Haplotypes
1
2
3
4
5
Number of
observation
T. platyphyllos
9
0
1
2
0
12
T.× europaea
8
1
2
13
0
24
T. cordata
0
0
18
64
2
84
17
1
21
79
2
120
Possible species
Total
The individual identification of Linden trees are given in Table 9-11. Detail descriptions
of the identified species are discussed here.
5.2.1
Summer Linden (T. platyphyllos)
The identification of SL tree was made on the basis of species specific alleles found in
Mnr-A and Lap-D, however other loci such as Skdh-B, Pgm-A, Pgi-B and Pgi-C
provided supplementary confirmation.
T. platyphyllos were the species of Tilia which were least in number in the region. There
were only 12 T. platyphyllos trees out of 294 trees identified. Nine SL trees were
distinguished at V1 and three were at V2 plots. There were not any SL trees at III-1
plot. The list of identified T. platyphyllos trees is given in Table 9.
Table 9: List of identified T. platyphyllos trees in Hainich
Research plots
No. of Trees
Individuals
III-1
0
V1
9
238, 240, 241, 246, 253, 256, 263, 282, 284
V2
3
132, 177, 180
45
Results
5.2.2
Hybrid Linden (T.× europaea)
Hybridization most commonly refers to mating by heterospecific individuals. There are
several T. cordata, T. platyphyllos and hybrid Tilia in the broad leaved deciduous forest
of Hainich. The fairly large numbers of different Tilia species growing together for
longer period provide better opportunity to hybridize with each other.
We found 27 Hybrid Linden (T.× europaea) out of identified 294 individuals. As per
identification, there are at least 5 Linden hybrids in III-1 plot, 13 in V1 (new) plot and 9
in V2 plot. Out of 27 hybrid Lindens, at least 16 Lindens were identified as advanced
generation hybrids. The prominent isozymes such as Mnr-A and Lap-D, did not match
with each other exactly to infer them as hybrids rather it was confirmed as the
introgressed hybrids (see Table 6 for the criteria to separate hybrid generation).
The introgressive hybridisation is the incorporation of genes of one species into the
genes of another species by repeated backcrossing (Anderson, 1949; Curtu, 2006). As
the results, the hybrids resemble a parental form (single species), however retain some
genetic information of other parent too (Curtu, 2006) as it was observed in either Mnr-A
or Lap-D allozymes in our study. One allozyme locus was demonstrating it as a pure T.
cordata or T. platyphyllos while another was displaying the presence of genetic
information from another species or hybrid. The list of identified hybrids is mentioned
in Table-10.
Table 10: List of identified T.× europaea trees in Hainich
Research plots
No. of Trees
Individuals
III-1
5
109, 134, 135, 200, 204
V1
9
131, 227, 233, 234, 239, 245, 252, 255, 283
V2
13
4, 5, 67, 68, 69, 78, 135, 142, 167, 168, 170,
171, 196
Both directional gene flows were estimated from the analysis of cpDNA haplotypes. To
produce Linden hybrids T. cordata and T. platyphyllos worked as the contributors of
pollens irrespective of their numbers.
46
Results
5.2.3
Winter Linden (T. cordata)
Among the Tilia species, T. cordata is the dominant species in Hainich National Park.
There were 255 T. cordata trees out of 294 trees investigated. There were 38 (at III-1
plot), 104 (at V1 plot) and 113 (at V2 plot) T. cordata trees. The list of identified T.
cordata trees is given in the Table-9.
Table 11: List of identified T. cordata trees in Hainich
Research
plots
III-1
V1
V2
No. of
Trees
38
Individuals
1, 2, 4, 6, 10, 15, 16, 17, 23, 25, 26, 27, 37, 38, 40, 47, 101,
102, 111, 112, 115, 117, 118, 122, 124, 125, 129, 130, 132,
133, 138, 140, 201, 202, 203, 103A, 103B, 134E
104
129, 130, 138, 144, 145, 148, 152, 153,
174, 175, 176, 177, 178, 182, 183, 184,
195, 197, 198, 199, 200, 206, 207, 208,
215, 216, 219, 220, 221, 222, 223, 225,
232, 235, 236, 242, 243, 244, 250, 257,
264, 265, 266, 267, 268, 270, 271, 273,
280, 285, 286, 287, 293, 294, 296, 297,
306, 307, 310, 506, 508, 511, 512, 513,
529, 530, 531, 532, 533, 535, 536, XO4
166,
185,
209,
226,
258,
274,
298,
516,
168,
186,
210,
228,
260,
275,
299,
519,
169,
187,
211,
229,
261,
276,
304,
520,
173,
188,
213,
230,
262,
277,
305,
524,
113
1, 3, 17, 19, 25, 28, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 45, 46, 47, 48, 49, 70, 71, 72, 73, 74, 76, 77,
101, 109, 110, 120, 121, 127, 128, 133, 134, 136, 137, 144,
145, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,
158, 160, 162, 163, 164, 165, 166, 172, 173, 174, 175, 176,
181, 188, 189, 190, 191, 192, 197, 200, 202, 203, 204, 205,
206, 213, 216, 218, 219, 222, 223, 224, 225, 226, 227, 228,
232, 233, 238, 239, 241, 243, 245, 246, 247, 248, 249, 250,
251, 252, 253, 255, 256, 257, 258, 259, 260, 261, 262, 500
47
Results
5.3
Genetic Patterns in Tilia spp.
The nuclear information obtained from isozyme study was applied in the estimation of
genetic patterns of Tilia spp. in Hainich NP. Eight gene loci from 6 enzyme systems
were considered during the analysis of genetic patterns of Tilia spp. The allelic
frequency of Tilia spp. in all eight isozyme loci is shown in Figure 19.
As per the available data, the average number of allele (Na) was more variable than
effective number of alleles (Ne) among all three Tilia spp. The average number of alleles
varied from 2.25 in T. platyphyllos to 3.25 in T.× europaea. The effective number of
alleles was the highest in T.× europaea and the least in T. cordata population. The gene
diversity or expected heterozygosity was also the highest in hybrid Linden. The
observed heterozygosity for SL was greater than Winter Linden. It was just due to the
extremely smaller sample sizes of SL than Winter Linden. The fixation value was
always close to zero or minimum for the hybrids. It was bigger (F=0.193) in Winter
Linden than Summer Linden (F=0.104) indicating that there was the higher influences
from inbreeding in WL than SL populations. The various private or rare alleles were
observed in each species. The largest numbers of private alleles were found in WL and
4.000
3.500
3.000
2.500
2.000
1.500
1.000
0.500
0.000
0.600
0.500
0.400
0.300
0.200
0.100
Heterozygosity
Mean
the least in SL. It was due to the variation of sample size analysed.
Na
Ne
No. Private
Alleles
He
0.000
SL
Hybrid
WL
Populations
Figure 18: Allelic patterns across populations recorded from isozyme study
48
Results
Allele Frequency for Lap
SL
Hybrid
WL
1
2
3
Frequency
Frequency
Allele Frequency for Mnr
1.000
0.800
0.600
0.400
0.200
0.000
1.000
0.800
0.600
0.400
0.200
0.000
4
SL
Hybrid
WL
1
2
Lap
Locus
Locus
SL
0.400
0.200
0.000
Hybrid
WL
2
3
1.200
1.000
0.800
0.600
0.400
0.200
0.000
SL
Hybrid
WL
4
1
Pgi-C
Locus
Locus
Allele Frequency for Pgm-D
SL
Hybrid
WL
Frequency
0.800
1.200
1.000
0.800
0.600
0.400
0.200
0.000
0.600
SL
0.400
Hybrid
0.200
WL
0.000
2
3
4
1
Pgm-D
Locus
Allele Frequency for Skdh
Allele Frequency for Adh
SL
Hybrid
WL
2
3
Locus
0.500
0.400
0.300
0.200
0.100
0.000
1
2
Pgm-A
3
4
Skdh
Locus
5
6
Frequency
Frequency
2
Pgi-B
Allele Frequency for Pgm-A
Frequency
4
Allele Frequency for Pgi-C
0.800
0.600
Frequency
Frequency
Allele Frequency for Pgi-B
1
3
Mnr
4
1.200
1.000
0.800
0.600
0.400
0.200
0.000
SL
Hybrid
WL
1
2
Adh
Locus
Figure 19: Allelic frequencies of Tilia spp. in all eight isozyme loci
3
Results
49
5.3.1 Specieswise Genetic Pattern in Tilia
5.3.1.1 Genetic Pattern of T. platyphyllos
The total numbers of alleles for SL trees were the least among all Tilia. The mean
effective number of alleles, expected heterozygosity and mean fixation index were the
highest at V1 plot for T. platyphyllos demonstrating highest genetic diversity at this
plot. There were not any SL trees at III-1. The important genetic patterns of SL are
mentioned in Appendix 17.
The pairwise comparison of Nei‟ genetic distance showed the difference between SL
from V1 and V2 was 0.135. So, it can be assumed that they are genetically closer (see
Appendix 18).
5.3.1.2 Genetic Pattern of T.× europaea
The hybrid Linden showed the largest total number of alleles. It was due to out crossing
between two species. During out crossing, hybrid Linden obtained alleles from both T.
cordata and T. platyphyllos making it the richest in allelic and genetic diversity. There
were altogether 22 alleles on average for eight gene loci. It showed the largest effective
number of alleles (Ne) and expected heterozygosity (He).
These Linden hybrids content the highest proportion of heterozygous genotypes. Few
gene loci such as Adh-A and Pgi-C did not produce variations in many cases, therefore
the PPL seems lower in hybrid Linden (T.× europaea) than pure T. cordata. The sample
size for hybrid Linden was smaller than WL, so the whole polymorphism in Adh and
Pgi-C loci could not be captured during the study. The important genetic parameters for
hybrid Linden is given at Appendix-19.
Nei genetic distance among hybrid Lindens distributed at III-1 and V1 plots was the
smallest and at III-1 and V2 plots was the farthest. The Nei‟s genetic distance between
V1 and V2 plots was bigger than III-1 and V2 plots despite of V1 and V2 plots are
situated close to each other (see Appendix 20).
