Honckenya peploides: Regional Gene Diversity
and Global Karyotype Investigations
Sigurður H. Árnason
Faculty of Life and Environmental Sciences
University of Iceland
2014
Honckenya peploides: Regional Gene
Diversity and Global Karyotype
Investigations
Sigurður H. Árnason
90 ECTS thesis submitted in partial fulfillment of a
Magister Scientiarum degree in Biology
Advisor
Kesara Anamthawat-Jónsson
Master’s program committee
Ægir Þór Þórsson
Borgþór Magnússon
Faculty of Life and Environmental Science
School of Engineering and Natural Sciences
University of Iceland
Reykjavik, May 2014
Honckenya peploides: Regional Gene Diversity and Global Karyotype Investigations
90 ECTS thesis submitted in partial fulfillment of a Magister Scientiarum degree in
Biology
Copyright © 2014 Sigurður H. Árnason
All rights reserved
Faculty of Life and Environmental Sciences
School of Engineering and Natural Sciences
University of Iceland
Askja-Sturlugata 7
101 Reykjavik, Reykjavik
Iceland
Telephone: 525 4000
Bibliographic information:
Sigurður H. Árnason, 2014, Honckenya peploides: Regional Gene Diversity and
Global Karyotype Investigations, MSc. thesis, Faculty of Life and Environmental
Sciences, University of Iceland.
Printing: Háskólaprent ehf., Fálkagata 2, 107 Reykjavík
Reykjavik, Iceland, May 2014
Abstract
Integration of classical ecological measurements with molecular, cytogenetic and
statistical analysis techniques is vital to a greater understanding of ecological and
evolutionary relationships in time and space. Such understanding is the key to reassembling and rehabilitating diversity in the face of current environmental and
climate changes. In this study, molecular and cytogenetic techniques were used to
evaluate both levels of genetic diversity and differentiation as well as karyotype
diversity in Honckenya peploides.
Populations from Surtsey were studied using Amplified Fragment Length
Polymorphisms (AFLP) and diversity measures were compared to populations from
other regions including the island Heimaey, the southern coast of Iceland, Greenland
and Denmark. Main results include: (i) Surtsey has the highest proportion of
polymorphic markers as well as a comparatively high genetic diversity and Denmark
the lowest, possibly indicating that H. peploides populations on Surtsey originate from
multiple colonization events from several source locations; (ii) the total genetic
differentiation (FST) among Surtsey and Heimaey populations was significant and less
than half of that found among mainland Iceland populations, indicating significant
gene flow within the islands; (iii) significant genetic distance was found within
Surtsey, among sites, this appears to correlate with the age of plant colonization ; iv)
most genetic variation was found within localities, possibly due to the outcrossing and
subdioecious nature of the species; and (v) there is a positive and significant
association between genetic differentiation and geographic distance at the broad scale
indicating isolation by distance has an effect on the Surtsey and Icelandic populations.
Through collaboration with investigators worldwide, seeds of H. peploides were grown for
karyotyping using the enzymatic root tip squash method. Results from Seltjarnarnes
samples show a tetraploid genome containing 68 metacentric and sub-metacentric
chromosomes, two of which contain satellites. The chromosome number found in the
current study validates some previous work conducted on mainland Iceland but still leaves
some questions regarding karyotype diversity both in Iceland as well as worldwide. Our
results coupled with a literature review point to a diverse genetic constitution for the
species, both within localities as well as worldwide. This warrants a deeper investigation
into the interplay of environmental variables and phenotypic diversity with the polyploid
nature of the species as well as its mating system.
iii
Yfirlit
Til að auka skilning á sambandi umhverfis og þróunar er nauðsynlegt að samþætta
hefbundnar umhverfismælingar við nútíma sameinda- og frumuerfðafræði og tölfræðilega
greiningatækni. Slík þekking er mikilvæg í viðhaldi og endurnýjun á breytileika á tímum
örra umhverfis- og loftslagsbreytinga. Í þessari rannsókn voru bæði sameindamerking og
frumuerfðagreining gerðar á völdum stofnum fjöruarfa, Honckenya peploides, til að
ákvarða umfang erfðabreytileika í stofnum og aðgreiningar á milli stofna, auk þess sem
breytileiki í litningagerð tegundarinnar var metinn.
AFLP aðferð var beitt á stofna frá Surtsey og breytileiki þeirra mældur og borinn
saman við stofna frá Heimaey, Stokkseyri, Garði, Grænlandi og Danmörku.
Eftirfarandi niðurstöður fengust: (i) stofnar frá Surtsey voru með mesta margleitni og
háan alhliða breytileika, en dönsku stofnarnir hins vegar með minnstan breytileika;
þetta bendir til að fjöruarfi hafi borist til Surtseyjar frá nokkrum svæðum; (ii) heildar
erfðaaðgreining (FST) milli stofna á Surtsey og Heimaey var tæplega helmingi lægri en
hjá stofnum á meginlandi Íslands sem gefur til kynna víðtækt genaflæði innan eyjanna;
(iii) marktækur erfðafræðilegur munur greindist innan Surtseyar og virðist hann hafa
fylgni við hve langt er liðið frá landnámi tegundarinnar á hverju svæði á eynni; (iv)
mestur erfðabreytileiki fannst innan svæða, hugsanlega vegna þess að tegundin er
víxlfrjóvguð og tvíbýl og (v) jákvæð og marktæk fylgni var á milli stofnaaðgreiningar
og landfræðilegrar fjarlægðar á stórum skala, sem bendir til að stofnar á Íslandi og á
eyjunum hafi lengi verið tiltölulega einangraðir.
Litningagreiningar voru gerðar á fjöruarfastofnum víðs vegar að úr heiminum, en fræ
af þeim var látið spíra. Notað var ensímaðferð við litningaeinangrun úr rótaendum til
að erfðamerkja og greina kjarnagerð. Stofn frá Seltjarnarnesi reyndist hafa fjórfalt
erfðamengi sem inniheldur 68 litninga, tveir þeirra hafa fylgihnött sem eru sæti
ríbósómgena. Niðurstöður okkar, ásamt ítarlegri könnun heimilda, benda til þess að
erfðasamsetning fjöruarfa sé afar fjölbreytt á Íslandi eins og annars staðar. Þörf er á
nákvæmari rannsókn á samspili umhverfisþátta og útlitsbreytileika við fjöllitnun og
æxlunarkerfi tegundarinnar.
iv
Acknowledgements
I would like to start out by thanking Dr. Kesara Anamthawat-Jónsson for her support
and great friendship. I am forever indebted to her for all her guidance. I would also like
to thank Dr. Ægir Þór Þórsson for his help with the initial phase of the research.
Furthermore I would like to thank Lilja Karlsdóttir for her support, great company and
good advice during the literature phase of the research. An extra special thanks goes
out to Magnea Ósk Sigrúnardóttir for all the hours of help and insight, I could not have
done it without you. Also I would like to thank parents, Árni and Guðrún and my
brother Arnar, for all their patience and support throughout the years.
v
Table of Contents
Abstract .............................................................................................................................. iii
Yfirlit ................................................................................................................................. iv
Acknowledgements ............................................................................................................. v
Table of Contents .............................................................................................................. vii
List of Figures .................................................................................................................... ix
General Introduction ........................................................................................ 13
1
1.1
1.2
Scope of Current Study ................................................................................... 13
Genetic Variation ............................................................................................ 14
1.2.1
Genetic Variation on Islands ............................................................................ 14
1.2.2
Island Biogeography and Gene Diversity ........................................................ 14
1.2.3
Dioecy and Genetic Variation .......................................................................... 15
1.2.4
Genetic Variation in Polyploids ....................................................................... 15
1.3
Quantifying Genetic Variation ........................................................................ 15
1.3.1
Amplified Fragment Length Polymorphism (AFLP) ...................................... 16
1.4
1.5
Karyotype Diversity ........................................................................................ 18
Natural Laboratories on Isolated Oceanic Islands .......................................... 18
1.5.1
Surtsey - A Natural Laboratory ....................................................................... 19
1.6
Honckenya peploides ...................................................................................... 23
1.6.1
Habitat and Distribution................................................................................... 23
1.6.2
Flower Morphology ......................................................................................... 24
1.6.3
1.6.3 Breeding System ..................................................................................... 25
1.7
Thesis Overview and Objectives .................................................................... 26
2
Spatial Genetic Structure of the Sea Sandwort ............................................... 27
2.1
2.2
Introduction ..................................................................................................... 27
Materials and Methods .................................................................................... 29
2.2.1
Study Species ................................................................................................... 29
2.2.2
Marker Choice ................................................................................................. 30
2.2.3
2.2.3 Plant Material and Collection Locations ................................................ 30
2.2.4
DNA extraction ................................................................................................ 32
2.2.5
AFLP Fingerprinting........................................................................................ 32
2.3
Data Analysis .................................................................................................. 34
2.3.1
Raw Data .......................................................................................................... 34
vii
2.3.2
Phylogenetic Relationships and Isolation by Distance ....................................... 34
2.3.3
Bayesian Clustering ......................................................................................... 34
2.3.4
Genetic Data Analysis ..................................................................................... 35
2.4
Results ............................................................................................................ 35
2.4.1
Paterns of Polymorphism and Heterozygosity ................................................ 35
2.4.2
Genetic Differentiation and Diversity ............................................................. 35
2.4.3
.Bayesian Analysis of Genetic Structure ......................................................... 36
2.4.4
Neighbor Joining Tree ..................................................................................... 38
2.4.5
Multidimensional Scaling ................................................................................ 38
2.4.6
Mantel Test - Isolation By Distance ................................................................ 39
2.5
Discussion ....................................................................................................... 42
2.5.1
Patterns of Diversity on Surtsey Versus Other Sites ...................................... 42
2.5.2
Genetic Differentiation Among Populations and Regions ............................. 43
2.5.3
Phylogeography of H. peploides in the Iceland Region ................................. 45
2.5.4
Present Research and Future Prospects ........................................................... 47
3
Karyotype Variation of Honckenya peploides Worldwide ............................ 49
3.1
3.2
Introduction..................................................................................................... 49
Materials and Methods ................................................................................... 52
3.2.1
Plant Material .................................................................................................. 52
3.2.2
Seed Stratification, Sterilization and Germination .......................................... 53
3.3
Results ............................................................................................................ 54
3.3.1
Seed Stratification, Sterilization and Germination .......................................... 54
3.3.2
Karyotype ........................................................................................................ 54
3.4
Discussion ....................................................................................................... 55
3.4.1
Significance of Chromosome Numbers ........................................................... 55
3.4.2
Future Prospects .............................................................................................. 56
4
Bibliography ................................................................................................... 59
viii
List of Figures
Figure 1: Outline of the methodology of Amplified Fragment Length Polymorphism
(AFLP) (Vos et al., 1995). (1) Total genomic DNA is digested with two
restriction enzymes (one frequent and one rare cutter). (2) During the
digestion reaction adapters with primer sites are ligated to the ends of the
fragments. (3) PCR using primers with one selective nucleotide extending
into the genome fragment is performed. This reduces the number of
fragments and improves reproducibility of final fingerprints. (4) A second
series of PCR follows using two or three selective nucleotides on each primer,
thus (5) generating a highly polymorphic DNA fingerprint. ©SHÁ ............ 17
Figure 2. Surtsey: a) West side of Surtsey, b) close up of the western crater, c) the
south coast of Surtsey, d) the east coast of Surtsey. Photo ©BM ............. 20
Figure 3: Map of the Vestmannaeyjar Archipelago ([email protected]) ........................... 21
Figure 5: Plant community on Surtsey with Leymus arenarius, Honckenya peploides
and Martensia maritima. The photo was taken July 2010 ©KAJ ............. 22
Figure 6: Honckenya peploides from Seltjarnarnes peninsula, Reykjavík, Iceland.
