Clines, species and eucalypts:
an evolutionary perspective
James Holman
Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy
Australian School of Environmental Science, Griffith University
October 2002
Summary
Summary
Two eucalypt clines were examined using morphological, ecophysiological and
molecular analyses. The species complexes examined were an ironbark complex
(Eucalyptus melanophloia x E. whitei) and a box complex (E. brownii x E.
populnea). Both of these complexes demonstrate continuous morphological
variation across their clines. The origin of these morphological clines has
previously been interpreted as the product of secondary contact between allopatric
species. In this study, an analysis of morphological variation across the clines did
not identify an increase in trait variance in the intermediate populations, which
suggests that previous theories concerning the origin of these clines may not be
valid.
Genetic structuring in nuclear and chloroplast DNA was examined across the
clines to investigate whether the morphological clines were the product of
secondary contact between two independent evolutionary lineages, or whether the
clines represent a single evolutionary lineage that has undergone primary
differentiation. The microsatellite analyses indicated that there was little genetic
structuring across either cline, and that there were only low levels of population
differentiation. The lack of hierarchical structuring in the distribution of nuclear
genetic variation suggests that these clines are unlikely to be the product of recent
gene flow between two formerly allopatric species/populations.
A nested clade analysis of the JLA+ region of the cpDNA provides additional
evidence to reject the null hypothesis that the morphospecies classifications
represent distinct evolutionary lineages. Instead the analyses indicate that each
cline represents a single cohesion species and a single evolutionary lineage. The
phylogeographic distribution of cpDNA haplotypes is likely to have resulted from
restricted seed mediated gene flow with isolation by distance. A more cogent
explanation for the clines, based on the genetic data, is that they have arisen
through the process of continuous morphological diversification that has been
promoted by a directional selection gradient.
I
Summary
Drought experiments were conducted in the glasshouse to investigate whether
differences in physiological performance under water stress helps to explain the
maintenance of the ironbark cline. Under increasing water stress, the
morphotypes showed differences in their ability to maintain water status and
photosynthetic rates, yet there was no obvious pattern to these differences across
the cline. Physiological differences are therefore inadequate to explain the
maintenance of the ironbark cline and highlight the compensatory role that
morphological variation may play in alleviating water stress.
The value of adopting the cohesion species concept and a hypothesis-testing
framework to assess species status is demonstrated in this study. This framework
provided a statistical approach to distinguish independent evolutionary lineages
from interspecific populations and provides evidence to refute the current species
status of the species complexes studied.
Eucalypt classification is predominantly based on morphology, which results in
taxonomic classification that may not reflect genealogical relationships. This is
due to the disparity between morphological and phylogenetic relationships. I
therefore suggest that current presumptions regarding the prevalence and
importance of hybridisation within the genus may reflect taxonomic
classification. An accurate assessment of the prevalence and importance of
hybridisation requires species classification to be based on genealogical
relationships.
II
Acknowledgements
Acknowledgements
Special thanks to Jane Hughes for giving me a perfect balance between
independence and guidance. Jane, thank you for always making time for me.
You always went out of you way to ensure that you were available to help and it
never ceased to amaze me how quickly and thoroughly you could return your
comments and suggestions when asked. I am grateful that you were part of this
project.
Rod Fensham has also been instrumental in providing direction and support for
this project. For the past 6 years you have not only been a great teacher but also a
good friend. I have enjoyed working with someone who is so enthusiastic about
his work, an ethic that has put me good stead for this project and for what should
follow. Our field trips have been a lot of fun and also provided me with a great
PhD project.
Raelene, I promise I will now spend a little less time at the computer, and a little
more time with you. In hindsight those really early morning glasshouse
experiments and long days in the field weren’t so bad were they?
Thanks to the GU genetic geeks, past and present, for making the endless hours
spent in the lab a hell of a lot more pleasurable. Jing Ma, your patience,
happiness and helpfulness were invaluable. Thanks Rachel King aka “Stats guru”
for helping me get my head around some of the statistical issues and for being my
eucalypt buddy. Rod Eastwood thanks for your help with the NCA. IP
132.234.Mark.Ponniah, it was great fun whinging in cyberspace despite the
technical setbacks…Houston we have a problem!!