50
Results
5.3.1.3 Genetic Patterns of T. cordata
There was not significant difference on total number of alleles in three studied plots
among WL however the largest numbers of alleles were found in V1 plot and the least
in the V2 plot. Similarly the allelic diversity was the highest in V1 and the least in V2.
T. cordata were polymorphic in almost all gene loci. The expected heterozygosity was
the highest at V2 plot however these differences between plots were relatively small.
The fixation values varied widely among demes of T. cordata. The highest value of
fixation was observed at III-1 plot and the least at V2 plot (Appendix 21).
The Nei genetic distance for T. cordata distributed among all three research plots were
low values. The Nei genetic distance (1978) for WL trees in between III-1 and V1 plots
were the closest despite of longer physical distance than V1 and V2 plots (Appendix 22)
5.3.2 Genetic Pattern of Tilia spp. across Study Plots
5.3.2.1 III-1 Plot
 Genetic Structure
The average number of alleles per locus was 2.9 for hybrid Linden and 2.25 for Winter
Linden at this plot. The larger number of alleles in Hybrids despite of its little number
of individuals was due to the additional contribution of particular alleles from SL tree.
Frequency
The relative frequency of alleles at various gene loci is shown at Figure 20.
1.200
1.000
0.800
0.600
0.400
0.200
0.000
WL
Hybrid
1 2 3 4 1 2 2 3 4 2 3 4 1 2 3 4 1 2 3 4 5 1 2 3 4 1 2 3
Pgi-B
Pgi-C Pgm-A
Pgm-D
Mnr
Skdh
Lap
Adh
Locus
Figure 20: Allelic frequency of Tilia spp. at III-1 plot in Hainich
The effective number of alleles was 2.04 for hybrid Linden and 1.479 for Winter
Linden. The observed heterozygosity was more than double in hybrid Linden in
51
Results
comparison to WL. Similarly the expected heterozygosity for T.× europaea was also
higher than T. cordata which was due to the out crossing between two different species.
The proportion of polymorphic loci was lesser in Hybrid. That was certainly underestimated due to the smaller sample size of hybrid Linden trees. The variation could not
be observed in Pgi-C and Adh-A loci in case of hybrid Linden whereas it was
polymorphic in the case of WL. The summary of important genetic parameters is given
in Appendix 23.
 Genetic Distance
The genetic difference between Winter Linden and Hybrid was smaller than SL and
Hybrid. The Nei genetic distance between WL and Hybrid was merely 0.116. This is
due to that the Hybrid constituted the genetic structure from Winter Linden itself and
SL. T. platyphyllos also shared many genetic constituents together with WL. Appendix24 shows the Nei genetic distances among studied populations.
5.3.2.2 V1 Plot
There were altogether 30 different alleles recorded at eight gene loci for three Tilia spp.
at this plot, yet the average number of observed alleles was just 20. Overall 21 alleles
were observed in WL, 21 alleles in Hybrid and 18 alleles in SL trees. There were few
very specific alleles to T. cordata and T. platyphyllos. The number of alleles ranged
from 2-6 in each enzyme systems. There were only 2 alleles in the case of Pgi-C locus
whereas 6 alleles in the case of Skdh-B locus. Relative frequency of distribution of
Frequency
alleles according to species is depicted in the Figure 21.
1.200
1.000
0.800
0.600
0.400
0.200
0.000
WL
Hybrid
SL
1 2 3 4 1 2 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 5 6 1 2 3 4 1 2 3
Pgi-B
Pgi- Pgm-A
C
Pgm-D
Mnr
Skdh
Lap
Adh
Locus
Figure 21: Allelic frequency of Tilia spp. at V1 plot in Hainich
52
Results
The disproportional sample sizes from all 3 species tremendously influenced the
differences in observed frequencies. As the consequences, the effective number of
alleles (Ne) varied from 1.518 in WL to 2.149 in Hybrid. The observed and expected
heterozygosity were the highest in Hybrids. Fixation value for the hybrid population
was negative. The summary of important genetic parameter is shown at Appendix 25.
The highest genetic differences were observed between WL and SL. The hybrids were
identified closer to SL than WL at this plot in genetic constitution, which might be due
to the higher genetic contribution from SL trees. Appendix 26 provides the detail
information about Nei Genetic distances for WL, SL and their hybrids at this plot.
5.3.2.3 V2 Plot
For all studied gene loci, altogether 19 alleles were recorded in WL, 22 in hybrid
Linden and 13 in SL trees at this plot. The lowest number of alleles was recorded in SL.
It was due to the small number of samples analysed, which reduced the chances of other
rare or private alleles to be observed. Distribution of relative frequency of alleles at all
Frequency
eight examined gene loci is shown at Figure 22.
1.200
1.000
0.800
0.600
0.400
0.200
0.000
WL
Hybrid
SL
2 3 4 1 2 2 3 2 3 1 2 3 4 5 1 2 3 4 5 1 2 3 4 1 2 3
Pgi-B
Pgi-C Pgm- PgmA
D
Mnr
Skdh
Lap
Adh
Locus
Figure 22: Allelic frequency of Tilia spp. at V2 plot in Hainich
T.× europaea had the highest number of alleles per locus and the largest proportion of
heterozygous genotype. The average number of alleles per locus was the minimum in T.
platyphyllos. The observed and expected heterozygosity were highest in hybrid Linden.
The fixation indixes were near to 0 for all species. Important genetic parameters are
given at Appendix-27.
53
Results
Nei Genetic distance was the longest between WL and SL. Although they were two
different species, there were many common alleles in both species at analysed gene loci
making smaller genetic distances (=0.595) than expected (=1). The Nei genetic distance
between Hybrid and WL was shorter than Hybrid and SL population (see Appendix-28).
5.4 cpDNA Genetic Patterns
5.4.1 Allelic Frequency
The ccmp3 marker showed 3 different alleles in the expected size range. There were
three different fragment size alleles namely 127 bp, 128 bp and 129 bp. The relative
frequency of the alleles (128 bp) was highest followed by the allele (127 bp). The allele
(129 bp) was very rare (p=0.024) and found only in two WL trees at the III-1 plot. The
allele 128 bp was predominantly found with Winter Linden trees whereas 127 bp was
found with SL trees. For the SL trees, the relative frequencies distribution of alleles was
0.75 for 127bp and 0.25 for 128bp fragment size. Similarly, the relative frequency for
WL trees has 0.976 for 128bp and 0.024 for 127bp.
The ccmp10 primer showed 4 different base pairs namely 103, 106, 112 and 113. The
113bp allele was predominantly occurred with WL (p>0.76) and their hybrids (p=0.542)
trees and 103bp with SL (p=0.75) and hybrids (p=0.33). As the area was dominated
with T. cordata, the most frequent allele was 113 bp. The base pair 106 was found only
in a hybrid Linden tree in V2 plot, so it was considered as the rare and private allele.
The frequency distribution of this rare or private allele was extremely small (p=0.042),
therefore it did not have great influence on genetic structure of the site.
1.200
Frequency
1.000
SL
0.800
Hybrid
0.600
0.400
WL
0.200
0.000
127
128
129
103
ccmp3
106
112
113
ccmp10
Locus
Figure 23: Relative frequency of alleles amplified with ccmp10 and ccmp3 primers
54
Results
5.4.2 Haplotypes Frequency
A haplotype is a finger print obtained from a set of alleles derived from linked loci.
For this study, five different haplotypes were found while we combined the alleles
obtained with the amplification from ccmp3 and ccmp10 primers.
The most frequent haplotype observed were haplotype 4 followed by haplotype 3 and
haplotype 1 respectively. The haplotypes 3 to 5 are specific to T. cordata and their
hybrid (T.× europaea). The haplotypes 1 and 2 are the specific haplotypes for T.
platyphyllos. Since T. cordata was largely distributed in the region, the haplotypes 4
and 3 were most frequently recorded.
The haplotypes 2 and 5 can be considered as private haplotypes due to its site specific
and infrequent occurrence. The haplotype-2 was found in T.× europaea at V2 site
whereas haplotype 5 was found only in T. cordata at III-1 site.
Table 12: Distribution of haplotypes at study sites
Haplotypes
Study plots
Number of
Observation
1
2
3
4
5
III-1
2
0
8
5
2
17
V1
13
-
9
9
-
31
V2
2
1
4
65
-
72
17
1
21
79
2
120
Total
The relative frequency of observed haplotypes can be visualised in Figure 24. The
distribution of haplotypes at V2 plot were one sided. There was the highest number of
haplotype 4 at V2 plot. Other haplotypes such a 1, 2, 3 and 5 were found in minimal
there. But there was relatively homogenous distribution of all haplotypes at V1 and
III-1 plots.
55
Results
The Relative Frequecy of Haplotypes
F
r
e
q
u
e
n
c
y
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
haplo-5
haplo-4
haplo-3
haplo-2
haplo-1
Hainich V1
Hainich V2
Hainich III1
Sites
Figure 24: Relative frequency of haplotypes in Tilia
The observed number of alleles, effective number of alleles and expected
heterozygosity were the greatest in hybrid Linden and the least in WL. The highest
number of alleles and heterozygosity in interspecies hybrids were due to contributions
of alleles from both of its parents. The expected heterozygosity was the least in WL.
The private alleles were observed only in WL and hybrid Linden. Figure 25 clearly
demonstrates the differences in allelic patterns across the population of T. cordata, T.
platyphyllos and T.× europaea. Both nuclear genetic parameters obtained from isozyme
study and the maternal information acquired from the analysis of cpDNA, showed that
5.000
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
Mean
4.000
3.000
2.000
1.000
0.000
SL
Hybrid
Heterozygosity
the Linden hybrids are genetically more variable than two other Tilia species.
Na
Ne
No. Private
Alleles
He
WL
Populations
Figure 25: Allelic patterns across populations recorded with the study of cpDNA
Discussion and Conclusions
6.