©SHÁ........................................................................................................ 24
Figure 7: Left figure shows Honckenya subspecies worldwide distribution (Hulten et
al., 1971). Right figure shows distribution in Iceland (floraislands.is). .... 24
Figure 8: Honckenya peploides; a) hermaphrodite flower showing large petals, b)
female flower showing smaller petals and c) fruit capsule. ©SHÁ. ......... 25
Figure 9: Schematic diagram of the dioceous pathway. ©SHÁ. ................................. 26
Figure 10: Bayesian inference clustering results for AFLP using the admixture
ancestral model in STRUCTURE software. The two genetic groups
detected (clusters I and II) are represented as pie charts (dark vs. light
blue) on the google earth maps of all sampling locations. ........................ 37
Figure 11: Un-rooted neighbor joining trees based on Nei´s genetic distances for; A)
The full data set and B) Surtsey only. Bootstrap values are shown at the
nodes (10,000 iterations). .......................................................................... 38
Figure 12: Multidimensional scale (MDS) plot based on FST values .......................... 39
Figure 13: Plot of pairwise estimates of FST versus geographic distance between; a) all
samples of Honckenya peploides, b) mainland Iceland, Surtsey, Heimaey
and Greenland, c) Mainland Iceland, Surtsey, Heimaey and Denmark .... 40
Figure 14: Plot of pairwise estimates of Nei´s genetic distance (Top 2) and FST
(Bottom 2) versus geographic distance between samples of Honckenya
peploides from; a & c) Mainland Iceland, Surtsey and Heimaey, b & d)
Surtsey only............................................................................................... 41
Figure 16: Honckenya peploides chromosomes stained with DAPI taken at 1000x
magnification (a), with drawn outlines (b) and karyotypically arranged.
Arrows indicate locations of satellites ...................................................... 54
ix
List of Tables
Table 1 Sampling locations, GPS coordinates, dates, ID’s and number of samples. ... 31
Table 2: Primers used in study (Applied Biosystems). ................................................. 33
Table 3 Genetic diversity in sampling locations of Honckenya peploides. ................. 36
Table 4: Nei´s genetic distances are displayed on the upper diagonal and FST values
on lower diagonal. *non-significant values .............................................. 36
Table 5: Analysis of Molecular Variance. .................................................................... 42
Table 6: All published chromosome numbers for Honckenya peploides. .................... 50
xi
1 General Introduction
1.1
Scope of Current Study
The following study is designed to test levels of genetic diversity and genetic
differentiation in populations of the dioecious, tetraploid beach plant Honckenya peploides
on the recently colonized, completely barren volcanic island of Surtsey and to compare
these patterns to those found in older populations from previously colonized locations. A
preliminary investigation into karyotype diversity was also started.
The focal location of the study is the island of Surtsey, a well protected UNESCO
World Heritage Site devoid of all human influence. The island provides the perfect
natural laboratory for a study of this kind as it is relatively young (50 years), plant
colonization and succession have been extensively recorded there since its inception
and the study organism, H. peploides was one of the first plant colonizers to arrive
(Friðriksson, 1964). Furthermore, H. peploides is the most abundant plant found on the
island and is a key facilitator of plant and animal colonization, providing substrate
binding for plants as well as nesting material for birds (Sigurðsson and Magnússon,
2009). Studies such as this which catalog and characterize existing systems and
identify the events that are critical in their evolution will play a pivotal role in their
conservation and future restoration. This will be of ever increasing importance as
anthropogenic habitat destruction and fragmentation along with global climate change
is currently causing a biodiversity crisis of a scale of magnitude not seen since the
Cretaceous, what some are calling the "Sixth Mass Extinction" (Barnosky et al., 2011).
Insight into the genetics of populations is of great significance as genetic variation is
the key to adaptation and exploitation of variable environmental niches, playing a vital
role in the survival of populations under changing environmental conditions (Lovelock
and Margulis, 1974; Frankel and Soulé, 1981; Berry, 1992). Reduced population size,
habitat area, chance events and inbreeding can have negative effects on genetic
variation and often lead to decreased fitness by increasing suseptability to diseases,
pests (Whiteman et al., 2006), infertility (Heber and Briskie, 2010), inbreeding
depression (Oakley and Winn, 2012) and population extinction (Franzen and Nilsson,
2009). As the detrimental impacts of reduced genetic variation become more apparent
it is of increasing importance to develop a comprehensive understanding of the factors
which mold and preserve it as well as of how such variation is structured in time and
space. If the distribution and subdivision of variation as we see it today reflects the
history of a population (Wright, 1969) then analysis of the spatio-temporal structure of
that variation should provide us with a deeper understanding of the processes which
have formed it.
In the following literature I will first discuss genetic variation and factors which influence
the genetics of populations as well as some methods used to asses certain parameters
relating to population genetic diversity. The relevance of cytological methods for
answering questions relating to ecotypic variation and population differentiation will then
be discussed briefly. Then the utility of islands for the study of the evolution of diversity
13
will be reviewed, followed by an introduction to the study system itself, the island of
Surtsey, Iceland. Some important aspects the study organisms, Honckenya peploides, life
history and morphology will also be discussed briefly. Lastly, the aims of the study will be
laid out followed by the respectable chapters.
1.2
Genetic Variation
The driving force behind genetic variation is mutation and recombination. The frequency
and intensity of which is influenced by events such as colonization, gene flow, genetic
drift, selection and breeding systems (Wright, 1969; Charlesworth, 2009). Establishment
of a new population starts with a founder event, the number of founders influences the
amount of variation in the new gene pool, with population reduction having a negative
effect on genetic diversity. Increased population differentiation and-or reduced fitness as a
result of random changes in alle frequency (genetic drift) sometimes follows after new
areas are colonized, again, with the effects being more pronounced in smaller populations
(Vandewoestijne and Van Dyck, 2010; Acquaah, 2012).
1.2.1
Genetic Variation on Islands
Island populations and endemic species overall are thought to have lower genetic
diversity than their mainland cousins, with genetic diversity decreasing with island size
and increasing degree of isolation (Frankham, 1997, 1998). The effects of limited
population size can be a constraint on levels of genetic variation. Typically only a
small portion of individuals (genetic variation) from the gene pool are sampled in the
initial founding event (Mayr, 2006). The result of this is a loss of rare alleles along
with changes in allele frequencies in the new population (Wright, 1948). These effects
can be mitigated if the distance for the migration of new genotypes into the population
(gene flow) is not too great, often leading to population cohesion (Dixon et al., 2011).
Reduced gene flow due to distance or ecological isolation can lead to population
differentiation, reproductive isolation and eventually speciation. Further isolation
along with reduction in population size can lead to inbreeding, reduction of within
population genetic variation (reduced heterozygosity) and decreased fitness. Also, if
ecological conditions vary between populations and gene flow is hampered then
selection can favor successful genotypes at each location, possibly leading to
population differentiation (Slatkin, 1987).
1.2.2
Island Biogeography and Gene Diversity
The Island Biogeography theory, first proposed by MacArthur and Wilson (1967)
offers a qualitative model for the relationship of species diversity with island isolation
and area. Simply put, as island distance from larger landmasses increases, species
number decreases, due to the effect of distance on immigration. Furthermore, species
number decreases as island size decreases, due to the heightened probability of
extinction. Jaenike (1973) proposes that isolation along with limited area should affect
genetic variation of populations on islands in a similar way. This is supported by many
studies showing a large number of taxa with significantly lower genetic diversity than
the same mainland species, with the effect increasing with decrease in island size
(Frankham, 1997). However, factors such as breeding systems, dispersal ability and
ploidy level all have a dynamic interplay, resulting in differences in genetic behavior
depending on the organism in question.
14
1.2.3
Dioecy and Genetic Variation
Dioecy, a condition in which sexes are on separate individuals, has been shown to
positively affect gene flow and greatly reduce inbreeding (Charlesworth, 2006; Obbard
et al., 2006). Nordal and Philipp (M. Philipp, pers. comm.) found that auto deposition
in hermaphrodites of H. peploides (bags were placed over individuals) in Greenland
did not produce any seeds and active selfing of hermaphrodites resulted in very low
seed set with very low seed weight, while active out-crossing in hermaphrodites
increased seed set. The occurrence of reduced fitness in seeds due to selfing of
hermaphrodites has been found for other subdioecious species as well and seems to be
a feature that has evolved to facilitate gene flow, prevent inbreeding and increase
heterozygosity (Tsukui and Sugawara, 1992; Delph and Carroll, 2001; Weller and
Sakai, 2004; Keller and Schwaegerle, 2006).
1.2.4
Genetic Variation in Polyploids
Genetic variation is often maintained through fixed heterozygosity in polyploid
species. Fixed heterozygosity has been reported in a number of polyploid arctic plants
and seems to be the norm rather than the exception in arctic polyploids. Brochmann et
al. (2004) found that all Svalbard polyploids were genetic autopolyploids with fixed
heterozygosity at isozyme loci. Furthermore they found that heterozygosity in 65 arctic
taxa increases dramatically from diploid to high-level polyploids. This high level of
heterozygosity has been proposed by other authors to be the motive force behind
polyploidy expansion into new habitats (Levin, 2002). Brochmann et al. (2004)
speculate that fixed-heterozygosity as a result of polyploidy formation, buffering
against inbreeding and drift, is the cause of the great evolutionary success of
polyploids in the arctic region. Out-crossing diploid populations tend to become
genetically homogeneous when confronted with bottlenecks and inbreeding, as is often
the case when colonization of isolated areas takes place (Aguilar et al., 2008; Yuan et
al., 2012). However, typical arctic plant populations (usually autopolyploids), despite
being faced with inbreeding and bottlenecks, have the possibility of maintaining
genetic variation due to fixed heterozygosity maintaining allelic diversity within each
individual (Otto and Whitton, 2000). The deposit of ancestral genetic material into
duplicated, fixed heterozygous genomes guarantees that genetic diversity is maintained
despite intense inbreeding and bottlenecks associated with long distance seed dispersal
events or re-colonization of recently barren areas (Tate et al., 2005).