The Queensland Herbarium is thanked for its continued support throughout the
project. John Thompson and Tony Bean provided useful field notes and species
information.
III
Acknowledgements
Thanks to my enthusiastic field assistants Raelene Sambrook, Louise Holman,
Michael King, Bernie Rasmussan and Barry Higgins for their considerable
contributions of time and effort in sampling and physiological experiments.
Finally, I would like to thank my parents for providing help, support and
enthusiasm throughout this project. Mum thanks for your help in the glasshouse
and for the weekly delicious focaccia. Dad thanks for reading all my drafts and
for trying to understand what I was doing.
IV
Declaration
Declaration
This work has not previously been submitted for a degree or diploma at this or
any other university. To the best of my knowledge and belief, this thesis contains
no material previously published or written by another person except as
acknowledged in the text.
James Holman
V
Table of Contents
Table of Contents
LIST OF FIGURES .................................................................................................... X
LIST OF TABLES ................................................................................................. XIII
1.0
GENERAL INTRODUCTION.......................................................................1
1.1
CLINAL VARIATION .........................................................................................1
1.2
PHYLOGEOGRAPHY AND COHESION SPECIES ...................................................2
1.3
PRIMARY DIFFERENTIATION ............................................................................3
1.3.1
Selection.................................................................................................3
1.3.2
Genetic drift ...........................................................................................5
1.3.3
Gene flow...............................................................................................5
1.3.4
Clinal patterns under primary differentiation ........................................6
1.4
SECONDARY CONTACT ....................................................................................8
1.4.1
Clinal patterns under secondary contact ................................................8
1.5
CLINES THROUGH MORPHOLOGICAL PLASTICITY ..........................................10
1.6
THESIS OBJECTIVES .......................................................................................11
2.0
STUDY AREA AND SPECIES ....................................................................12
2.1
IRONBARK CLINE: E. MELANOPHLOIA X E. WHITEI COMPLEX .........................12
2.2
BOX CLINE: E. BROWNII X E. POPULNEA COMPLEX ........................................16
3.0
MORPHOLOGICAL VARIATION............................................................19
3.1
INTRODUCTION .............................................................................................19
3.2
OBJECTIVES ..................................................................................................22
3.3
METHODS......................................................................................................23
3.3.1
Sampling ..............................................................................................23
3.3.2
Measurements ......................................................................................25
3.3.2.1
Foliar morphology ...........................................................................25
3.3.2.2
Seedling Morphology: E. melanophloia & E. whitei only...............26
3.3.2.3
Fruit Morphology: E. melanophloia & E. whitei only.....................26
3.3.3
Analysis................................................................................................27
3.3.3.1
True Breeding ..................................................................................28
3.3.3.2
Trait variance ...................................................................................28
VI
Table of Contents
3.4
RESULTS .......................................................................................................29
3.4.1
3.4.1.1
Mature Leaf characteristics..............................................................29
3.4.1.2
Fruit Characteristics .........................................................................35
3.4.1.3
Seedling Leaf Characteristics ..........................................................36
3.4.1.4
True breeding ...................................................................................40
3.4.2
3.5
Box complex: E. populnea x E. brownii (north-south transect) ..........42
DISCUSSION ..................................................................................................48
3.5.1
4.0
Ironbark complex: E. melanophloia & E. whitei (east-west transect) 29
Primary differentiation vs. Secondary contact.....................................49
ECOPHYSIOLOGY......................................................................................51
4.1
INTRODUCTION .............................................................................................51
4.2
OBJECTIVES ..................................................................................................53
4.3
METHODS......................................................................................................54
4.3.1
Geology and rainfall of study area.......................................................54
4.3.2
Field experiments.................................................................................54
4.3.2.1
Soil Moisture....................................................................................56
4.3.2.2
Water relations .................................................................................56
4.3.3
4.4
Glasshouse ...........................................................................................57
4.3.3.1
Water relations .................................................................................58
4.3.3.2
Chlorophyll fluorescence .................................................................59
RESULTS .......................................................................................................60
4.4.1
Field .....................................................................................................60
4.4.2
Glasshouse ...........................................................................................65
4.5
4.4.2.1
Water Potential.................................................................................65
4.4.2.2