56
The isozyme study provided us strong basis to differentiate the species for selected
individual Linden trees. Mnr-A and Lap-D loci were considered primarily to identify
the species as these loci show species specific alleles in T. cordata and T. platyphyllos.
Out of 332 sampled trees, 294 trees could be assigned particular species. The remaining
38 trees were not assigned due to lack of sufficient and good quality materials.
There were 255 (87%) Winter Linden, 27 (9%) hybrid Linden and 12 (4%) Summer
Linden trees among identified 294 Linden trees. Out of 27 hybrid (T.× europaea), 16
were identified as the F2 hybrids. The presence of hybrid Lindens in all three research
plots prove that the species admixture of T. cordata and T. platyphyllos spontaneously
taking part in natural hybridisation and introgression. Therefore, the first hypothesis of
the study articulating that species admixture of T. cordata and T. platyphyllos does not
lead to species hybrids in natural stands, is rejected.
A survey conducted by other group tried to find out the species differentiation based on
morphological traits. Both morphological and genetic methods of species identification
produced 39.2% identical results and 29.4% partially identical results. The partially
identical means the identification of pure species as hybrid or vice versa. The
morphological identification between pure species and their hybrid is more difficult due
to the intermediary characteristics shown by hybrids. Due to the overlapping and
continuous characteristics, the morphological identification has been difficult in
differentiating species. There is not any distinct point to separate species grouping. The
problems for morphological identification aggravated more when only few
morphological traits are considered. Whereas the genetic traits are always discrete,
discriminating and not influenced by genetic-environment interaction.
The maternally inherited chloroplast DNA information was compared with nuclear
information obtained through isozyme study. The cpDNA information was variable
according to species hence can be differentiated in Tilia spp. All five cpDNA
haplotypes were rightly grouped according to species identified from isozyme study.
CpDNA haplotypes of 117 samples out of 120 were rightly assigned to species group.
The haplotypes 1 and 2 were the specific to T. platyphyllos and haplotypes 3, 4, and 5
were specific to T. cordata. It shows that the cpDNA haplotypes were differentiated
57
according to species in Tilia therefore the hypothesis such as „no evidence for species
differentiation in cpDNA haplotypes‟ is rejected.
There was both directional gene flow during hybridisation of T. cordata and T.
platyphyllos. Both T. cordata and T. platyphyllos have acted as pollen and seed parents.
It was confirmed by comparing nuclear and chloroplast information. The maternally
inherited cpDNA haplotypes were successfully grouped separately for hybrid Linden
(see Table 8) indicating that the effective gene flow occurs in both directions.
Irrespective of their numbers, both T. cordata and T. platyphyllos contributed pollens to
produce their putative hybrids. On this background, the hypothesis of uni directional
gene flow towards less frequent species during hybridisation is rejected.
All the sampled trees have been distributed in smaller areas. There were approximately
5-15 meters distances between the sampled Tilia trees. This proximity of the trees might
have greatly influenced the genetic structure of the species. Further the sample sizes of
all three Tilia spp. were extremely different to each others which have also great
influence on genetic patterns.
The gene diversity of hybrid Linden (T. × europaea) is the highest among all three
species. It has the highest observed and expected heterozygosity. Consequently, the
fixation index for Hybrid is found near to zero or negative. Since the gene diversity and
genetic variation is the highest in T.× europaea, they are considered superior to either of
pure Tilia species. The fixation index (F) for all three Tilia spp. are either minimal or
negative. It clearly indicates that the absence or minimal cases of inbreeding. This is
taken as the beneficial situation from the management perspective since the case of
inbreeding depression is not the problem there in Tilia.
The precise identification of species in Tilia can be done on the basis of genetic
markers. There is the availability of species specific alleles/variants which can be
rightly used in isozyme study to identify the species.
The number of identified SL is quite small in comparison to WL and hybrid Linden. It
indicates about the high risk of losing T. platyphyllos trees and their particular
genotypes in future. The situation supports the need of urgent action to be taken to
conserve the species.
Summary
7.
58
Summary
There are around 50 genera under the Tiliaceae family. Tilia is one out of them. Genus
Tilia includes 35 to 50 different species. The species are distributed in Europe, America
and Asia.
Among these species, the study concentrates only in three species of Tilia such as T.
cordata, T. platyphyllos and T.× europaea. These species are mainly distributed all over
Europe. T. cordata is widely distributed in Europe whereas T. platyphyllos has the
distribution range in South and Central Europe. The distribution of T. platyphyllos is
quite limited in comparison to T. cordata. T. × europaea are distributed naturally only in
those areas where both of T. cordata and T. platyphyllos grow sympatrically.
Tilia species are considered as European noble tree species. They are generally grown
in association with other species but they never constitute pure stands in larger areas.
The species are important from the ecological, cultural and environmental perspectives;
however they are neglected from the economic utilization.
T. cordata, T. platyphyllos and T.× europaea are similar in their outer appearance;
however they are different in many morphological traits. Earlier studies about these
species tried to distinguish the species based on morphological traits. Many
morphological traits are found different among these species. The mosaic of
characteristics of leaves, inflorescences, fruits and pollen has been applied in identifying
the species. But the precise identification of the species in Tilia is difficult and
misleading on the basis of morphological traits alone.
The morphological traits are highly influenced with local environment. The same
materials collected from different parts of the tree and in different days might have
different characteristics which are very difficult to compare and to take the decision
about species identification. Besides, during the winter, it is almost impossible to get
these materials for the identification purposes.
To overcome these problems and correct identification of the species in Tilia, the
genetic approach can be rightly applied. In these contexts, we tried to distinguish the
species with genetic analysis for 294 out of 332 Tilia trees in Hainich National Park,
Western Thuringia, Germany. Besides, we tried to access the genetic patterns of these
Summary
59
Tilia trees. The samples were collected from 3 plots established by Albrecht von Haller
Institute of Plant Sciences at Hainich National park.
To identify precisely the species in Tilia, the genetic markers such as isozyme and
chloroplast microsatellite were applied. For the isozyme analysis, overall 8 gene loci
from 7 enzyme system were studied under three different running systems. GOT and
PGI enzymes from Ashton and Braden, LAP, MNR and SKDH from Histidine-Citrate
and PGM and ADH from Tris-Citrate were applied. Among these three running systems,
Histidine-Citrate was most efficient to analyse the species. MNR and LAP were vital to
discriminate the species in Tilia since MNR and LAP used to produce non overlapping
species specific alleles for T. cordata and T. platyphyllos. Additionally PGI, PGM and
SKDH also used to produce species specific variants for T. platyphyllos only. MNR and
LAP gene loci mainly considered while determining the species.
The analysis of cpDNA was held to get additional information regarding the intra and
inter species differentiation. Polymerase Chain Reaction was held in Peltier
Thermocycler using ccmp3 and ccmp10. The PCR products were scanned in ABI
Genetic Analyser 3100. With the gene scanning and genotyping, five different
haplotypes were formed by combining linked size fragments obtained from ccmp10 and
ccmp3. The ccmp10 primer produced 3 alleles and ccmp3 produced 4 alleles in the
expected size ranges.
The haplotypes 1 and 2 were observed only with Summer Linden and hybrid Linden
trees. These haplotypes confirmed the genetic contribution from SL tree. On the other
side, haplotypes 3, 4 and 5 were recorded mainly from Winter Linden and Hybrids.
Both directional pollen flows was occurred during hybridization. Both T. cordata and T.
platyphyllos were contributing pollens to cross fertilisation to produce hybrids. The
fragment size 128 bp and 129 bp could be taken as an allele at least contributed from the
WL parent. In contrast to this logic, 1 tree from haplotype 3 and 2 from haplotype 4
were identified as SL trees. These might be due to some externalities or due to the
occurrence of back crossing for longer period.
Precise identification of species could be made on total 294 trees out of 332 samples.
Among the identified species, 255 were Winter Linden trees, 27 were Hybrid and 12
Summary
60
were SL trees. Out of 27 hybrid Linden trees, 16 were identified as the F2 generation
hybrid Linden trees.
The genetic identification of the Linden trees did not match perfectly with
morphological identification. In morphological identification, there was the larger
number of SL and hybrids than genetically identified. The morphological traits are
continuous, overlapping and highly influenced by locality factors where as genetic traits
are discriminating, non-overlapping and not effected by environment. Morphological
traits are continuous traits, therefore getting the cut point is always difficult while
identifying species, but genetic traits are discrete hence undoubtedly identified the
species on the basis of availability or absence of certain genetic trait or genotype.
Hybrid Lindens are the most genetically diversified (He=0.473) species among three
analysed species of Tilia. It is taken as normal since it acquires genetic contribution
from both Tilia spp. The gene diversity is higher in T. platyphyllos (He=0.411) than T.
cordata (He=0.28). There is minimal or negative Fixation index (F) in Tilia species at
Hainich, which describes it as the predominantly out crossing species.
The SL trees are very rare in the region. Therefore, the genetic conservation of this
species should be initiated immediately.
61
References
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69
Appendixes
Appendixes
Appendix 1: Recipe for preparation of homogenization buffer pH 7.5
(With Rinder Serum for 100 ml)
 Tris
 Saccharose
 Titriplex III or EDTA
(Ethylene diamine tetraacetic acid.Na2-Salt)
C10H14N2O8.Na2.2 H2O
1.600 g
10.00 g
0.15 g
 PVP (Polyvinylpyrrolidone)
3.00 g
 Rinder Serum (Albumin Bovine Fraction V)
 DTT (Dithiothreitol) C4H10O2S2
0.15 g
0.375 g
The buffer pH 7.5 prepared with 1 N HCl. We can use homogenization buffer for
1-2 weeks.
Appendix 2: Recipe for preparation of gel buffer
A. Gel buffer for Histidine-Citrate pH 6.2
 Histidine- Base
10 g /l
 Maleic acid / H2O
~1.9 g/l
The prepared gel buffer should be mixed with double distilled water at the ration of
1:3 to prepare gel buffer for Histidine-Citrate.
B. Gel buffer for Tris-Citrate pH 7.4
 75% double distilled water and 25% Electrode Buffer as prepared according to
above mentioned (3B) recipe.