1.3
Quantifying Genetic Variation
Molecular marker techniques are often used to directly examine genetic variation as
the interplay of genetic and environmental factors on phenotypic variation can be
challenging to untangle (reviewed in Agarwal et al., 2008). The varying number and
frequency of alleles per locus is used to determine the genetic variation in a population,
typically separated into hierarchical levels such as the individual, the population or the
region. Variation within these parameters is quantified using the number of
polymorphic loci, the number of alleles at a locus and heterozygosity levels from
which within-population heterozygosity (HW) and total heterozygosity (HT) can be
determined. Between populations, variation is typically quantified as the variation in
15
allele frequency differences between them, a measure denoted as population
differentiation or fixation index, FST (Wright, 1965).
1.3.1
Amplified Fragment Length Polymorphism (AFLP)
A tool that has provided great insight into the genetics of populations is AFLP-PCR (Vos
et al., 1995; Maughan et al., 1996; Muluvi et al., 1999; Kim et al., 2002; Bonin et al.,
2007; Reeves and Richards, 2009; Sánchez-Vilas et al., 2010). AFLPs are selectively
neutral, highly polymorphic molecular markers which have been used to evaluate patterns
of genetic variation and genetic structure in a number of Arctic, Subarctic and Antarctic
plant populations as well as isolated island plant populations (Sharma et al., 2000;
Holderegger et al., 2003; Schonswetter et al., 2003; Alsos et al., 2007; Migliore et al.,
2011). The method quickly generates large numbers of markers, prior knowledge of the
genomic sequence in question is not necessary, very little starting template is required and
it can be used for most organisms (van Haeringen et al., 2002; Groot et al., 2003; Brooker
et al., 2008). Several studies have compared the effectiveness of AFLP to RFLP
(Restriction Fragment Length Polymorphism), SSR (simple sequence repeats or
microsatellites), and RAPD (Random Amplified Polymorphic DNA) at producing
polymorphic loci and all have found AFLP to be the most effective and reliable method, in
every case producing a much higher amount of polymorphic bands per primer used
(Maughan et al., 1996; Nakajima et al., 1998; Barker et al., 1999; Xu et al., 1999; Meudt
and Clarke, 2007; Reeves and Richards, 2009).
Through PCR amplification, AFLP methods allow the detection of polymorphisms of
genomic restriction fragments (Vos et al., 1995). AFLP markers have been particularly
useful in investigations of population structure and differentiation (Tohme et al., 1996;
Heun, 1997; Triantaphyllidis et al., 1997; Arens et al., 1998). The power of AFLP
analysis derives from its ability to quickly generate large numbers of marker fragments
from minute pieces of any organism with no prior knowledge of the genomic
sequence. Other advantages of the AFLP method are that the PCR is very fast and high
numbers of different genetic loci can be simultaneously analyzed during each
experiment (Powell et al., 1996). This high multiplex ratio guarantees that the whole
genome is being sampled rather than just one piece of it. Another huge advantage of
AFLP is its reproducibility, Jones et al. (1997) used a number of labs throughout
Europe to show that through rigorous control of all variables it was possible to
reproduce the same banding patterns in all the laboratories.
Using PCR the AFLP technique amplifies pieces of genomic restriction fragments
(Figure 1). The first step of the AFLP process involves the cutting of DNA into
small fragments using restriction enzymes and the ligation of double-stranded
adapters of a specific sequence to both ends of all restriction fragments to create
primer binding sites for the pre-selective and selective DNA amplification in the
steps that follow. A pre-selective amplification is then performed in which specifically designed primers containing only one nucleotide at the 3’ end are ligated to
the restriction fragments. Exact matching of the 3’ end of a primer is necessary for
amplification because the polymerase can only copy DNA molecules which have a
primer attached. Therefore only restriction fragments in which the 3’ primer
extensions match the sequences flanking the restriction sites will be amplified.
This phase of the AFLP process is called pre-selective due to the fact that only DNA
16
Figure 1: Outline of the methodology of Amplified Fragment Length Polymorphism (AFLP) (Vos
et al., 1995). (1) Total genomic DNA is digested with two restriction enzymes (one frequent and
one rare cutter). (2) During the digestion reaction adapters with primer sites are ligated to the ends
of the fragments. (3) PCR using primers with one selective nucleotide extending into the genome
fragment is performed. This reduces the number of fragments and improves reproducibility of final
fingerprints. (4) A second series of PCR follows using two or three selective nucleotides on each
primer, thus (5) generating a highly polymorphic DNA fingerprint. ©SHÁ
molecules that have base pair sequences that are complementary to the primer plus the one
selective nucleotide will be amplified. To verify amplification, the products of the preselective amplification are checked by running them on a 1.5% agarose gel. A selective
17
amplification is then performed. This amplification uses two primers with three selective
nucleotides attached. The attachment of three nucleotides to the 3’ end of the primers
makes this step in the AFLP process even more selective because now only DNA
molecules which have sequences complementary to the primer plus the three nucleotides
will be amplified. One of the selective amplification primers (EcoRI) includes a
fluorescent label on the 5’ nucleotide. Selective amplification with an EcoRI and an MseI
primer amplifies EcoRI-MseI ended fragments. The EcoRI-EcoRI fragments do not
amplify well, the MseI-MseI fragments are not visualized because they do not contain
fluorescent dye labels, however, the EcoRI-containing strands are detected because they
contain the dye labels.
1.4
Karyotype Diversity
The overall form of the nuclear genome is quite variable among plants. A rich diversity in
genetic constitution and morphology is known to be found between and thought to also be
present within species (Watanabe et al., 1999; Bennett, 2008; Young et al., 2012).
Differences in chromosome number, form and size can have profound phenotypic
consequences as the nuclear DNA content itself is known to influence every stage of
development, from the cellular to the somatic and reproductive phases. These differences
often lead to differential survival of favorable ecotypes, resulting in variable spatiotemporal distributions both globally and locally (Ray, 2010; Bennett and Leitch, 2011;
Leitch and Leitch, 2012).
The enzymatic root tip squash method for plant chromosome preparation has been used in
order to obtain chromosomes suitable for various cytogenetic studies such as kayotyping,
chromosome banding and fluorescence in situ hybridization (FISH) since as early as the
nineteen fifties (Jacobsen, 1954; Malling, 1957; Schwarzacher and Leitch, 1994;
Anamthawat-Jónsson, 2001; Valárik et al., 2004). Karyotypes combined with molecular
phylogenetic analysis can provide information about taxonomic relationships, ecotypic
variation, genetic peculiarities and the evolutionary origins of species (Watanabe et al.,
1999; Leitch and Leitch, 2008; Chokchaichamnankit et al. 2007; Anamthawat-Jónsson et
al., 2009; Lysak and Koch, 2011; Wolny et al. 2013). Furthermore, chromosome size
differences, arm-length ratios and FISH can be used to distinguish identifying features and
loci between ecotypes (Truta et al., 2010; Ebadi-Almas et al., 2012; Young et al., 2012;
Catalán et al. 2012). This may provide a good basis for distinguishing between
subpopulation, providing insight into mechanisms relating to population differentiation.
1.5
Natural Laboratories on Isolated Oceanic
Islands
The utility of isolated oceanic islands for the study of evolutionary processes is not
without justification. They are both centers of biodiversity and evolution as well as the
setting for the majority or over 80% of known extinctions since the year 1500
(Rodriguez et al., 2012). This makes islands valuable tools in understanding the
fundamental mechanisms involved in speciation and can provide insight into the
causes and consequences of reduced diversity. Given that they make up such a small
amount of the earth’s land area (5%), island populations, compared to their continental
18
counterparts, display prolific and extraordinary radiations (Cody and Overton, 1996;
Givnish and Sytsma, 1997; Gemmill, 2002; Kapralov et al., 2009; Schmitz and
Rubinoff, 2011). Attested to by the fact that islands contain a very high proportion of
all known species, with 20% of plant species as well as 15% of known mammal, bird
and amphibian species being found on islands (Brooks et al., 2002; Brooks, 2006).
Therefore, conditions presented by the island environment (isolation, niches, and etc.)
must favor species diversification. This is particularly clear on older, more isolated
islands such as the Hawaiian, Galapagos and Canary islands (McCullen, 1987; Hortal
et al., 2007; Cowie and Holland, 2008; Baldwin and Wagner, 2010). It can be argued
then that islands themselves present us with a basic, replicable system whose finite
size, isolated nature, niche diversity and sheer number offer an ideal environment in
which to study the processes of evolution.
1.5.1
Surtsey - A Natural Laboratory
In the turbulent month of November 1963 the Nordic fire god Surtur plotted with the
Hawaiian goddess Pele to produce an empty frame upon which evolution could weave
its web, Surtsey was born (Figure 2). The island, located at 63.4°N, 20.3°W, was
created during almost four years of continuous volcanic activity. Surtsey is in the
Vestmannaeyjar Archipelago (Figure 3), an archepelago that was created just south
(20-30 km) of the mainland of Iceland by a series of submarine volcanic eruptions
following the last ice age. The initial size of Surtsey after the eruption was measured at
2.65 km2, however today, due to the constant battering by wind and sea the island has
been reduced to 1.47 km2 by 1998 (Jakobsson et al., 2000). The highest point on the
island is 155 m above sea level (Austurbunki). The climate is humid with cool
summers, relatively warm winters, strong winds and is often overcast. The average
winter temperature is 1.5 - 2°C with some 80 days below freezing. The summer
average is around 10°C, but temperatures below -15°C or higher than 20°C are
uncommon. The average annual rainfall is 1,600 mm, falling mostly between October
and March, a third of it as a mixture of rain and snow. Winds prevail from the east off
the mainland and are of hurricane strength at least 15 days a year. Waves are driven
from the southwest and can be very high: 16.68 m was recorded at a measuring buoy,
and the lava cliffs are heavily eroded. Like all the islands in the archipelago, Surtsey is
of volcanic origin with palagonite tuff or basaltic lava (Steinthorsson, 1965).
In 1965, Surtsey was declared a protected area and secured for the sake of science.