Chlorophyll Fluorescence ................................................................66
DISCUSSION ..................................................................................................71
4.5.1
Water relations .....................................................................................71
4.5.2
Chlorophyll Fluorescence ....................................................................71
4.5.3
Adaptation, Persistence and Distribution.............................................73
5.0
GENETIC VARIATION...............................................................................76
5.1
INTRODUCTION .............................................................................................76
5.1.1
Population structuring ..........................................................................77
VII
Table of Contents
5.1.1.1
Contemporary structuring ................................................................77
5.1.1.2
Historical structuring .......................................................................80
5.2
OBJECTIVES ..................................................................................................83
5.3
METHODS......................................................................................................84
5.3.1
Sampling ..............................................................................................84
5.3.2
DNA extraction ....................................................................................85
5.3.3
Microsatellite Protocol.........................................................................85
5.3.4
cpDNA Protocol...................................................................................87
5.3.4.1
DNA sequencing ..............................................................................87
5.3.4.2
Outgroup heteroduplex analysis using temperature gradient gel
electrophoresis (TGGE) ...................................................................................87
5.3.5
5.3.5.1
Microsatellite analysis .....................................................................89
5.3.5.2
cpDNA analysis ...............................................................................90
5.4
RESULTS .......................................................................................................92
5.4.1
E. melanophloia x E. whitei.................................................................92
5.4.1.1
Microsatellite results........................................................................92
5.4.1.2
Chloroplast results ...........................................................................97
5.4.2
5.5
Statistical analysis................................................................................89
E. populnea x E. brownii ...................................................................103
5.4.2.1
Microsatellite results......................................................................103
5.4.2.2
Chloroplast results .........................................................................108
DISCUSSION ................................................................................................115
5.5.1
Deviations from Hardy-Weinberg equilibrium..................................115
5.5.2
Pollen versus seed mediated gene flow .............................................118
5.5.3
Contemporary Population Structuring ...............................................118
5.5.4
Historical and phylogeographic interpretation...................................119
6.0
GENERAL DISCUSSION ..........................................................................122
7.0
REFERENCES.............................................................................................125
8.0
APPENDIX...................................................................................................137
8.1
DISTRIBUTION MAPS AND CLIMATIC ENVELOPE METHODOLOGY ...............137
8.1.1
Distribution Maps ..............................................................................137
8.1.2
Climatic Envelope methodology........................................................138
VIII
Table of Contents
8.2
P-VALUE FOR LINKAGE DISEQUILIBRIUM BETWEEN LOCI.............................139
8.2.1
Ironbark Complex ..............................................................................139
8.2.2
Box Complex .....................................................................................139
8.3
IRONBARK CPDNA SEQUENCES ..................................................................140
8.4
IRONBARK CPDNA HAPLOTYPE NETWORK WITH NESTING (INDEL INCLUDED)
....................................................................................................................143
8.5
RESULTS OF NESTED CLADE ANALYSIS FOR THE IRONBARK COMPLEX WITH
INDELS INCLUDED. ...............................................................................................144
8.6
IRONBARK NESTED CLADE INTERPRETATION WITH INDELS INCLUDED......145
8.7
BOXES CPDNA SEQUENCES........................................................................146
8.8
BOX CPDNA HAPLOTYPE NETWORK WITH NESTING WITH INDELS INCLUDED
....................................................................................................................151
8.9
RESULTS OF NESTED CLADE ANALYSIS FOR THE BOX COMPLEX INDELS
INCLUDED. ..............................................................................................................152
8.10
BOX NESTED CLADE INTERPRETATION WITH INDELS INCLUDED. ..............153
IX
List of Figures
List of Figures
Figure 1-1 Morphological cline parallel to environmental gradient .......................7
Figure 1-2 Trait variance across the cline formed through primary
differentiation.......................................................................................................7
Figure 1-3 Increased quantitative trait variance due to secondary contact ...........8
Figure 2-1 Ironbark species distribution .................................................................13
Figure 2-2 Clinal transition from E. whitei (left) to E. melanophloia (right)........14
Figure 2-3 MDS of annual rainfall (R) and mean annual temperature (T) for
E. melanophloia and E. whitei ................................................................................ ..15
Figure 2-4 Distribution of E. brownii and E. populnea...........................................17
Figure 2-5 Clinal transition from E. brownii (left) to E. populnea (right) ............17
Figure 2-6 MDS of annual rainfall (R) and mean annual temperature (T) for
E. brownii and E. populnea..................................................................................... ..18