C. Gel buffer for Ashton and Braden pH 8.1
 H2O (double distilled)
 Tris
 Citric acid
1l
6.18 g
1.68 g
We used to maintain the pH 8.1 with addition of citric acid. To prepare gel buffer for
this system, we need to make solution by adding 10% electrode buffer from Ashton and
Braden pH 8.1 and 90% gel buffer Ashton and Braden pH 8.1.
70
Appendixes
Appendix 3: Recipe for preparation of electrode buffer
A. Electrode buffer for Histidine-Citrate pH 6.2
 Histidine- Base
 Citric acid
1l
10 g /l
~1.9 g/l
B. Electrode buffer for Tris-Citrate pH 7.4
 Tris
 Citric acid
1l
16.35 g
9.04 g
C. Electrode buffer for Ashton and Braden pH 8.1
 Boric acid
 LiOH
1l
~1.6 g/l
0.9 g
Appendix 4: Recipe for preparation of starch gel (for 30 samples)
A. Tris-Citrate





Starch
(C6H10O5)n
Saccharose (C12H22O11)
Urea
(NH2)2CO
Electrode buffer
H2O (Double distilled)
23 g
9g
1g
60 ml
170 ml
B. Histidine-Citrate




Starch
(C6H10O5)n
Sachharose (C12H22O11)
Gel buffer
H2O (Double distilled)
23 g
9g
60 ml
170 ml
C. Ashton and Braden




Starch
(C6H10O5)n
Saccharose (C12H22O11)
Electrode buffer
Gel buffer
23 g
9g
23 ml
210 ml
71
Appendixes
Procedure:
Step 1: Pour 1/3 of gel buffer + bidest water solution into Erlenmeyer flask
containing starch and other ingredients and stir.
Step 2: Heat rest 2/3 of gel solution into microwave oven for 3-5 minutes and
poured back to Erlenmeyer flask.
Step 3: Well stir the solution and cook the starch gel for 1 minute.
Step 4: Degassing with Aspirator tape.
Step 5: Pour slurry evenly into Plexiglass.
Step 6: Cool the gel for awhile in room temperature then on cooler pad for 15 m.
Appendix 5: Recipe for preparation of staining buffer
A. Alcohol Dehydrogenase
 Tris HCl buffer with pH 8.0
 Ethanol absolute*
 MTT*
[Thiazolyl Blue Bromide (C18H16BrN5S)]
 NAD solution
 PMS* solution
*Add immediate before pouring into staining tray.
50 ml
3.5 ml
10 mg
5.33 ml
1.33 ml
B. Phosphoglucomutase
 Tris HCl Buffer with pH 8.0
 a-D Glucose-1-Phosphate
(Disodium salt: E.C. No. 260-154-1)
 MTT*
Bromide
50 ml
60 mg
10 mg
 NADP (C21H29N7O17P3) solution
2.7 ml
 MgCl2 solution
1.33 ml
 PMS* solution
1.33 ml
 Glucose-6-phophatedehydrogenase solution (G-6-PDH) 100 µl
*Add immediate before pouring it into staining tray.
C. Phosphoglucose Isomerase
 Tris HCl Buffer with pH 8.0
 D-Fructose-6-Phosphate
(Barium salt: EC No. 227-914-4)
 MTT* Bromide
 MgCl2 Solution
50 ml
15 mg
10 mg
1.33 ml
72
Appendixes
5.33 ml
 PMS* solution
1.33 ml
 Glucose-6-phophatedehydrogenase solution(G-6-PDH) 100µl
D. Shikimate Dehydrogenase
 Shikimic acid
(C7H10O5)
(EC N. 205-334-2)
 MTT*
Bromide
50 ml
46.7 mg
10 mg
5.33 ml
 PMS* solution
1.33 ml
E. Menadione Reductase
 Menadione Sodium Bisulphate [C11H8O2.NaHSO3]*
(2-methyl-1, 4-naphthoquinone sodium bisulfite)
 NADH * (Disodium Salt Hydrate C21H27N7O14P2.2Na aq.)
 MTT*
Bromide
50 ml
70 mg
30 mg
10 mg
F. Leucine Amino Peptidase
 Tris maleat buffer 5.4
 L Leucine (H-Leu-ß Na:HCl)
 Fast Black K Salt [C14H12N5O4.1/2Zn-Cl4]*
E.C. No. 248-648-5
 Alanine (H-Ala-ßNa.HBr)
50 ml
30 mg
30 mg
20 mg
G. Glutamate Oxalacetate Transaminase
 GOT buffer
 Fast Blue B. B.
Where, GÖT buffer contents,
o H2O (double distilled)
o Asparagine acid
o Oxaglu
acid
o PVP (Polyvinylpyrrolidone)
o Na2.EDTA
50 ml
110 mg
500 ml
2.5 g
1.2 g
2.0 g
0.19 g
73
Appendixes
Appendix 6: The electric parameters used for isozyme electrophoresis
Enzyme
System
Ashton and
Braden
(Also called
Tris-borate)
Tris-citrate
Histidinecitrate
Voltage
(V)
400
Current
(I)
*
Duration
4 hours
*
160 mA
4 hours
≤300
70 mA
4 hours
Remarks
Initially, we turned on up to
maximum current, then we set
voltage at 400. Finally, we
maintained current to be stable at
that level.
In the beginning, we provided
maximum voltage, and then we
set C at 160 mA. After that we
allow the constant voltage at the
level when C will remain 160 mA
throughout.
Appendix 7: The protocol for running PCR for cpDNA
Steps
1.
Temperature Duration Cycles Remarks
95°C
15 m
94°C
1m
50°C
1m
72°C
1m
3.
72°C
10 m
1
4.
16°C
∞
1
2.
1
35
Activation or initial denaturation
Denaturation
Annealing
Elongation
Final extension
74
Appendixes
Appendix 8: Isozyme and cpDNA data for individual trees at III-1 plot
Tree
no.
Pgi-B
Pgi-C
Pgm-A
Pgm-D Mnr
Skdh
Lap
Adh
Spp
1
2
2
2
3
3
1
1
1
2
2
2
2
2
3
2
3
2
3
3
3
3
4
4
4
4
2
2
2
2
2
2
2
2
4
6
3
3
3
3
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
4
1
4
4
4
2
2
2
2
-1
-1
-1
-1
10
15
3
3
3
3
1
1
1
2
2
2
2
2
2
2
3
3
3
4
4
4
4
2
4
4
2
2
2
2
-1
-1
-1
-1
16
17
3
2
3
2
1
1
2
1
2
2
2
2
2
3
3
3
4
3
4
3
2
4
4
4
2
2
2
2
-1
-1
-1
-1
23
25
2
2
3
3
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
2
2
2
2
2
-1
2
-1
26
27
3
3
3
3
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
37
38
3
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
4
4
4
4
2
2
2
2
2
-1
3
-1
40
47
2
3
3
3
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
3
4
2
4
2
2
2
2
2
-1
-1
-1
-1
101
102
109
3
2
3
3
1
1
1
1
2
2
2
2
2
3
3
3
4
4
2
2
2
2
2
2
2
2
111
4
3
-1
1
-1
1
2
3
3
3
3
2
3
3
3
3
3
4
3
1
2
3
3
2
3
3
4
3
4
3
1
2
2
2
2
2
2
2
112
115
2
2
3
3
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
2
1
2
1
2
2
2
2
117
118
2
1
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
4
4
1
4
4
1
2
2
2
2
2
2
2
122
124
2
2
3
3
1
1
1
1
3
2
3
2
3
3
3
3
3
3
3
3
4
2
4
4
1
2
2
2
2
2
2
2
125
129
2
3
3
3
1
1
1
1
2
2
2
2
3
2
3
2
3
4
3
4
2
3
4
4
2
2
2
2
2
2
2
2
130
132
3
3
3
3
1
1
1
1
2
2
2
2
2
3
2
3
4
3
4
3
3
4
4
4
2
2
2
2
2
1
2
2
133
134
3
4
3
4
1
1
1
1
2
3
2
3
3
3
3
4
3
1
3
4
4
3
4
5
2
3
2
4
1
2
2
2
135
4
2
1
1
1
1
2
3
3
3
3
2
3
3
2
3
3
4
4
4
3
2
3
2
138
3
1
1
2
2
2
2
2
140
200
3
3
3
4
1
1
1
1
2
2
2
3
3
3
3
3
3
2
3
3
1
3
4
4
1
2
2
4
2
2
2
2
201
202
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
4
3
2
4
2
2
2
2
2
2
2
2
2
203
204
2
3
3
4
1
1
1
1
3
3
3
4
3
2
3
3
3
1
3
3
3
4
4
4
2
2
2
2
2
2
2
2
103A
103B
3
3
3
3
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
2
2
2
2
2
2
2
2
134E
3
3
1
1
2
2
3
3
3
3
4
4
2
2
2
2
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
H
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
H
H
WL
WL
H
WL
WL
WL
H
WL
WL
WL
ccmp
10
ccmp3
113
128
113
112
128
128
112
112
112
128
128
128
113
128
103
129
112
128
103
129
113
128
103
112
127
128
112
103
113
128
127
128
113
128
75
Appendixes
Appendix 9: The isozyme and cpDNA data for individual trees at V1 plot
Tree
no.