Since then it has been highly revered as a natural living laboratory for scientific
discovery and was named a Unesco World Heritage Site in 20 0 8.
The first biological observations were performed on Surtsey in the spring of 1964
(Friðriksson, 1964). Molds, bacteria and fungi were among the first organisms found
(Brock, 1966). The first vascular plant, the sea rocket (Cakile arctica), was observed in
1965 and plants have been investigated annually since. A year later lymegrass (Leymus
arenarius) began to grow on the island and by 1967 the sea sandwort (Honckenya
peploides) joined the flora (Friðriksson and Johnsen, 1968). Six years after its arrival,
H. peploides began to produce seed locally, making the population increase no longer
dependent upon accidental seed transport. By 2008 H. peploides had managed to
distribute itself all over the island and is currently the most frequent of all vascular
plants found in study plots on Surtsey (Magnússon et al., 1996, 2009).
19
Figure 2. Surtsey: a) West side of Surtsey, b) close up of the western crater, c) the south coast
of Surtsey, d) the east coast of Surtsey. Photo ©BM
20
Figure 3: Map of the Vestmannaeyjar Archipelago ([email protected])
Similar to other barren seashore habitats in the circumpolar north, H. peploides has
formed a cohabiting community with lymegrass, L. arenarius, and lungwort or sea
bluebells, Mertensia maritima (Friðriksson, 1987) (Figure 4). In 1978 H. peploides and
L. arenarius began overlapping and spreading over sandy areas in the east part of the
island. These two species and M. maritima have developed into a primitive, coastal
community. In this relationship, H. peploides and L. arenarius serve as pioneer plants,
holding the ground stratum, and later arriving species grow above it, taking advantage
of the moist sand media held in place by the former species. By 2008 the number of
vascular plant species colonizing the island had reached 69, of which 63 or 91% were
found alive that year and 32 or 46% had formed viable populations.
As stated above, the initial colonization of the island was characterized by the arrival
of shore plants such as H. peploides, C. arctica and L. arenarius. These are said to
have facilitated the arrival of seabirds which used the plant material for nest building,
especially H. peploides, which is said to have provided the nesting material for the first
pair of the great black-backed gulls (Larus marinus) to breed on the island. Most of the
first plant species had a high survival rate and formed viable populations. This initial
period of colonization (1965 – 1975) was followed by a lag in new species
colonization (1975 – 1984) in which relatively few new species entered the island and
survival dropped. During this time, the shore plant community, mainly H. peploides
21
and L. arenarius, became more established and spread out. This was coupled by an
increase in nesting gulls which seek out the developing patches of shore plants as
nesting sites and enhance their growth through nutrient deposition (Magnússon and
Magnússon, 2000; Sigurdsson and Magnusson, 2010).
Figure 4: Plant community on Surtsey with Leymus arenarius, Honckenya peploides and
Martensia maritima. The photo was taken July 2010 ©KAJ
The formation of a bird community on Surtsey has been thoroughly documented
(Petersen, 2009). After a sharp increase in the gull population around 1985 and the
formation of a dense breeding colony on the southern part of the island, a new wave of
vascular plant invasion was facilitated (through seed dispersal and nutrient deposition
by the birds) and survival rates increased. The period from 1985 – 1994 is
characterized by the invasion of various sea gulls, mainly the herring gull (Larus
argentatus), the lesser black-backed gull (L. fuscus) and the great black-backed gull (L.
marinus). This invasion of gulls facilitated a big increase in new plant colonizers, the
seeds of which are mostly dispersed by the gulls themselves. During this time,
mycorrhizal fungi are first detected and are said to have facilitated the colonization and
spread of rudimentary species such as annual meadow grass (Poa annua), chickweed
(Stellaia media) and pearlwort (Sagina procumbens). At the end of this period,
vegetative cover on the southern part of the island had reached 3 ha.
The period from 1995 – 2008 has been characterized by the development of a
secondary plant community and the formation of a forb rich grassland in which the
meadow-grass (Poa pratensis) and the arctic fescue (Festuca richardsonii) are the
dominant species. This period has shown a large increase in soil organic matter and
plant biomass within the gull colony, as well as an increase in the overall area of the
vegetative material making up the bird colony from 3 ha to 10 ha. Also during this
time, invertebrate species number and abundance have increased, facilitating the
arrival of passerine, land bird species such as the snow bunting (Plectrophenax
22
nivalis), the white wagtail (Motacilla alba) and the meadow pipit (Anthus pratensis),
which all feed their young on insects. Along with the arrival of the land birds, came the
arrival of ravens (Corvus corax), which feed their young mainly on the eggs and young
of fulmars (Fulmarus glacialis), gulls and kittiwakes (Rissa tridactyla).
Dispersal methods of new seedlings to the island vary. Based on observations of the
colonization sequence and locations of first encounters, the majority, around 75% of
new colonists arrived by birds, 16% by wind and 9% by sea currents. It is also relevant
to note that all species found on Surtsey so far are also found on the mainland of
Iceland and about 80% may have derived from the other Vestmanna Islands
(Magnússon et al., 2009). The vascular plant species brought by sea are all coastal
plants adapted to sea dispersal such as C. arctica, L. arenarius and H. peploides and
these species were among the initial colonizers of the island. These initial colonizers
are all adapted to infertile habitats along sandy shores and barren inland areas with
little nutrient content. It is also interesting to note that these species are all clonal
perennials with high stress tolerance and large seeds.
Being a newly formed island Surtsey is a perfect natural laboratory for the study of this
type. The island provides the rare opportunity to study the flora and fauna of an island
as colonization takes place. This could bring novel understanding of the effects that
colonization of barren island ecosystems has on the genetic behavior of populations,
shedding further light on the mechanisms involved in population differentiation,
reproductive isolation, speciation and extinction.
1.6
Honckenya peploides
The Sea Sandwort, Honckenya peploides (Figure 5) is the only species within the
genus Honckenya (L.) Ehrh., belonging to the subfamily Alinoideae in the carnation
family Caryophyllaceae. The plant reproduces both sexually and asexually and
displays a large amount of genetic variation within spatially segregated unisexual
populations in northwestern Spain (Sánchez-Vilas et al., 2010), Greenland and
Svalbard (M. Philipp, pers. comm.). Typical of plants found in northerly and recently
de-glaciated or bare areas, it is a tetraploid (Brochmann et al., 2004), with a relatively
high chromosome number (2n = 4x = 66, 68, 70) (Malling, 1957) and large genome
(8.57 to 10.66 pg/cell) when compared to other members of Caryophyllaceae
(Kapralov et al., 2009).
1.6.1
Habitat and Distribution
Honckenya is an early colonizer of barren seashores, facilitating both plant
colonization and ecosystem development by binding soil particles together with its
roots and providing nesting material for sea birds (Houle, 1997; Gagne and Houle,
2002; Sigurðsson and Magnússon, 2009). Adapted to habitats such as fore-dunes, drift
lines, seashores and lake shores (Brysting et al., 2007), it was one of the pioneer plant
species colonizing Surtsey only a few years after the island appeared (Friðriksson and
Johnsen, 1968). The plant sometimes grows in large mats or clumps, forming small
dunes through the spreading of leafy rhizomes whose shape and orientation facilitate
sand accumulation (Gagne and Houle, 2002).
23
Figure 5: Honckenya peploides from Seltjarnarnes peninsula, Reykjavík, Iceland. ©SHÁ
Four subspecies of Honckenya have been identified (Figure 6) (Jonsell, 2001): subsp.
peploides, distributed from northern Norway to northern Portugal; subsp. diffusa, which
has circumpolar distribution mainly in Arctic and northern Boreal zones; subsp. major,
found in the North West Pacific area; and subsp. robusta, found in Northeastern North
America. According to ult n (
), subsp. diffusa is a variety of subsp. peploides and
this is the subspecies I will be focusing on in the present research. Subsp. peploides occurs
mainly in the northern parts of Norway, Svalbard, Greenland, Iceland and subarctic
Canada (Houle, 1997; Jonsell, 2001). In Iceland H. peploides can be found on most all
shorelines from sea level up to 50m above sea level with Surtsey being the only place
where the plant can be found growing up to 100 m above sea level (Figure 6).
Figure 6: Left figure shows Honckenya subspecies worldwide distribution (Hulten et al.,
1971). Right figure shows distribution in Iceland (floraislands.is).
1.6.2
Flower Morphology
Honckenya peploides flowers are axillary and solitary with one to six flowered
terminal chymes. There are two sexual morphs, the hermaphrodites having large petals
and stamens and the females whose flowers have very small stamens and petals
24
(Brysting et al., 2007) (Figure 7). Such a condition is termed dioecy and is rare, being
found only in roughly 6% of known angiosperm species (14,620 of 240,000) (Renner
and Ricklefs, 1995). Some individuals (pistillate) have non-functional anthers, long
styles and short petals while others (staminate) have long stamens that produce pollen
grains, short styles and long petals (Tsukui and Sugawara, 1992). Individuals with
pistillate flowers are females and those with staminate flowers are hermaphrodites.
Females never produce pollen and are constant in their expression. Hermaphrodites
(pollen producing morphs) rarely produce seeds and when they do, the number of
seeds is very low compared to female flowers (Malling, 1957). This is characteristic of
a subdioecious breeding system (reviewed by Delph and Wolf, 2005). Flower
pollination occurs by insects (Lelej et al., 2012) and pollen is sometimes carried from
flower to flower with the assistance of sand grains (Baillie, 2012). The fruit (Figure 7)
produced is spherical or ovoid in shape; with three teeth/valves, which pop open to
release seeds at maturity, after which the fruit is shed (Brysting et al., 2007). Later,
seeds are spread by sea currents and wind (Jonsell, 2001).
Figure 7: Honckenya peploides; a) hermaphrodite flower showing large petals, b) female
flower showing smaller petals and c) fruit capsule. ©SHÁ.
1.6.3
Breeding System
Hermaphrodite seeds develop into female and hermaphrodite plants in the approximate
ratio 1:3. Seeds of female flowers produce about as many hermaphrodites as females
(Malling, 1957; Sánchez-Vilas et al., 2010), implying that the sex determination system is
heterogamous for the hermaphrodite; female = XX, hermaphrodite = XY or YY.
The evolutionarily relevant criterion for classifying breeding systems is fitness-based
functional gender, i.e. the proportion of haploid genomes transmitted through pollen or
through ovules (Lloyd, 1980). Examples of these fitness based systems in plants
include gynodioecy (female and hermaphrodite), subdioecy (female, male and
hermaphrodite), androdioecy (male and hermaphrodite) and dioecy (male and female
functional parts). On the evolutionary pathway from co-sexuality to dioecy lie the
intermediate breeding systems of gynodioecy and androdioecy (Charlesworth and
Laporte, 1998), with subdioecy occupying an intermediate position between
andro/gynodioecy and complete dioecy (Delph and Wolf, 2005; Wagner et al., 2005).