Figure 3-1 Sampling sites across both species complexes.......................................24
Figure 3-2 Leaf traits measured. ..............................................................................25
Figure 3-3 Fruit traits measured ..............................................................................26
Figure 3-4 Variation in mature leaf length and width (from west to east across
the ironbark complex.........................................................................................29
Figure 3-5 Variation in apex and basal leaf angles from west to east across the
ironbark complex ...............................................................................................30
Figure 3-6 Variation in petiole length from west to east across the ironbark
complex. ..............................................................................................................30
Figure 3-7 Variation in node l and node 2 lengths from west to east across the
ironbark complex ...............................................................................................31
Figure 3-8 UPGMA dendrogram of site average foliar characteristics................32
Figure 3-9 Two-dimensional MDS plot of mature foliar characteristics. Vector
direction indicates the how the traits differentiate the morphotypes. ..........33
Figure 3-10 CV of petiole length across the study area ..........................................34
Figure 3-11 CV of N1 length across the study area ................................................35
Figure 3-12 UPGMA of site average fruit characteristics......................................35
X
List of Figures
Figure 3-13 Variation in seedling leaf length and width across the east west
transect................................................................................................................36
Figure 3-14 Variation in petiole length across the east west transect ...................37
Figure 3-15 Variation in apex and basal leaf angles across the east west transect.
..............................................................................................................................37
Figure 3-16 Variation in node l and node 2 lengths across the east west transect
..............................................................................................................................38
Figure 3-17 UPGMA of seedling site average foliar characteristics .....................38
Figure 3-18 Two-dimensional MDS plot of seedling foliar characteristics. .........39
Figure 3-19 Correlation between parent and offspring leaf width........................40
Figure 3-20 Correlation between parent and offspring leaf width to length ratio
..............................................................................................................................40
Figure 3-21 Correlation between parent and offspring basal angle......................41
Figure 3-22 Correlation between parent and offspring petiole length..................41
Figure 3-23 Variation in mature leaf length and width across the north-south
transect................................................................................................................43
Figure 3-24 Variation in apex and basal leaf angles across the north-south
transect................................................................................................................43
Figure 3-25 Variation in petiole length across the north-south transect ..............44
Figure 3-26 UPGMA of site average foliar characteristics. ...................................44
Figure 3-27 Two-dimensional MDS plot of mature foliar characteristics............45
Figure 3-28 CV of leaf W:L from north to south across the box complex ...........47
Figure 3-29 CV of leaf Dmax: L from north to south across the box complex.......47
Figure 3-30 CV of leaf apex angle from north to south across the box complex .48
Figure 4-1 Sampling sites across ironbark complex. Rain stations (aI, III, III).
..............................................................................................................................55
Figure 4-2 Average monthly rainfall at rain stations (I, II, III) across cline........55
Figure 4-3 September soil moisture content............................................................61
Figure 4-4 December soil moisture content.. ...........................................................62
Figure 4-5 Field water potentials. Box plot of predawn water potential obtained
across the study area in September. .................................................................63
XI
List of Figures
Figure 4-6 Field water potentials. Box plot of predawn water potential obtained
across the study area in December. ..................................................................64
Figure 4-7 Monthly rainfall relative to long-term average at the rain stations.. .64
Figure 4-8 Predawn water potential versus Field water capacity (FWC) ............66
Figure 4-9 Minimum fluorescence versus field water capacity.. ...........................67
Figure 4-10 Maximum fluorescence versus field water capacity...........................68
Figure 4-11 Maximum photochemical efficiency versus field water capacity......69
Figure 4-12 Daily Maximum Electron Transport Rate regression curves versus
Photosynthetic Photon Flux Density (PPFD) ..................................................70
Figure 5-1 Haplotype type network under primary differentiation and secondary
contact. ................................................................................................................83
Figure 5-2 Sampling across the ironbark complex .................................................84
Figure 5-3 Sampling across the box complex ..........................................................85
Figure 5-4 MDS plot of RST values for microsatellite data from the ironbark
complex. ..............................................................................................................95
Figure 5-5 MDS plot of FST values for microsatellite data from the ironbark
complex. ..............................................................................................................96
Figure 5-6 Spatial distribution of cpDNA haplotypes across the ironbark
complex. ..............................................................................................................99
Figure 5-7 (Left) Haplotype network for the ironbark complex. ........................101
Figure 5-8 MDS plot of RST values for microsatellite data for the box complex.