129
130
131
Pgi-B
Pgi-C
Pgm-A
Pgm-D
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
138
3
3
4
3
1
1
1
2
2
2
3
2
2
3
2
3
144
145
3
2
3
3
1
1
2
2
2
2
2
2
3
2
148
152
153
-1
3
3
-1
3
3
-1
1
1
-1
1
1
2
3
2
3
3
2
166
168
2
2
3
3
1
1
1
2
2
2
169
173
2
2
3
2
1
1
2
1
174
175
-1
-1
-1
-1
-1
-1
176
177
2
-1
2
-1
178
180
3
-1
182
183
Mnr
3
3
2
4
3
2
3
3
3
3
3
3
2
2
2
2
-1
-1
1
-1
3
-1
3
2
184
185
Skdh
4
4
4
4
4
1
3
4
4
3
3
3
4
3
2
3
3
3
-1
3
3
-1
3
3
2
2
3
3
3
3
2
2
2
2
3
3
2
2
2
2
3
-1
1
-1
2
2
2
2
1
-1
2
-1
2
-1
3
2
1
1
1
1
2
2
2
2
1
1
186
187
2
3
2
3
188
195
2
3
197
198
Lap
2
2
2
2
2
4
2
4
4
3
2
4
3
3
3
3
3
-1
3
-1
2
-1
2
2
1
1
1
1
3
3
3
2
199
200
Adh
1
-1
2
-1
2
2
2
2
2
2
1
2
2
1
2
3
2
4
3
4
-1
2
2
-1
2
2
2
2
2
3
2
2
2
1
4
1
1
2
2
2
2
2
2
2
3
4
1
3
1
3
2
2
2
2
2
-1
2
-1
3
3
3
3
3
3
4
4
2
-1
2
-1
-1
-1
-1
-1
3
-1
3
3
3
3
3
3
4
4
2
-1
2
-1
1
-1
1
-1
3
-1
3
-1
3
-1
3
-1
3
-1
3
-1
1
-1
2
-1
1
-1
2
-1
2
2
2
3
3
3
3
3
3
4
3
3
4
3
2
2
2
2
2
1
2
2
2
2
2
2
3
2
3
3
3
3
4
4
3
3
3
3
2
2
2
2
1
-1
2
-1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
3
2
3
4
2
2
2
2
1
1
2
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
4
3
2
4
4
4
1
2
2
2
1
2
2
2
3
3
1
1
1
1
2
2
2
2
2
3
3
3
3
3
4
3
3
4
4
4
2
2
2
2
2
-1
2
-1
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
4
4
4
4
2
2
2
2
2
-1
2
-1
206
207
3
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
3
2
3
3
2
2
2
2
2
1
2
2
208
209
3
3
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
3
4
3
4
2
1
2
2
2
2
2
2
210
211
2
2
2
2
1
1
1
1
2
2
2
2
2
2
3
3
3
3
4
4
3
3
4
4
2
2
2
2
-1
-1
-1
-1
213
215
3
3
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
1
1
4
4
1
1
2
2
2
2
2
2
216
219
2
2
3
3
1
1
1
1
2
2
2
2
2
3
2
3
3
3
3
4
2
4
4
4
1
2
2
2
2
2
2
2
220
221
2
3
3
3
1
1
1
1
2
2
2
2
2
2
3
2
3
3
3
4
3
2
4
4
2
2
2
2
2
2
2
2
222
223
2
3
3
3
1
1
1
1
2
2
2
2
2
2
2
3
3
3
4
4
2
3
4
4
1
1
2
2
2
2
3
2
225
226
3
3
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
1
1
4
4
2
-1
2
-1
2
2
2
2
Spp
WL
WL
H
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
×
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
ccmp ccmp
10
3
103
127
13
128
112
112
113
112
113
112
128
128
128
x
128
128
112
112
112
113
113
112
113
113
113
128
128
128
128
128
128
128
128
x
76
Appendixes
Tree
no
Pgi-B
Pgi-C
Pgm-A
Pgm-D
Mnr
Skdh
Lap
227
3
4
-1
-1
3
3
2
3
1
228
229
3
3
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
230
232
233
234
3
2
3
3
1
1
1
2
2
2
2
2
3
2
3
3
3
3
4
4
1
1
1
1
3
2
4
3
3
3
4
3
235
236
238
239
3
3
3
3
1
1
1
1
2
2
2
2
2
2
3
3
3
3
4
4
1
1
1
1
3
2
4
2
1
3
3
4
240
241
4
4
4
4
1
1
1
1
2
-1
4
-1
2
3
2
3
242
243
2
3
3
3
1
1
1
1
2
2
2
2
3
2
244
245
2
3
3
4
1
1
2
1
2
3
2
4
246
3
2
4
3
1
1
1
1
2
2
2
4
6
2
4
2
2
3
3
4
4
4
4
2
2
2
2
2
2
2
2
3
3
2
1
3
3
3
2
2
1
4
4
2
2
2
2
2
-1
2
-1
3
3
5
4
4
2
4
2
-1
-1
-1
-1
3
3
2
3
3
2
4
4
4
4
-1
-1
1
1
1
2
3
5
2
2
4
4
-1
-1
3
3
2
2
3
2
-1
-1
-1
-1
1
1
4
5
4
5
-1
3
-1
3
2
2
2
2
3
2
3
3
3
3
4
1
4
4
-1
2
-1
2
2
2
2
2
2
2
2
3
3
3
3
4
4
3
4
4
2
2
-1
3
-1
3
4
4
2
2
2
2
2
2
3
3
-1
-1
-1
-1
1
3
3
3
4
2
2
1
3
2
1
-1
4
2
2
3
2
1
-1
3
-1
-1
-1
-1
-1
-1
-1
-1
-1
3
-1
4
2
2
2
2
2
2
3
2
3
1
3
1
-1
3
2
-1
4
1
1
-1
2
2
-1
2
3
3
4
4
1
1
3
3
4
4
-1
3
-1
4
2
2
2
2
257
258
2
3
3
3
1
1
1
1
2
2
2
2
2
2
3
2
3
3
4
3
3
4
4
4
1
2
2
2
2
2
2
2
260
261
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
1
2
2
2
2
2
2
2
262
263
3
3
3
4
1
1
1
1
2
3
2
3
3
1
3
1
3
1
3
1
1
4
2
4
2
3
2
4
2
2
2
2
264
265
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
2
2
2
2
1
1
2
2
266
267
2
3
3
3
1
1
2
1
2
2
2
2
3
3
3
3
3
3
3
3
-1
-1
-1
-1
2
1
2
2
1
2
2
2
268
270
3
2
3
3
1
1
1
1
2
2
2
2
3
2
3
3
3
3
3
3
3
3
4
4
1
2
2
2
2
2
2
2
271
273
1
2
3
3
1
1
1
1
2
2
2
2
3
2
3
3
3
3
3
3
2
-1
3
-1
2
-1
2
-1
2
2
2
2
274
275
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
1
1
4
4
2
2
2
2
2
2
2
3
276
277
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
1
1
4
4
2
2
2
2
2
2
2
2
280
282
2
3
3
4
1
1
1
1
2
3
2
3
3
1
3
1
4
5
2
3
2
4
2
2
2
2
4
4
4
4
1
1
1
1
2
2
3
3
3
3
3
3
3
-1
3
2
1
4
283
284
3
-1
2
1
4
4
5
5
-1
-1
-1
-1
2
2
2
2
285
286
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
1
1
4
4
2
2
2
2
2
2
2
2
287
293
2
3
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
3
3
1
3
4
4
2
2
2
2
2
2
2
2
250
252
253
255
256
Adh
Spp ccmp
10
H
112
WL
WL
WL
WL
H
103
H
113
WL
WL
SL
103
H
103
SL
103
SL
103
WL
WL
WL
H
103
SL
103
WL
H
WL
H
103
SL
103
WL
WL
WL
WL
WL
SL
103
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
SL
103
H
103
SL
103
WL
WL 112
WL
WL
ccmp3
128
127
128
127
127
127
127
127
127
x
127
127
127
127
127
128
77
Appendixes
Tree
no.
Pgi-B
Pgi-C
Pgm-A
Pgm-D
Mnr
Skdh
Lap
294
2
3
1
1
2
2
3
3
3
296
297
2
2
3
3
1
1
1
1
2
2
2
2
3
3
3
3
3
3
298
299
2
3
3
3
1
1
1
2
2
2
2
2
3
3
3
3
304
305
2
3
3
3
1
1
1
1
2
2
2
2
3
3
306
307
-1
3
-1
3
-1
1
-1
1
-1
2
-1
2
310
506
3
-1
3
-1
1
-1
1
-1
2
-1
508
511
2
3
3
3
1
1
1
1
512
513
2
3
3
3
1
1
516
519
3
3
3
3
520
524
2
2
529
530
3
3
4
2
2
2
2
3
3
2
2
4
4
2
2
2
2
2
2
2
3
3
3
3
3
2
2
4
4
2
2
2
2
2
2
3
2
3
3
3
3
3
3
3
4
3
4
2
2
2
2
2
2
2
2
-1
3
-1
3
3
3
3
3
2
4
4
4
-1
2
-1
2
2
2
2
2
2
-1
2
3
3
3
3
3
3
3
2
3
2
5
2
-1
2
-1
2
2
2
2
2
2
2
2
3
2
3
3
3
3
3
3
3
2
4
2
2
2
2
2
1
2
2
2
1
1
2
2
2
2
-1
2
-1
3
3
3
3
3
3
4
4
4
1
2
2
2
2
2
2
2
1
1
1
1
2
2
2
2
2
3
3
3
3
3
3
3
4
1
4
4
2
2
2
2
2
2
2
2
3
3
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
3
1
3
3
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
2
2
2
2
-1
3
-1
3
3
3
3
3
1
-1
2
-1
1
2
2
2
2
2
2
2
531
532
2
3
2
3
1
1
1
1
2
2
2
2
3
2
3
3
3
3
3
3
1
1
3
3
1
2
2
2
2
2
2
2
533
535
3
2
3
2
1
1
1
1
2
2
2
2
3
2
3
2
3
3
3
3
4
1
4
4
2
1
2
2
2
2
2
2
536
XO4
1
3
3
3
1
1
1
1
2
2
2
2
2
-1
3
-1
3
3
3
3
3
2
4
2
2
2
2
2
2
2
2
2
Note: ( -1) means the unclear or absence of isozyme data
Adh
Spp ccmp ccmp3
10
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL 113 128
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
78
Appendixes
Appendix 10: Isozyme and cpDNA data for individual trees at V2 plot
Tree
no.