This pathway (Figure 8) starts out with the invasion of a unisexual mutant, either
female sterile (male) or male sterile (female). This in turn causes the hermaphrodites to
allocate more of their sexual function towards the gender that is lacking, eventually
leading, in some cases, to hermaphrodites becoming unisexual, hence making the
population dioecious (Charlesworth and Laporte, 1998).
25
Figure 8: Schematic diagram of the dioceous pathway. ©SHÁ.
1.7
Thesis Overview and Objectives
There are two chapters in the thesis, each of them utilizing different tools to investigate
the genetic behavior of Honckenya peploides. The focal point of this thesis, chapter
two, examines questions regarding the genetic effects that the colonization of the
island of Surtsey has had on the genetics of H. peploides populations. This chapter,
entitled Spatial Genetic Structure of The Sea Sandwort, looks at genetic diversity
patterns on island and mainland populations using AFLP markers. The main focus in
this chapter is to determine if within population genetic diversity has changed
following colonization to the island of Surtsey. Patterns of genetic differentiation are
also compared to mainland areas and conservation implications are discussed briefly.
Chapter three, Karyotype Variation in Honckenya peploides Worldwide, deals with
finding some evolutionary trends in karyotype diversity. This chapter sets the stage for
further investigations into relationships between karyotypic variation within the
species and environmental variables.
26
2 Spatial Genetic Structure of the
Sea Sandwort
S. H. Árnason 1 , Æ.Þ. Þórsson 1 , B. Magnússon 2 , M. Philipp 3 , H. Adsersen 3 and K.
Anamthawat-Jónsson 1
[1] Institute of Life and Environmental Sciences, University of Iceland, Askja,
Sturlugata 7, Reykjavík, IS-101, Iceland
[2] Icelandic Institute of Natural History, Urriðaholtsstræti 6-8, Gardabær, IS-212,
Iceland
[3] Department of Biology, University of Copenhagen, Universitetsparken 15, DK2100 Copenhagen, Denmark
2.1
Introduction
On the 14th of November 1963, just 30 km off the southern coast of Iceland, the young
island of Surtsey arose violently to the sea surface, instantly producing a natural
laboratory for scientific exploration. Surtsey surfaced out of the frigid North Atlantic
Ocean during almost four years (November 1963 - June 1967) of continuous spewing
of volcanic material from the mid-Atlantic ridge at lat. 63° 8´ 22’’ N, long. 20° 36’
5’’ W, just 3 nautical miles west-south-west of Geirfuglasker, the southernmost island
in the Vestmannaeyjar Archipelago (Þórarinsson, 1967). Plant seeds were already
being documented washing up on shore during the first survey of the island on May
214st 1964. The first seedlings of the searocket, Cakile arctica, were recorded there on
June 3rd 1965, later to be buried under layers of ash. It seems almost certain that the
first plants to arrive to the island grew from seed carried either by floating debris or
wind to the island, as the seedlings of the first colonizers, C. arctica, Leymus arenarius
and Honckenya peploides, were found growing in a row at the high tide line
(Friðriksson and Johnsen, 1968).
The meticulously documented history of colonization and relatively recent geologic
origin of Surtsey offer a unique perspective for the scientist. The island provides a
natural system whose community development can be monitored and studied devoid of
all human influence. The focal species and the first plant to establish a viable seed
producing population on the island, Honckenya peploides, was first recorded there in
1967 (26 individuals, Friðriksson and Johnsen, 1968), set seed first in 1968 and has
grown on and dominated the vegetation on the island to this day (Magnússon et al.,
1996, 2009). By 1968 there were 103 individuals, by 1971 their number was reduced
to 52, five of which flowered, with one producing seed (most likely a female). In 1972,
71 plants were recorded, then a major colonization event took place before the summer
of 1973 in which 548 H. peploides individuals were recorded (along with a dramatic
27
increase in both L. arenarius and Cochlearia officinalis individuals). This is thought to
be due to an exceptionally good seed production seasons preceding this period.
The study of evolutionary processes has long been focused on isolated oceanic islands
as their finite size, isolated nature and niche diversity provide a basic, replicable
system in which to quantify and study the evolution of populations (Darwin, 1859;
Wallace, 1880; MacArthur and Wilson, 1967; Whittaker et al., 2010). Although they
make up only a very small fraction of the earths land surface area (5%), islands are
home to roughly 15% of all known mammal, bird and amphibian species and 20% of
all known vascular plant species (Brooks, 2006). Newly formed oceanic islands as well
as areas recently affected by cataclysmic events such as volcanic eruptions present
scientists with the unprecedented opportunity to collect data on and analyze
evolutionary processes at spatial and temporal scales not possible with ecosystems in
higher successional stages (Friðriksson, 1970; Fridriksson and Magnússon, 1992; Bush
and Whittaker, 1991; Tsuyuzaki, 2009). Due to the relative isolation and novel
ecological niches that these islands provide, colonization by small populations often
leads to rapid evolutionary change, adaptive radiation and speciation (Wagner and
Funk, 1995; Cody and Overton, 1996; Givnish and Sytsma, 1997; Gemmill, 2002;
Ziegler, 2002; Wagner et al., 2005; Kapralov et al., 2009; Schmitz and Rubinoff,
2011). Such radiations are thought to be a result of mutational spread under different
selection pressures as well as allele fixation and differential survival of genotypes best
suited for the particular environment (Huxley et al., 1958; Barton and Mallet, 1996).
The use of molecular techniques along with greenhouse experiments has been
instrumental in illuminating patterns of biogeography, genetic diversity and dispersal
as well as in providing answers regarding genome size changes, polyploidy, breeding
systems and speciation on oceanic islands (Bush and Whittaker, 1991; Givnish and
Sytsma, 1997; Sakai et al., 1997; Nielsen, 2004; Cowie and Holland, 2008; Green et
al., 2011; Himmelreich et al., 2012).
The distribution and segregation of current genetic variation is thought to be a reflection of
the history of populations through time (Wright, 1969). Inbreeding and increased
homozygosity are often the results of reduced variation within a population, frequently
leading to infertility, increased sensitivity to disease and in the most extreme cases
inbreeding depression and extinction (Charlesworth and Charlesworth, 1987; Ellstrand and
Elam, 1993; Frankham, 1998; Futuyma, 1998; Eldridge et al., 1999; Ellis et al., 2006;
Dostálek et al., 2009). Islands that are geologically older and extremely isolated from
mainland sources of variation often times display an extraordinary propensity for species
radiations and evolution. This evidence in the immense diversity is found within the
Hawaiian (0.4 - 4.7 million years or Myr) flora and fauna (Cowie and Holland, 2008;
Baldwin and Wagner, 2010), the Galapagos (0.7 - 4.2 Myr) vascular flora (McCullen,
1987) and the forest biota of the Canary Islands (0.8 - 20 Myr) (Hortal et al., 2007). The
dynamic interplay of spatio-temporal factors, isolation-distance, breeding systems and
dispersal ability results in differences in genetic behavior depending on the organism in
question. Could it then be feasible to hypothesize that we will see any evidence of
evolutionary processes on young islands such as Surtsey, where time and space have not
existed until relatively recently (50 yrs.)?
A vast amount of theory and investigative methods have been established which
illuminate how genetic variation in particular has been shaped by evolutionary
processes over time (Huxley et al., 1958; Wright, 1969; Nei et al., 1975; Maynard
28
Smith, 1989; Dawkins, 1982). In light of the rapid anthropogenic habitat destruction
and fragmentation that is currently causing an unparalleled loss of biodiversity around
the planet, an analysis of the distribution of variation and an understanding of the
processes which shape and conserve it at small spatial and short temporal scales is of
great significance (Brooks et al., 2002; Travis, 2003; Brooks, 2006; Cardinale et al.,
2006, 2009; Brittain and Craft, 2011; Rodriguez et al., 2012). In the following study,
amplified fragment length polymorphism (AFLP) data along with classical and
Bayesian statistical analysis methods were used to deduce the genetic structure of
Honckenya peploides in sampling locations on Surtsey, Heimaey (Vestmannaeyjar),
mainland Iceland, Greenland and Denmark. Due to its young geologic age, the fact that
colonization and succession have been thoroughly studied there since its inception and
the fact that H. peploides was one of the first and most prolific colonizers of the island,
Surtsey provides the perfect natural laboratory for a study of this type and Honckenya
peploides an excellent model species. This study is the first of its kind conducted on
the island of Surtsey and provides the unprecedented opportunity to study the
processes of evolutionary change at the molecular level as a population develops on a
young (50 yrs.), small (< 2 km2) isolated (30 km from mainland) oceanic island located
in the sub-arctic region (63°N, 20°W).
The objectives of this study were as follows: (i) to assess the genetic diversity and
population genetic structure of Honckenya peploides on the island of Surtsey, (ii) to
compare the genetic structure of H. peploides at locations on Surtsey to the genetic
structure on older, more established locations on Heimaey, mainland Iceland, Denmark
and Greenland and (iii) to examine the level and effects of isolation by distance on the
sample locations.
2.2
2.2.1
Materials and Methods
Study Species
The autotetraploid (2n = 4x = 66, 68, 70), gynodioceous, perennial, maritime, eudicot,
H. peploides can reproduce both sexually with pistillate (female) and staminate
(hermaphrodite) flowers as well as asexually using its rhizomes. Often daughter clones
remain attached to parent clones via rhizomes whose connections sometimes run up to
2 m from the parent plant (Sánchez-Vilas et al., 2010). Hermaphrodite seeds develop
into female and hermaphrodite plants in an approximate 1:3 ratio. Seeds of female
flowers produce about as many hermaphrodites as females (Malling, 1957), implying
that the sex determination system is heterogamous for the hermaphrodite. An
experiment in which bags were placed over individual H. peploides plants in
Greenland (M. Philipp, pers comm.) showed that hermaphrodites did not produce any
seeds and active selfing of hermaphrodites resulted in very low seed set with very low
seed weight, while out-crossing in hermaphrodites increased seed set. This indicates
that although rare, self-fertilization is indeed possible for H. peploides and that, similar
to most arctic species, the plant has a mixed mating system (Brochmann et al., 2004).
However similar to most male heterogamous subdioecious species, the hermaphrodite
seed production and fitness is quite low, possibly indicating some level of inbreeding
depression (Tsukui and Sugawara, 1992; Delph and Carroll, 2001; Weller and Sakai,
2004; Keller and Schwaegerle, 2006). This seems to be a feature that has evolved to
prevent inbreeding and reduced heterozygosity.