............................................................................................................................106
Figure 5-9 MDS plot of FST values for microsatellite data from the box complex.
............................................................................................................................107
Figure 5-10 Spatial distribution of cpDNA haplotypes across box complex. .....111
Figure 5-11 Haplotype network for the box complex. ..........................................113
XII
List of Tables
List of Tables
Table 3-1 Adult leaf trait dimensions for UPGMA groups. See Fig 3-2 for leaf
traits. ...................................................................................................................32
Table 3-2 Principal Axis Correlation (PCC) of tree average foliar characters in
the 2D ordination space. ....................................................................................33
Table 3-3 R2 and P-values for the regression of trait CV with distance from cline
geographic mid-point and distance from site 1, the most westerly site.........34
Table 3-4 Principal Axis Correlation (PCC) of site average foliar characters in
the 2D ordination space. ....................................................................................39
Table 3-5 Average dimensions for UPGMA groups ...............................................45
Table 3-6 Principal Axis Correlation (PCC) of tree average foliar characters in
the 2D ordination space. ....................................................................................46
Table 3-7 R2 and P-values for regression of trait CV with distance from cline
geographic mid-point and also distance from site 1, the most northern site..
..............................................................................................................................46
Table 5-1 Primer designation, type of repeat and optimised annealing
temperature ........................................................................................................86
Table 5-2 Levels of genetic variation at 5 Embra microsatellite from 7
populations across the ironbark complex. .......................................................93
Table 5-3 Weir & Cockerham (1984) estimation of FIT, FST and FIS for the
ironbark complex. ..............................................................................................94
Table 5-4 Pairwise estimates of multilocus RST and FST between populations
across the ironbark complex. ............................................................................94
Table 5-5 Analysis of molecular variance for ironbark complex. The three
regions are: west, intermediate and east..........................................................96
Table 5-6 List of JLa+ haplotypes for the ironbark complex when INDELs are
included in the analysis......................................................................................98
Table 5-7 Haplotype distribution across the ironbark complex............................98
Table 5-8 Haplotype distribution across the ironbark populations when INDEL
mutations are excluded from the analysis. ......................................................99
XIII
List of Tables
Table 5-9 Analysis of molecular variance for ironbark complex ........................100
Table 5-10 The permutational chi-squared probabilities for geographical
structure of the clades identified in Fig. 5-7 for the ironbark complex ......101
Table 5-11 Results of nested clade analysis for the ironbark complex ...............102
Table 5-12 Levels of genetic variation at 5 Embra microsatellite for the box
complex. ............................................................................................................104
Table 5-13 Weir & Cockerham (1984) estimation of FIT, FST and FIS for the box
complex. ............................................................................................................105
Table 5-14 Pairwise estimates of multilocus RST and FST between box
populations........................................................................................................105
Table 5-15 Analysis of molecular variance for the box complex. The three
regions are: northern, intermediate and southern........................................107
Table 5-16 List of JLa+ haplotypes for the box complex when INDELs were
included in the analysis....................................................................................109
Table 5-17 Distribution of cpDNA haplotypes across box populations ..............110
Table 5-18 List of revised JLa+ haplotypes for the box complex when INDELs
were excluded from the analysis.....................................................................110
Table 5-19 Analysis of cpDNA molecular variance for the box complex ...........111
Table 5-20 The exact permutation contingency test probabilities for geographical
structure of the clades identified in Fig. 5-11 for the box complex. ............113
Table 5-21 Results of nested clade analysis for the box complex ........................114
XIV