1
3
4
5
17
19
25
27
28
31
32
33
34
35
36
37
38
39
40
41
45
46
47
48
49
67
68
69
70
71
72
73
74
76
77
78
101
109
110
120
121
127
128
132
133
134
135
Pgi-b
3
3
3
3
3
3
2
3
2
3
3
3
3
3
3
2
2
3
3
3
2
2
3
3
2
3
3
3
3
3
3
2
3
2
2
3
3
3
3
2
2
3
2
4
2
2
3
3
3
4
4
3
3
3
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
3
3
3
3
3
3
3
4
3
3
3
3
3
3
3
4
3
3
4
Pgi-C Pgm-A
1
1
-1
-1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
-1
-1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
-1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
-1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
3
Pgm-D
3
3
-1
2
3
2
2
-1
2
3
2
2
2
2
2
3
2
3
3
2
3
3
3
2
2
3
3
3
2
2
3
3
2
2
-1
-1
3
3
3
2
2
2
2
3
2
2
-1
3
3
-1
2
3
2
3
-1
3
3
2
2
2
3
3
3
3
3
3
2
3
3
3
2
3
3
3
3
2
2
3
3
3
2
-1
-1
3
3
3
2
2
2
2
3
2
3
-1
Mnr
Skdh
3
3
1
-1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
3
3
3
3
2
4
3
2
-1
4
3
3
3
3
4
3
3
3
3
3
3
3
4
4
3
4
3
3
3
4
4
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
3
3
-1
4
4
3
3
3
4
4
3
3
4
3
3
3
3
3
4
3
1
3
3
-1
2
1
5
5
2
1
2
4
4
2
1
2
1
1
1
2
3
1
1
1
3
4
2
1
2
3
1
1
3
3
3
1
4
2
1
3
2
4
4
4
4
2
4
3
4
4
3
3
4
5
5
4
3
3
4
4
3
4
4
4
3
3
4
3
3
3
4
4
4
3
4
2
3
1
1
4
4
4
2
4
4
3
4
3
4
4
4
4
4
4
5
4
4
4
Lap
Adh
2
2
2
2
2
2
2
-1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
-1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
2
2
2
2
2
2
2
2
2
2
3
3
2
2
2
2
2
2
2
4
2
2
2
2
2
2
1
4
2
2
2
2
2
2
2
2
2
2
4
2
2
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
1
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
1
1
2
1
2
2
2
2
Spp
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
WL
WL
H
H
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
H
H
H
WL
WL
WL
WL
WL
WL
WL
H
WL
WL
WL
WL
WL
WL
WL
SL
WL
WL
H
ccm
p10
113
113
113
113
113
113
113
ccm
p3
128
128
128
128
128
128
128
X
113
113
113
113
113
113
113
113
113
113
128
128
128
128
128
128
128
128
128
128
128
113
128
113
113
113
128
128
128
106
127
112
128
103
127
79
Appendixes
Tree
no.
136
137
142
144
145
147
148
149
150
151
152
153
154
155
156
157
158
160
162
163
164
165
166
167
168
170
171
172
173
174
175
176
177
180
181
188
189
190
191
192
196
197
200
202
203
204
205
206
213
216
Pgi-b
2
2
3
2
2
2
2
2
2
3
-1
2
2
2
3
3
3
3
3
3
3
3
2
3
3
3
3
2
2
2
3
2
3
3
3
2
2
2
2
3
3
3
3
3
2
3
3
3
2
2
3
3
4
3
3
3
3
3
3
3
-1
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
3
3
3
3
2
4
4
3
3
2
2
3
3
3
3
3
3
2
3
3
3
3
3
Pgi-C Pgm-A
-1
-1
1
1
1
1
1
1
1
1
-1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
-1
-1
1
1
1
1
1
1
2
1
-1
1
1
2
1
1
1
1
1
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
2
2
2
2
2
2
2
2
2
2
-1
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
-1
-1
2
2
-1
2
2
2
2
3
2
2
2
2
2
2
2
-1
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
2
2
2
2
2
3
3
2
2
2
2
2
2
2
2
2
-1
-1
2
2
-1
2
2
Pgm-D
3
3
3
2
3
2
3
2
-1
3
2
-1
2
2
2
3
3
3
3
3
3
3
3
3
-1
2
-1
3
2
2
2
2
-1
-1
2
2
2
2
2
2
2
2
3
-1
-1
2
2
-1
3
2
3
3
3
2
3
3
3
3
-1
3
3
-1
3
3
3
3
3
3
3
3
3
3
3
3
-1
3
-1
3
3
3
3
3
-1
-1
3
2
3
3
3
3
3
3
3
-1
-1
3
3
-1
3
2
Mnr
3
3
2
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
4
3
3
3
3
3
3
3
2
2
1
1
3
3
3
3
3
2
2
4
4
4
4
4
4
3
4
4
4
4
4
4
3
3
3
3
3
3
3
3
3
4
5
4
4
3
3
3
2
2
3
3
3
3
3
-1
-1
3
-1
3
3
3
3
3
3
3
3
3
3
3
4
-1
-1
3
-1
4
3
3
3
4
3
4
Skdh
Lap
3
3
3
3
-1
-1
-1
-1
2
3
3
3
-1
-1
3
-1
-1
1
4
4
3
3
2
3
3
3
4
1
3
3
2
4
3
3
1
3
3
3
-1
-1
-1
2
-1
4
2
2
2
2
2
3
2
2
2
2
2
4
2
2
2
2
2
2
2
1
-1
2
2
2
1
-1
2
2
2
2
2
2
3
3
3
3
2
2
2
2
2
2
2
2
-1
2
2
2
2
-1
2
2
2
2
2
2
4
4
4
4
2
2
2
-1
2
3
4
2
2
2
2
2
2
2
2
-1
-1
-1
1
1
-1
2
2
2
2
2
-1
2
3
4
2
2
2
2
2
2
3
2
-1
-1
-1
2
2
-1
2
2
4
4
3
4
-1
-1
-1
-1
4
4
3
4
-1
-1
4
-1
-1
2
4
4
4
4
4
5
5
4
5
2
4
4
2
4
5
5
3
3
4
4
-1
-1
-1
4
-1
4
4
4
4
2
4
4
Adh
2
2
2
2
2
2
2
2
2
3
-1
3
2
2
2
2
2
3
2
2
2
2
2
2
3
2
2
2
2
2
2
3
2
2
-1
-1
-1
2
2
-1
-1
-1
2
-1
-1
1
1
-1
2
2
2
2
2
2
2
2
2
2
2
3
-1
3
2
2
2
2
2
3
2
2
2
2
2
2
3
2
2
2
2
2
2
3
2
2
-1
-1
-1
2
2
-1
-1
-1
2
-1
-1
2
2
-1
2
2
Spp
WL
WL
H
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
H
H
H
H
WL
WL
WL
WL
WL
SL
SL
WL
WL
WL
WL
WL
WL
H
WL
WL
WL
WL
WL
WL
WL
WL
WL
ccm
p10
ccm
p3
103
127
112
113
113
113
113
128
128
128
128
128
113
113
128
128
113
128
113
113
113
113
113
113
113
113
128
128
128
128
128
128
128
128
80
Appendixes
Tree
no.