29
H. peploides habitat is mainly limited to dunes, drifts lines, lake-shores and seashores.
It has a circumpolar distribution extending from the arctic to the temperate zone in
Western Europe, North America and North Eastern Russia down to Japan (Jonsell,
2001). The plant is an early colonizer, contributing to the anchorage of the soil and is
the first plant to set seed on Surtsey only a few years after the island first appeared
(Friðriksson and Johnsen, 1968). H. peploides has been shown to exhibit a large
amount of within population genetic variation in both northwestern Spain (SánchezVilas et al., 2010), Greenland and Svalbard (M. Philipp, pers. comm.). However, little
to nothing is known about the genetic makeup of populations located in Surtsey,
Vestmannaeyjar or mainland Iceland.
2.2.2
Marker Choice
The selectively neutral, highly polymorphic technique, Amplified Fragment Length
Polymorphism (AFLP) (Vos et al., 1995), combines restriction digestion and PCR to
produce Mendelian inherited, multi-locus, dominant markers which are highly
reproducible and can be used for analysis of genetic structure (Jones et al., 1997).
AFLP has been used in a number of studies of Arctic, Subarctic and Antarctic plant
populations as well as isolated island plant populations to evaluate patterns of genetic
variation and genetic structure (Sharma et al., 2000; Holderegger et al., 2003;
Schonswetter et al., 2003; Alsos et al., 2007; Migliore et al., 2011; ). AFLPs have also
shown that recently formed populations can have a propensity towards decreased
genetic diversity after introduction to un-colonized areas (Muluvi et al., 1999;
Amsellem et al., 2000). Also, with AFLPs, a positive relationship has been
demonstrated between genetic diversity and populations size (Morden and Loeffler,
1999; Gaudeul and Till-Bottraud, 2008). Additionally AFLPs can reveal genetic
structure in numerous plant species including Honckenya peploides the species under
study (Sánchez-Vilas et al., 2010).
In the last decade, AFLP has become one of the most widely used molecular markers
to study the genetic structure of natural populations (Meudt and Clarke, 2007; Foll et
al. 2010). The method quickly generates large numbers of markers, prior knowledge of
the genomic sequence in question is not necessary, very little starting template is
required and it can be used for most organisms. The performance of AFLPs compared
to other dominant, genome-wide markers or compared to traditional co-dominant
markers like microsatellites or allozymes have been investigated on numerous
occasions (Powell et al., 1996; Jones et al., 1997; Barker et al., 1999; Mariette et al.,
2002; Nybom, 2004). All have found AFLP to be the most effective and reliable
method, in every case producing a much higher amount of polymorphic bands per
primer used. Due to the extensive polymorphisms generated, the AFLP method is very
powerful in revealing intraspecific variation, hence applicable also for genotyping,
fingerprinting and barcoding purposes (Meudt and Clarke, 2007; Huang et al., 2013;
Cornille et al. 2014).
2.2.3
2.2.3 Plant Material and Collection Locations
During July of 2010 and May of 2011, a total of 397 samples were collected, of these
347 samples from 12 locations in the north Atlantic region were analyzed (Table 1, see
also Figure 9). This includes; five locations on Surtsey: SC (63°18.246N /
20°36.751W), located inside the western crater Surtungur at 70 m above sea level,
30
growing on volcanic tuft: SD (63°18.500N / 20°35.987W), located on the northern
sand spit at 16 m above sea level, growing on volcanic sand: SE (63°18.197N /
20°35.632W), located on the eastern slope at 104 m above sea level, growing on
volcanic tuft and gravel: SF (63°17.942N / 20°36.348W), located in the gull colony on
the southern shore: and SK (63°18.416N / 20°36.00W), located on the steep eastfacing slope of the old tephra crater Surtur leading to the northern sandbar at 36 m
above sea level, growing on volcanic tuft and gravel; two locations on Heimaey: AH
(63°24.506/20° / 16.792W), located on the south side of the island, growing on
volcanic sand and gravel at 1-3 m above sea level: and BH (63°26.911N / 20°16.250W
), located on the northern side of the island, growing on volcanic sand and gravel at 12
m above sea level; two locations on mainland Iceland: Stokkseyri (ST - 63°50.376N /
21°04.636W), located at the end of a fresh water marsh in between two large glacial
rivers at 1-3 m above sea level on the southern coast, growing on glacial deposits and
fine sand: and Gardur (G - 64°04.963N / 22°41.548W), located at the western-most tip
of the Reykjanes peninsula at 1-3 m above sea level, growing on a rocky coast; two
locations in Denmark: Skansehage (DKS - 55°58N / 11°46E), located on the north
eastern tip of the island of Zealand at 1-3 m above sea level, growing above the high
tide line on a limestone sand beach: and Knarbo (DKK - 55°50N / 11°22E), also
located on the island of Zealand in very similar habitat at 1-3 m above sea level; and
one location in Greenland: Qeqertarsuaq (GR - 69°15N/53°30W), located on the south
coast of Disko Island on the west coast of Greenland at 1-3 m above sea level, growing
on a rocky seashore in glacial till.
Table 1 Sampling locations, GPS coordinates, dates, ID’s and number of samples.
Locality
GPS Coordinates
Date
Sample ID
N
Surtsey
63°18.24N/20°36.75W
12-15.07.10
SC 1-30
30
Surtsey
63°18.50N/20°35.98W
12-15.07.10
SD 1-30
30
Surtsey
63°18.19N/20°35.98W
12-15.07.10
SE 1-30
30
Surtsey
63°17.94N/20°36.34W
12-15.07.10
SF 1-30
30
Surtsey
63°18.416N/20°36W
12-15.07.10
SK 1-30
30
Heimaey
63°24.50/20°16.79W
9.7.10
AH(1-12)(16-30)
27
Heimaey
63°26.91N/20°16.25W
9.7.10
BH 1-30
30
Garður
64°04.96N/22°41.54W
17.7.10
G(1-12)(16-27)
24
Stokkseyri
63°50.37N/21°04.63W
17.7.10
ST(1-6)(10-30)
27
Qeqertarsuaq
69°15N/53°30W
25.7.10
GR 1-29
29
Knarbo
55°50N/11°22E
15.5.11
DKK 1-30
30
Skansehage
55°58N/11°46E
15.5.11
DKS 1-30
30
Samples were collected by placing a marker in a random location surrounded by
clumps of H. peploides and sampling clumps within a 20 meter radius of the marker.
At each locality 30 spatially segregated clumps were sampled, avoiding sampling of
clones as best as possible. Fresh leaf samples were collected and stored in plastic bags
with silica beads until further laboratory analysis could be conducted.
31
2.2.4
DNA extraction
Genomic DNA was extracted from dehydrated leaf tissue using the CTAB method as
described by Aldrich and Cullis (1993), slightly modified for smaller samples. CTAB
extraction buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA pH 8.0, 100 mM TrisHCl
pH 8.0, 0.2% 2-mercaptoethanol) was pre-warmed to 65°C. Approximately 15 - 20 mg
of dried leaf tissue from each individual was placed in 1.5 mL eppendorf tubes and
manually pulverized to a fine powder with sterile plastic pestles in liquid nitrogen.
Warm extraction buffer (0.5 mL) was then added to the tube and mixed well until all
material was suspended in the liquid, this was then incubated at 65°C in a water bath
for 1 to 2 hours (longer incubation results in cleaner pellets) and mixed occasionally by
inversion. An equal volume chloroform-isoamyl alcohol (24:1) was then added and
mixed well before centrifuging at 1400 RPM for 10 minutes. The supernatant was
recovered and placed in a 1.5 mL Eppendorf tube and the DNA precipitated using 1.5
mL (2.5 volume) of cold 96% ethanol for 25 minutes at room temperature. The tube
was then centrifuged again at 3000 RPM for 10 minutes and the pellet washed twice
with 0.5 mL wash buffer (76% ethanol and 10 mM ammonium acetate). The tube was
then centrifuged again briefly at 3000 RPM, the supernatant removed and pellet air
dried. Once dry, 200 µl of 1x TE buffer (10 mM TrisHCl, pH 8.0 and 1 mM EDTA,
pH 8.0) and 1 µl of RNase (10 mg/ml) was added, the solution mixed well and
incubated at 37°C for 30 minutes. After incubation, 78 µl of 5 M NaCl was added, the
tube inverted several times and solution mixed well. DNA was then precipitated again
by adding 0.9 mL of ice cold 96% ethanol, inverting and mixing. The tube was then
centrifuged again at 3000 RPM for 10 minutes and the supernatant removed. The pellet
was then washed with 0.5 mL of wash buffer (76% ethanol and 10 mM ammonium
acetate) quickly spun, the supernatant removed and the pellet air dried. 100 µl of 1x
TE buffer was then added and samples stored in a -27°C freezer until AFLP work
could be done. This method works well for extraction of H. peploides DNA and when
performed correctly can result in a large quantity of high quality DNA. The quality and
concentration of DNA in all samples was measured using Thermo Scientifics
NanoDrop 1000 Spectrophotometer at OD 260nm/280nm. Of the 347 samples
collected, 300 were used for the AFLP analysis.
2.2.5
AFLP Fingerprinting
The AFLP technique was performed with fluorescent dye labeling and detection
technology. Fragment detection was performed on the ABI Prism 3730 Genetic
Analyzer (Applied Biosystems), an automated capillary electrophoresis device. The
AFLP analysis followed the standard procedure as described by Vos et al. (1995) on
quality Honckenya peploides DNA (250 ng in 5.5 µl) that was isolated from dry plant
leaf tissue using the CTAB method. Prior screening for primers suitable for use with
H. peploides was previously performed on individuals of both sexes across multiple
localities (Sánchez-Vilas et al., 2010). Restriction and ligation reactions were carried
out in accordance with the AFLP Plant Mapping Protocol (Applied Biosystems) for
average sized genomes.
32
Table 2: Primers used in study (Applied Biosystems).
Primer Name
Primer Sequence
EcoRI
5’-fam-GAC TGC GTA CCA ATT CAC T-3’
EcoRI
5’-joe- GAC TGC GTA CCA ATT CAG G-3’
MseI
5’-GAT GAG TCC TGA GTA ACT A-3’
MseI
5’-GAT GAG TCC TGA GTA ACT C-3’
Two primer pairs were chosen (MseI-CTA/EcoRI-ACT & MseI-CTC/EcoRI-AGG)
and selective amplification was carried out on all samples (Table 2). Single PCRs were
performed for each primer combination, and the products from two primer pairs (with
different dyes) were multiplexed for electrophoresis. In all the reactions, only the
EcoRI primers were 5´ labeled with a fluorescent dye (Applied Biosystems).