218
219
222
223
224
225
226
227
228
232
233
238
239
241
243
245
246
247
248
249
250
251
252
253
255
256
257
258
259
260
261
262
500
Pgi-b
2
3
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
3
2
2
3
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
2
3
3
3
3
3
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Pgi-C Pgm-A
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
-1
2
2
2
2
2
2
-1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
-1
2
2
2
2
2
2
-1
2
2
Pgm-D
2
2
2
2
2
2
3
3
2
2
2
2
3
3
2
2
2
2
3
3
2
2
2
3
3
3
3
3
2
3
3
3
2
2
2
3
2
3
2
3
3
3
3
3
3
3
3
2
3
3
3
3
3
3
2
3
3
3
3
3
3
2
3
3
3
2
Mnr
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Skdh
4
3
3
4
3
3
3
3
3
4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
4
3
3
4
4
4
3
3
3
3
4
3
3
3
3
3
4
1
2
4
4
1
3
1
3
2
4
1
1
1
1
1
1
2
4
1
1
3
4
1
1
3
4
1
4
2
2
3
4
3
4
2
2
3
4
3
3
1
3
3
3
2
4
-1 -1
3
3
2
4
3
3
1
2
-1 -1
Lap
2
2
1
2
1
2
1
1
2
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
-1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
-1
2
2
2
2
2
Adh
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
3
2
2
2
2
3
2
1
Spp
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
2
3
2
2
2
2
3
2
2
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
WL
ccm
p10
113
113
113
113
113
112
112
ccm
p3
128
128
128
128
128
128
128
113
113
113
128
128
128
113
113
113
128
128
128
113
113
113
113
113
113
113
113
113
113
113
113
113
113
113
113
113
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
81
Appendixes
Appendix 11: Allelic structure of T. cordata in Hainich
Enzyme
PGI-B
PGI-C
PGM-A
PGM-D
MNR
SKDH
LAP
ADH
Allele
N
1
2
3
4
N
1
2
N
2
3
N
2
3
N
3
4
N
1
2
3
4
5
N
1
2
N
1
2
3
III-1
Counts
76
2
20
54
0
76
72
4
76
68
8
76
25
51
76
64
12
76
3
8
20
45
0
76
6
70
56
2
53
1
RF
38
0.03
0.26
0.71
0.00
38
0.95
0.05
38
0.89
0.11
38
0.33
0.67
38
0.84
0.16
38
0.04
0.11
0.26
0.59
0.00
38
0.08
0.92
28
0.04
0.95
0.02
Research plots
V1
Counts
RF
196
98
2
0.01
68
0.35
126
0.64
0
0.00
98
196
186
0.95
10
0.05
204
102
201
0.99
3
0.01
196
98
41
0.21
155
206
190
16
200
25
24
52
98
1
192
20
172
182
15
160
7
0.79
103
0.92
0.08
100
0.13
0.12
0.26
0.49
0.01
96
0.10
0.90
91
0.08
0.88
0.04
V2
Counts
226
0
70
155
1
222
214
8
214
214
0
214
87
127
224
186
38
202
31
36
57
78
0
210
12
198
210
8
187
15
Average
RF
113
0.00
0.31
0.69
0.00
111
0.96
0.04
107
1.00
0.00
107
0.41
Counts
166
1.33
52.67
111.67
0.33
164.67
157.33
7.33
164.67
161.00
3.67
162.00
51.00
RF
83
0.01
0.31
0.68
0.00
82.33
0.95
0.05
82.33
0.96
0.04
81.00
0.31
0.59
112
0.83
0.17
101
0.15
0.18
0.28
0.39
0.00
105
0.06
0.94
105
0.04
0.89
0.07
111.00
168.67
146.67
22.00
159.33
19.67
22.67
43.00
73.67
0.33
159.33
12.67
146.67
149.33
8.33
133.33
7.67
0.69
84.33
0.86
0.14
79.67
0.11
0.13
0.27
0.49
0.00
79.67
0.08
0.92
74.67
0.05
0.91
0.04
82
Appendixes
Appendix 12: Allelic structure of T.× europaea in Hainich
Enzyme
PGI-B
PGI-C
PGM-A
PGM-D
MNR
SKDH
LAP
ADH
Allele
N
1
2
3
4
N
1
N
2
3
4
N
2
3
4
N
1
2
3
4
N
1
2
3
4
5
6
N
1
2
3
4
N
2
3
III-1
Counts
10
1
0
3
6
8
8
10
3
6
1
10
1
8
1
10
2
3
4
1
10
0
1
2
6
1
0
10
1
5
1
3
10
10
0
RF
5
0.10
0.00
0.30
0.60
4
1.00
5
0.30
0.60
0.10
5
0.10
0.80
0.10
5
0.20
0.30
0.40
0.10
5
0.00
0.10
0.20
0.60
0.10
0.00
5
0.10
0.50
0.10
0.30
5
1.00
0.00
Research plots
V1
Counts
RF
16
8
0
0.00
0
0.00
7
0.44
9
0.56
14
7
14
1.00
18
9
7
0.39
9
0.50
2
0.11
16
8
4
0.25
10
0.63
2
0.13
18
9
4
0.22
8
0.44
5
0.28
1
0.06
16
8
1
0.06
0
0.00
5
0.31
6
0.38
3
0.19
1
0.06
10
5
0
0.00
5
0.50
0
0.00
5
0.50
12
6
12
1.00
0
0.00
V2
Counts
26
0
0
14
12
22
22
26
16
10
0
16
4
12
0
20
3
7
4
6
24
4
0
9
4
7
0
26
1
10
7
8
24
20
4
Average
RF
13
0
0.00
0.54
0.46
11
1.00
13
0.62
0.38
0.00
8
0.25
0.75
0.00
10
0.15
0.35
0.20
0.30
12
0.17
0.00
0.38
0.17
0.29
0.00
13
0.04
0.38
0.27
0.31
12
0.83
0.17
Counts
17.33
0.33
0.00
8.00
9.00
14.67
14.67
18.00
8.67
8.33
1.00
14.00
3.00
10.00
1.00
16.00
3.00
6.00
4.33
2.66
16.67
1.67
0.33
5.33
5.33
3.67
0.33
15.33
0.67
6.67
2.67
5.33
15.33
14.00
1.33
RF
8.66
0.03
0.00
0.43
0.54
7.33
1.00
9.00
0.43
0.49
0.07
7.00
0.20
0.73
0.08
8.00
0.19
0.36
0.29
0.16
8.33
0.08
0.03
0.30
0.38
0.19
0.02
7.67
0.05
0.46
0.12
0.37
7.67
0.94
0.06
83
Appendixes
Appendix 13: Allelic structure of T. platyphyllos in Hainich
Enzyme
PGI-B
PGI-C
PGM-A
PGM-D
MNR
SKDH
LAP
ADH
Alleles
N
3
4
N
1
N
2
3
4
N
1
2
3
N
1
2
N
2
3
4
5
N
3
4
N
2
III-1
Counts RF
-----------------------------------------------------
Research plots
V1
V2
Counts
RF
Counts
16
8
6
5
0.31
2
11
0.69
4
14
7
6
14
1.00
6
16
8
6
6
0.38
2
8
0.50
4
2
0.13
0
14
7
2
7
0.50
0
2
0.14
0
5
0.36
2
14
7
6
8
0.57
2
6
0.43
4
18
9
6
1
0.06
0
4
0.22
3
9
0.50
0
4
0.22
3
14
7
6
8
0.57
2
6
0.43
4
16
8
6
16
1.00
6
No T. platyphyllos tree are grown in III-1 plot.
Average
RF
3
0.33
0.67
3
1.00
3
0.33
0.67
0
1
0
0.00
1.00
3
0.33
0.67
3
0.00
0.50
0.00
0.50
3
0.33
0.67
3
1.00
Counts
11
3.5
7.50
10
1.00
11
4.00
6.00
1.00
8
3.50
1.00
3.50
10
5.00
5.00
12
0.50
3.50
4.50
3.50
10
5.00
5.00
11
11
RF
5.50
0.32
0.68
5
1.00
5.50
0.36
0.59
0.07
4
0.25
0.07
0.68
5
0.45
0.56
6
0.03
0.39
0.25
0.39
5
0.45
0.55
5.5
1.00
84
Appendixes
Appendix 14: Genotypic structure of T. cordata in Hainich
Genotypes
PGI-B
1x2
1x3
2x2
2x3
3x3
3x4
PGI-C
1x1
1x2
PGM-A
2x2
2x3
3x3
PGM-D
2x2
2x3
3x3
MNR
2x3
3x3
3x4
4x4
SKDH
1x1
1x2
1x3
1x4
2x2
2x3
2x4
3x3
3x4
3x5
4x4
LAP
1x1
1x2
2x2
2x3
ADH
1x1
1x2
1x3
2x2
2x3
3x3
III-1
Counts
RF
Research plots
V1
Counts RF
V2
Counts
RF
Average
counts
Average
RF
1
1
1
17
18
0
0.03
0.03
0.03
0.45
0.47
0.00
0
2
12
44
40
0
0.00
0.02
0.12
0.45
0.41
0.00
0
0
6
58
48
1
0.00
0.00
0.05
0.51
0.42
0.01
0.33
1.00
6.33
39.67
35.33
0.33
0.01
0.02
0.07
0.47
0.44
0.00
34
4
0.89
0.11
88
10
0.90
0.10
103
8
0.93
0.07
75.00
7.33
0.91
0.09
34
0
4
0.89
0.00
0.11
100
1
1
0.98
0.01
0.01
107
0
0
1.00
0.00
0.00
80.33
0.33
1.67
0.96
0.00
0.04
5
15
18
0.13
0.39
0.47
8
25
65
0.08
0.26
0.66
25
37
45
0.23
0.35
0.42
12.67
25.67
42.67
0.15
0.33
0.52
30
4
4
0
0.79
0.11
0.11
0.00
0
87
16
0
0.00
0.84
0.16
0.00
0
76
34
2
0.00
0.68
0.30
0.02
10.00
55.67
18.00
0.67
0.26
0.54
0.19
0.01
0
0
0
3
2
0
4
7
6
0
16
0.00
0.00
0.00
0.08
0.05
0.00
0.11
0.18
0.16
0.00
0.42
2
2
3
16
3
3
13
10
25
1
22
0.02
0.02
0.03
0.16
0.03
0.03
0.13
0.10
0.25
0.01
0.22
5
5
10
6
5
5
16
9
24
0
16
0.05
0.05
0.10
0.06
0.05
0.05
0.16
0.09
0.24
0.00
0.16
2.33
2.33
4.33
8.33
3.33
2.67
11.00
8.67
18.33
0.33
18.00
0.02
0.02
0.04
0.10
0.04
0.03
0.13
0.12
0.22
0.00
0.27
1
4
33
0
0.03
0.11
0.87
0.00
1
0
18
77
0.01
0.00
0.19
0.80
0
12
0
93
0.00
0.11
0.00
0.89
0.67
5.33
17.00
56.67
0.01
0.07
0.35
0.56
0
2
0
25
1
0
0.00
0.07
0.00
0.89
0.04
0.00
1
11
2
72
5
0
0.01
0.12
0.02
0.79
0.05
0.00
0
8
0
89
1
7
0.00
0.08
0.00
0.85
0.01
0.07
0.33
7.00
0.67
62.00
2.33
2.33
0.00
0.09
0.01
0.84
0.03
0.02
85
Appendixes
Appendix 15: Genotypic structure of T.× europaea in Hainich
Genotype
PGI-B
1x4
3x3
3x4
4x4
PGI-C
1x1
PGM-A
2x2
2x3
3x3
3x4
PGM-D
2x2
2x3
3x3
3x4
MNR
1x1
1x2
1x3
1x4
1x5
2x2
2x3
2x4
3x4
SKDH
1x1
1x4
2x4
3x3
3x4
3x5
4x4
4x5
4x6
5x5
LAP
1x2
1x4
2x2
2x3
2x4
3x4
4x4
ADH
2x2
3x3
III-1
Counts
1
0
3
1
RF
0.20
0.00
0.60
0.20
Research plots
V1
Counts
RF
0
0.00
0
0.00
7
0.88
1
0.13
0.00
0.08
0.92
0.00
Average
Counts
0.33
0.33
7.33
0.67
Average
RF
0.07
0.03
0.80
0.11
V2
Counts
RF
0
1
12
0
4
1.00
7
1.00
11
1.00
7.33
1.00
0
3
1
1
0.00
0.60
0.20
0.20
1
5
1
2
0.11
0.56
0.11
0.22
4
8
1
0
0.31
0.62
0.08
0.00
1.67
5.33
1.00
1.00
0.14
0.59
0.13
0.14
0
1
3
1
0.00
0.20
0.60
0.20
1
2
3
2
0.13
0.25
0.38
0.25
1
2
5
0
0.13
0.25
0.63
0.00
0.67
1.67
3.67
1.00
0.08
0.23
0.53
0.15
0
0
1
1
0
3
0
0
0
0.00
0.00
0.20
0.20
0.00
0.60
0.00
0.00
0.00
1
2
0
0
0
1
4
0
1
0.11
0.22
0.00
0.00
0.00
0.11
0.44
0.00
0.11
0
1
0
1
1
1
2
2
2
0.00
0.10
0.00
0.10
0.10
0.10
0.20
0.20
0.20
0.33
1.00
0.33
0.67
0.33
1.67
2.00
0.67
1.00
0.04
0.11
0.07
0.10
0.03
0.27
0.21
0.07
0.10
0
0
1
0
1
1
2
0
0
0
0.00
0.00
0.20
0.00
0.20
0.20
0.40
0.00
0.00
0.00
0
1
0
0
3
2
0
1
1
0
0.00
0.13
0.00
0.00
0.38
0.25
0.00
0.13
0.13
0.00
2
0
0
2
3
2
0
1
0
2
0.17
0.00
0.00
0.17
0.25
0.17
0.00
0.08
0.00
0.17
0.67
0.33
0.33
0.67
2.33
1.67
0.67
0.67
0.33
0.67
0.06
0.04
0.07
0.06
0.28
0.21
0.13
0.07
0.04
0.06
1
0
1
0
2
1
0
0.20
0.00
0.20
0.00
0.40
0.20
0.00
0
0
0
1
3
0
1
0.00
0.00
0.00
0.20
0.60
0.00
0.20
0
1
2
3
3
4
0
0.00
0.08
0.15
0.23
0.23
0.31
0.00
0.33
0.33
1.00
1.33
2.67
1.67
0.33
0.07
0.03
0.12
0.14
0.41
0.17
0.07
5
0
1.00
0.00
6
0
1.00
0.00
10
2
0.83
0.17
7.00
0.67
0.94
0.06
86
Appendixes
Appendix 16: Genotypic structure of T. platyphyllos in Hainich
Genotypes
PGI-B
3x4
4x4
PGI-C
1x1
PGM-A
2x2
2x3
2x4
3x3
3x4
PGM-D
1x1
1x3
2x2
2x3
3x3
3x4
MNR
1x1
1x2
1x4
1x5
2x2
2x3
2x4
3x4
SKDH
2x4
3x3
3x4
3x5
4x5
5x5
LAP
3x3
3x4
4x4
ADH
2x2
Research plots
V1
III-1
V2
Average
counts
Average
RF
3.50
2.00
0.65
0.35
Counts
---
RF
---
Counts
5
3
RF
0.23
0.38
Counts
2
1
RF
0.67
0.33
--
--
8
1.00
3
1.00
5.50
1.00
------
------
1
3
1
2
1
0.13
0.38
0.13
0.25
0.13
0
2
0
1
0
0.00
0.66
0.00
0.33
0.00
0.50
2.50
0.50
1.50
0.50
0.06
0.52
0.06
0.29
0.06
-------
-------
3
1
1
0
2
0
0.43
0.14
0.14
0.00
0.29
0.00
0
0
0
0
1
0
0.00
0.00
0.00
0.00
1.00
0.00
1.50
0.50
0.50
0.00
1.50
0.00
0.21
0.07
0.07
0.00
0.64
0.00
---------
---------
3
2
0
0
2
0
0
0
0.43
0.29
0.00
0.00
0.29
0.00
0.00
0.00
0
1
1
1
1
2
2
2
0.00
0.10
0.10
0.10
0.10
0.20
0.20
0.20
1.50
1.50
0.50
0.50
1.50
1.00
1.00
1.00
0.21
0.19
0.05
0.05
0.19
0.10
0.10
0.10
-------
-------
1
1
2
2
2
1
0.11
0.11
0.22
0.22
0.22
0.11
0
0
0
3
0
0
0.00
0.00
0.00
1.00
0.00
0.00
0.50
0.50
1.00
2.50
1.00
0.50
0.06
0.06
0.11
0.61
0.11
0.06
----
----
1
6
0
0.14
0.86
0.00
1
0
2
0.33
0.00
0.66
1.00
3.00
1.00
0.24
0.43
0.33
--
--
8
1.00
3
1.00
5.50
1.00
87
Appendixes
Appendix 17: Genetic parameters for T. platyphyllos in Hainich
S.N.
Plots
Mean of all observed gene loci
No. of
Alleles
A/L
PPL
Ne
Ho
He
F
1.
III-1
0
0
0
0
0
0
0
2.
V1
18
2.250
0.750
1.936
0.386
0.407
0.011
3.
V2
13
1.625
0.625
1.525
0.292
0.285
0.000
15.5
1.938
1.731
0.687
0.339
0.346
0.006
Average
Appendix 18: Pairwise population matrix of Nei genetic distance for
T. platyphyllos
Populations
V1
V1
0.000
V2
0.135
V2
0.000
Appendix 19: Genetic parameters for T. × europaea in Hainich
S.N.
Plots
No. of
alleles
A/L
PPL
Ne
Ho
He
F
1.
III-1
23
2.875
0.750
2.044
0.550
0.418
-0.309
2.
V1
21
2.625
0.750
2.142
0.566
0.438
-0.299
3.
V2
22
2.750
0.875
2.323
0.504
0.472
0.006
22
2.750
0.79
2.170
0.540
0.442
-0.190
Average
88
Appendixes
T.× europaea.
Populations
III-1
V1
III-1
0.000
V1
0.032
0.000
V2
0.086
0.051
V2
0.000
Appendix 21: Genetic parameters for T. cordata in Hainich
S.N.
Plots
No. of
alleles
A/L
PPL
Ne
Ho
He
F
1.
III-1
20
2.500
1.000
1.484
0.203
0.284
0.328
2.
V1
21
2.625
0.875
1.518
0.251
0.267
0.111
3.
V2
19
2.375
1.000
1.632
0.262
0.286
0.081
20
2.500
0.958
1.545
0.239
0.279
0.177
Average
T. cordata
Populations
III-1
V1
III-1
0.000
V1
0.008
0.000
V2
0.010
0.011
V2
0.000
89
Appendixes
Appendix 23: Various genetic parameters for Tilia spp. at III-1
S.N.
Species
No. of
Alleles
A/L
PPL
Ne
Ho
He
F
1.
WL
18
2.25
1.0
1.479
0.207
0.280
0.264
2.
Hybrid
23
2.86
.75
2.044
0.55
0.418
-0.309
20.5
2.688
0.875
1.762
0.379
0.349
0.018
Average
Appendix 24: Pairwise population matrix of Nei genetic distance at III-1
Populations
Winter Linden
Winter Linden
0.000
Hybrid
0.116
Hybrid
0.000
Appendix 25: The genetic parameters for Tilia spp. at V1 in Hainich
S.N.
Species
No. of
Alleles
A/L
PPL
Ne
Ho
He
F
1.
WL
21
2.625
1.00
1.518
0.251
0.267
0.111
2.
Hybrid
21
2.625
0.75
2.142
0.566
0.438
-0.299
3.
SL
18
2.25
0.75
1.936
0.407
0.092
0.011
Average
20
2.500
0.833
1.865
0.401
0.370
-0.042
90
Appendixes
Appendix 26: Pairwise population matrix of Nei Genetic Distance for V1
Populations
Winter Linden
Hybrid
Winter Linden
0.000
Hybrid
0.243
0.000
Summer Linden
0.555
0.139
Summer Linden
0.000
Appendix 27: Genetic parameters for Tilia spp. at V2 plot in Hainich
S.N.
Species
No. of
Alleles
A/L
PPL
Ne
Ho
He
F
1.
WL
19
2.375
0.875
1.632
0.262
0.286
0.081
2.
Hybrid
22
2.750
0.875
2.323
0.504
0.472
0.006
3.
SL
13
1.625
0.625
1.525
0.292
0.285
0.000
18
0.119
2.250
1.827
0.353
0.348
0.032
Average
Appendix 28: Pairwise population matrix of Nei Genetic Distance for V1
Populations
Winter Linden
Hybrid
Winter Linden
0.000
Hybrid
0.243
0.000
Summer Linden
0.555
0.139
Summer Linden
0.000
91
Erklärung
ERKLÄRUNG
“Hiermit versichere ich gemäß § 26 Abs. 6 der Master-Prüngsordnung vom
27.08.2002, dass ich die vorliegende Arbeit selbständig verfasst und keine
anderen als die angegebenen Quellen und Hilfsmittel benutzt habe.”
……………………
Rajendra K.C.
Göttingen, den 26 January 2009
Curriculum Vitae
Personal Data
Name
Date of Birth
Place of Birth
Marital status
Sex
Nationality
Rajendra K.C.
27 March 1972
Butwal, Nepal
Married
Male
Nepalese
Email
[email protected]
Education
2006-2009
Candidate, M. Sc. Tropical and International Forestry,
Faculty of Forest Sciences and Forest Ecology, GeorgAugust University, Goettingen, Germany
1997-1998
M. A. Sociology, Trichandra
University, Kathmandu, Nepal
1993-1996
B. Sc. Forestry, Institute of Forestry, Tribhuvan
University, Pokhara, Nepal (Gold Medal)
1990-1991
Intermediate Science in Forestry, Institute of Forestry,
Tribhuvan University, Hetauda, Nepal (Gold Medal)
1979-1989
School Leaving Certificate, Kanti Secondary School,
Butwal, Nepal
College,
Tribhuvan
Occupation
1998 to present
Forest Officer, Department of Forests, Ministry of Forests
and Soil Conservation, Nepal
1997-1998
Programme Officer, District Development Committee,
Myagdi, Nepal
Awards
Mahendra Education Medal-III (1997) (Presently known
as Nepal Education Medal-III), for scholastic
achievement for B.Sc. Forestry
Foresters‟ Memorial Award (1997), Nepal Foresters
Association, Kathmandu, Nepal
Languages
Nepalese, English, Hindi, Urdu (Spoken), Deutsch
(Rudimentary)

Species Differentiation in Tilia

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