A total of 250 ng of DNA was added to a digestion-ligation mix in a final volume of
11 µL containing digestion ligation buffer (50 mM NaCl, 50 ng/µL BSA), 1 µL of
both MseI and EcoRI adapters (Applied Biosystems), 1U T4 DNA ligase, 5 U EcoRI
(Applied Biosystems), 1 U MseI (Applied Biosystems) and digested at 37°C for 2
hours. Following digestion-ligation, pre-amplification PCR was performed with
EcoRI-A/MseI-C primer pairs (Applied Biosystems) having one selective nucleotide
with 19 cycles of: 2 min at 72°C, 1 sec at 94°C, 30 sec at 56°C, 2 min at 72°C;
followed by a final extension for 30 min at 60°C. Each 10 µL of the reaction contained
2 µL of the restriction/ligation dilution (1:20), 7.5 µL of AFLP core mix from the
Applied Biosystems Amplification Core Mix Module (buffer, nucleotides, and
AmpliTaq® DNA Polymerase) and 25 mM of each primer pair containing one
selective nucleotide.
The PCR product was then diluted (1:20) and used for the selective amplification with
two primer combinations containing three selective nucleotides this time (MseICTA/EcoRI-ACT and MseI-CTC/EcoRI-AGG). The selective amplification PCR
reaction entailed: 1 cycle of 2 min at 94°C, 30 sec at 65°C, 2 min at 72°C; 8 cycles of
1 sec at 94°C, 30 sec at 64°C and 2 min at 72°C; 23 cycles of 1 sec at 94°C, 30 sec at
56°C, 2 min at 72°C; followed by a final extension for 30 min at 60°C. Each 10 µL of
the reaction contained 1.5 µL of the pre selective amplification dilution (1:20), 7.5 µL
of the AFLP Applied Biosystems® core mix, 0.5 µL EcoRI primer at 1µM
(flourecently labeled) and 0.5 µL MseI primer at 5µM. For analysis using capillary
electrophoresis, each 11 µL reaction contained 1 µL of each of the two selective
amplification products, 9.5 µl HiDiFormamid and 0.5 µl Genescan-500 Rox-labeled
internal size standard (Applied Biosystems).
After preparation, samples were denatured for 2 min at 94°C and chilled on ice.
Capillary electrophoresis was carried out on the 3730 series DNA Analyzer using POP
7 capillary polymer and ABI Prism 3730 electrophoresis buffer (Applied Biosystems).
The capillaries were 47 cm in length with 50 µm ID. Samples were injected electrokinetically for 5-20 sec at 15 kV and were run at 15 kV for 24 min at 60°C. Fragments
of each primer combination were then scored as described in the next section. Initial
fragment analysis was performed at the center for Macroecology, Evolution and
Climate at the University of Copenhagen, Denmark. Further data analysis was
performed at home university, in the Laboratory of Plant Genetics at Askja, Reykjavík.
33
2.3
2.3.1
Data Analysis
Raw Data
In the AFLP analysis, only amplified fragments with sizes between 50 and 500 base
pairs range that could be scored unambiguously and showed clear presence or absence
patterns in all H. peploides samples were included. Fragments outside this range cannot be
accurately sized, hence excluded. The raw AFLP data were scored using the programs
GeneMapper 3.7 (Applied Biosystems) and the presence or absence of each fragment was
scored for each individual manually in GeneMarker® (Softgenetics). As is usual for
AFLPs, we assumed that each AFLP band corresponded to a separate locus with two
alleles (Maguire et al., 2002). Therefore, the presence of a band indicates that the
individual sample may have originated from a heterozygote or a dominant homozygote,
not a recessive homozygote of the corresponding allele. Analysis of the AFLP data was
based on the phenotypic frequency at a particular locus (i.e. the proportion of individuals
having a band). We applied the band based approach due to the polyploidy nature of our
study species and set the allele frequency equal to band frequency.
2.3.2
Phylogenetic Relationships and Isolation by
Distance
The relationship among populations and individuals was first explored by using Nei´s
(1972) genetic distances among populations estimated in AFLP-SURV 1.0 (Vekemans et
al., 2002) (available at http://www.ulb.ac.be/sciences/lagev/) with 10,000 bootstrapped
distance matrices. These matrices were then graphically displayed as un-rooted
neighbour-joining (NJ) trees with the NEIGHBOR and CONSENSUS programs in PHYLIP
3.65 (Felsenstein, 1989). Nei´s pairwise genetic distance matrices as well as pairwise
FST matrices calculated with AFLP-SURV 1.0 were also plotted against geographic
distances in order to test for the effects of isolation by distance. All locations in the full
data set as well as select regions and locations were compared. Significance was tested
with a Mantel correlation test in R with the ade4 package (Chessel et al., 2004).
Multidimensional scaling (Kruskal, 1964) was also used to visualize patterns of
relatedness within the FST matrices.
2.3.3
Bayesian Clustering
A Bayesian clustering approach, implemented in STRUCTURE 2.2 (Pritchard et al.,
2000) was used to determine the number of genetic clusters (K) in the data set without
any prior information on the sampling locations, using the admixture model. The
number of genetically distinct clusters (K) was set to vary from 1 to 12 (total number
of populations). Twenty independent simulations were run for each value of K with a
burn-in length of 104 and a run length of 105 Monte-Carlo Markov Chain generations.
The optimal value of K was estimated by using the R script STRUCTURE-Sum-2009
(part of AFLPdat; Ehrich, 2006) to summarize the output files, calculate similarity
coefficients between replicate runs and to plot the means of the estimated log posterior
probability of the data over the replicate runs for each K value or L(K) in order to
determine the highest level of hierarchical structure (Evanno et al., 2005). After
detection of genetic divergence among populations, the analysis was repeated for each
group to determine if any substructure existed below the highest hierarchical level.
Graphical output was generated using CLUMP version 1.1.1 (Jakobsson and Rosenberg,
2007) and DISTRUCT (Rosenberg, 2003) software.
34
2.3.4
Genetic Data Analysis
Genetic differentiation among populations was calculated as Wright´s FST (Lynch and
Milligan, 1994) in AFLP-SURV 1.0 (Vekemans et al., 2002) using 10,000 permutations
to test for significance. The percentage of polymorphic loci (PLP), gene diversity (HT),
expected heterozygosity (HE) and average gene diversity (HW) were also estimated in
AFLP-SURV 1.0. To test for significant difference in PLP and HT between locations, a
Wilcoxon rank sum test was implemented in R. The relationship between PLP and HT
as well as between population size and PLP or HT was also tested with a Spearman´s
rank correlation test in R. Analysis of molecular variance (AMOVA) was performed to
assign components of genetic variation to sets of populations defined by location in
Arlequin 3.5 (Excoffier and Lischer, 2010).
2.4
2.4.1
Results
Paterns of Polymorphism and Heterozygosity
Using 300 individuals (Table 1) and two primer combinations (Table 2), 173 AFLP
loci were scored across all sampling locations. Of these, 65 were polymorphic (37.6%)
and used for our analysis (Table 3). In general, highly reproducible AFLP patterns
were obtained. An average error rate of 2.1% was estimated across the 10 pilot
samples for all primer pairs. This value fell below the maximum error rate percentage
accepted for good AFLP reproducibility (5%) (Piñeiro et al., 2007). The proportion of
polymorphic loci per population (PLP) varied from 31.8% to 55.5%. The total
heterozygosity (HT) was 0.2118. The expected heterozygosity within populations
varied from 0.1397 in the Denmark locations to 0.2130 in the SK Surtsey location
which had the highest heterozygosity. Vestmannaeyjar south (AH) and Stokkeyri (ST)
showed similar values, 0.1964 and 0.1972 respectively. The average genetic diversity
within the populations (HW) was 0.1746.
A significant difference was found between the Denmark and Surtsey populations for both
the PLP (DKK = 31.8%, DKS = 31.8%, SC = 48%, SD = 48%, SE = 46%, SF = 44.5%,
SK = 55.5%) and the total heterozygosity HT (Denmark = 0.1362, Surtsey = 0.1974, with
p = 0.0243 and p = 0.0264 respectively). Although PLP and HT differed slightly between
all the populations, none of the other populations were found to differ significantly. PLP
and HT values were also moderately correlated with each other (Spearman’s rank
correlation, R = 0.64, p < 0.05) but were not correlated with population size (R = 0.12 and
R = - 0.12, respectively, p > 0.05) over the whole geographic range.
2.4.2
Genetic Differentiation and Diversity
The total genetic differentiation among all 12 Honckenya peploides sampling locations
was moderate (FST = 0.1769, Table 3) and highly significant (permutation test, P <
0.001) in all collection locations except Denmark (Table 4). The FST value for
Denmark was found to be non-significant (p = 0.367). The total FST among the
Surtsey populations (0.0714) as well as the Vestmannaeyjar populations (0.0550) were
less than half of that found among the mainland populations (0.1747). As seen in Table
4, the largest FST values were found when comparing any population with the
Denmark populations (DKK and DKS), especially Greenland (GR: 0.431 and 0.440),
Garður (G: 0.356 and 0.367) and location SC on Surtsey (0.348 and 0.357).
35
Table 3 Genetic diversity in sampling locations of Honckenya peploides.
ID
Polymorphic
Bands
PLP
HE
SC
83
48
0.1740
SD
83
48
0.1808
SE
81
46.8
0.1789
SF
77
44.5
0.1699
SK
96
55.5
0.2130
AH
86
49.7
0.1964
BH
82
47.4
0.1696
G
78
45.1
0.1765
ST
89
51.4
0.1972
GR
76
43.9
0.1663
DKK
55
31.8
0.1397
DKS
55
31.8
0.1328
Total
65
37.6
FST
HT
HW
0.0714
0.1974
0.1833
0.0550
0.1938
0.1830
0.1747
0.2265
0.1869
-0.0003
0.1362
0.1362
0.1769
0.2118
0.1746
Total means at the species level. PLP - percentage of polymorphic loci, HE - Nei´s (1973) gene diversity
(expected heterozygosity), FST - Wright´s FST, HT - total gene diversity (total heterozygosity), HW average gene diversity within populations (Nei´s gene diversity).
Table 4: Nei´s genetic distances are displayed on the upper diagonal and FST values on lower
diagonal. *non-significant values
AH
AH
BH
BH
DKK
DKS
G
GR
SC
SD
SE
SF
ST
SK
0.013
0.095
0.102
0.005
0.020
0.009
0.006
0.026
0.018
0.025
0.037
0.128
0.125
0.006
0.025
0.011
0.018
0.050
0.047
0.061
0.067
0.000*
0.109
0.147
0.104
0.081
0.059
0.070
0.053
0.050
0.112
0.148
0.105
0.085
0.070
0.083
0.062
0.062
0.019
0.012
0.018
0.046
0.040
0.050
0.058
0.020
0.030
0.054
0.045
0.060
0.064
0.004
0.027
0.024
0.036
0.039
0.015
0.012
0.023
0.028
0.004
0.004
0.008
0.005
0.014
0.056
DKK 0.312
0.396
DKS
0.331
0.398
0.000*
G
0.021
0.029
0.356
0.367
GR
0.080
0.109
0.431
0.440
0.083
SC
0.037
0.050
0.348
0.356
0.052
0.089
SD
0.026
0.078
0.291
0.305
0.077
0.124
0.020
SE
0.100
0.188
0.232
0.269
0.174
0.202
0.109
0.065
SF
0.073
0.185
0.271
0.309
0.158
0.180
0.102
0.051
0.017
ST
0.092
0.209
0.206
0.236
0.175
0.207
0.134
0.088
0.018
0.022
SK
0.123
0.216
0.189
0.227
0.190
0.212
0.138
0.103
0.031
0.056
2.4.3
0.002
0.009
.Bayesian Analysis of Genetic Structure
In the Bayesian cluster analysis obtained from STRUCTURE, the model with the highest
K satisfactorily explained the data obtained from the AFLP analysis (Figure 9).
36
Figure 9: Bayesian inference clustering results for AFLP using the admixture ancestral model
in STRUCTURE software. The two genetic groups detected (clusters I and II) are represented
as pie charts (dark vs. light blue) on the google earth maps of all sampling locations.
In the estimated model based on the AFLP data set when K = 2, most individuals in the
Denmark (DKK, DKS), Stokkseyri (ST), Surtsey E, Surtsey K and Surtsey F locations
were assigned to cluster I; most of the individuals in the Greenland (GR), Gardur (G),
Surtsey C, Surtsey D, as well as Vestmannaeyjar north and south (BH & AH) locations
were assigned to cluster II. A similar pattern can also be seen in Figures 10 and 11. No
sub-structures were detected within any cluster as a result of further analysis.
37
Furthermore, the sample locations on the island of Surtsey showed a clear genetic split
between those on the north-west coast and those on the south-east coast, also supported
by the NJ Tree (Figure 10) and the multidimensional scale plot (MDS) (Figure 11).
2.4.4
Neighbor Joining Tree
The neighbor joining (NJ) tree based on genetic distances produced from the full data
set (Figure 10A) shows a split into two clusters (cluster I dark blue; cluster II light
blue). DKK, DKS, SK, ST, SE and SF group together in one cluster with bootstrap
values above 88% while GR, G, BH, SC, AH and SD group together in another cluster
with bootstrap values above 53%. This clustering shows a pattern similar to both the
MDS plot (Figure 11) and the Bayesian analysis (Figure 9), with the south-eastern side
of Surtsey (SK, SE and SF) clustering tightly together with Stokkseyri (ST) and closer
to the Denmark populations (DKK & DKS) than the populations sampled on the northwestern side of the island (SD & SC) which cluster closer to the Reykjanes peninsula
population (G), the Vestmannaeyjar populations (AH & BH) and the Greenland
population (GR). Looking closer at only the Surtsey population (Figure 10B), two
clusters, supported by bootstrap values over 98% can be seen in which the populations
on the north-west side of the island (SC & SD) cluster together (Nei´s D = 0.0044) and
clearly separate (Nei´s D = 0.0115) from the populations located on the south-east side
of the island (SF, SE and SK).
Figure 10: Un-rooted neighbor joining trees based on Nei´s genetic distances for; A) The full
data set and B) Surtsey only. Bootstrap values are shown at the nodes (10,000 iterations).
2.4.5
Multidimensional Scaling
The multidimensional scaling plot based on FST (Figure 11) shows SD, AH, SC and
GR clustering closely. A similar clustering can also be seen in the neighbor joining
(NJ) tree (Figure 10A) with SC, GR, BH and G forming a cluster that splits from the
other sample locations. Similarly, SK, ST, SE and SF cluster together in both the MDS
plot and the NJ tree. Furthermore, the genetic distance (Table 4) between SD and SC
(0.004) is less than half the genetic distance between SD and SF (0.012) and the
genetic differentiation (FST) between SD and SC (0.020) is also less than half the FST
between SD and SF (0.051), indicating a clear genetic split between the locations
sampled on Surtsey (Figures 10A & 10B).
Figure 11: Multidimensional scale (MDS) plot based on FST values
38
2.4.6
Mantel Test - Isolation By Distance
A mantel test on the full data set (not shown) indicated a positive and significant
association between Nei´s (1972) genetic distance and geographic distance (r = 0.6331,
p = 0.001) as well as genetic differentiation (FST) and geographic distance (r = 0.8083,
p = 0.001) (Figure 12a). This indicates isolation by distance with geographic distance
explaining roughly 80% of the variation in genetic differentiation. Non-significant
associations between genetic distance (Nei´s) and geographic distance as well as
between genetic differentiation (FST) and geographic distance were detected when the
populations within each site or region were compared (Figures 12b & 12c; Figures 13a
& 13c). This is an indication of significant gene flow and the limited effects of
isolation by distance at the regional and local scale.
However within the island of Surtsey, there appears to be a negative yet non-significant
correlation between geographic and genetic distances (Figures 13b & 13d). Although not
significant this is of some interest as negative spatial autocorrelations are indicative of a
pattern of dissimilar values (genotypes) (Manni et al., 2004) appearing in close spatial
association, a pattern also seen in the STRUCTURE analysis as well as the NJ Tree (Figures
9 and 10). Also, the mantel test between all locations (Denmark excluded, Figure 12b) and
Greenland revealed a smaller and less significant correlation than the mantel test between
all locations (Greenland excluded, Figure 12c) and the Denmark locations. This points to a
pattern of greater differentiation between the Denmark and Icelandic populations than
between the Greenland and Icelandic populations.
39
Figure 12: Plot of pairwise estimates of FST versus geographic distance between; a) all
samples of Honckenya peploides, b) mainland Iceland, Surtsey, Heimaey and Greenland, c)
Mainland Iceland, Surtsey, Heimaey and Denmark
40
Figure 13: Plot of pairwise estimates of Nei´s genetic distance (Top 2) and FST (Bottom 2)
versus geographic distance between samples of Honckenya peploides from; a & c) Mainland
Iceland, Surtsey and Heimaey, b & d) Surtsey only.
2.4.7 AMOVA
The AMOVA (Table 5) showed that variation within populations (sample locations)
accounted for most (77.01%) of the genetic variance. Variation among groups (areas or
regions) accounted for 17.40% of the total genetic variance and variation among
populations (sample locations) within groups (areas) accounted for only 5.59%.
Genetic differentiation among groups was significant (p < 0.01) and highly significant
among populations within groups as well as within populations (p < 0.001).
41
Table 5: Analysis of Molecular Variance.
Source of Variation
df
Variance
Variation (%)
Fixation Index
P
Among groups
4
3.23899
17.40
FCT = 0.17402
<0.01
Among populations within groups
7
1.04037
5.59
FSC = 0.06767
<0.001
Within populations
288
14.33320
77.01
FST = 0.22992
<0.001
Total
299
18.61256
5 geographic groups
Populations were arranged into five groups according to location; G1) DKK & DKS, G2) G & ST, G3)
All Surtsey (SC, SD, SE, SF, SK), G4) AH & BH, and G5) GR.
2.5
Discussion
Due to the isolated nature and restricted size of island habitats, population
establishment is predicted to result in some reduction of genetic diversity and/or a
population bottleneck (Barton and Charlesworth, 1984). Levels of genetic variation in
such systems are thought to be dictated mostly by the loss of genetic variation at
foundation, further loss due to finite population size since colonization and addition of
genetic variation due to immigration and mutation (Jaenike, 1973). Also, the number
of initial founders is predicted to have strong effects on levels of genetic variation,
especially if the initial number is low and the populations stay small for several
generations (Nei et al., 1975). As a result, island populations usually have lower
genetic diversity than their mainland counterparts. This relationship however, depends
on the intensity of isolation, island size and dispersal capabilities of the organism in
question (Frankham, 1997). Although in general there are significant losses of both
allelic richness and heterozygosity in introduced populations, and large gains in
diversity are rare, multiple introductions have been shown to rescue the losses in
diversity in a wide range of plant, animal and fungal taxa (reviewed in Dlugosch and
Parker, 2008). Genetic diversity or allelic variation also appears to increase over long
time scales, suggesting a role for gene flow in augmenting diversity over the long-term
(e.g. Désamoré et al., 2012).
2.5.1
Patterns of Diversity on Surtsey
Versus Other Sites
Contrary to original expectations, genetic diversity (HE) was found to be significantly
higher in the Surtsey and Iceland populations than in the Denmark populations.
Furthermore, when compared to Iceland, genetic diversity remains quite similar and is
even increased in some Surtsey populations. The greater population size, age and
wider geographic range of H. peploides in both Iceland and Denmark is expected to
lead to higher genetic variation in those areas (Nei et al., 1975). However, the findings
of this study contradict this and shed some doubt on the long held view that genetic
diversity is generally lower in oceanic island populations (Frankham, 1997; Franks,
2010). Furthermore the present results, in parallel with a growing number of studies,
find either similar (Su et al., 2010; García-Verdugo et al., 2013) or greater (FernándezMazuecos and Vargas, 2011; Désamoré et al., 2012) genetic diversity in oceanic island
species when comparing them to their mainland counterparts. We therefore postulate
that the resulting genetic structure on Surtsey and overall patterns of genetic diversity
42
during colonization of oceanic islands for that matter, are a more complex than
previously thought (Jaenike, 1973) and are likely driven by a dynamic interaction
between multiple spatial, temporal and life history variables.
Facilitated by the species long distance dispersal capabilities, the colonization of H.
peploides on Surtsey starts with a few individuals and increases rather quickly to cover
most of the empty niches on the island (Sigurdsson and Magnusson, 2010). The
dispersal distance from mainland Iceland or any other islands in the archipelago
(<35km) is most likely not a huge barrier to dispersal in this group (Jonsell, 2001).
Furthermore, seeds of H. peploides are relatively small and are not prone to desiccation
by salt water as the plant is a beach dune halophyte and actually requires some form of
cold stratification and surface abrasion in order to break dormancy (Baskin and Baskin,
2001; see chapter 3). These dispersal characteristics likely promote enough gene flow
between populations to negate the effects of isolation by distance at the regional scale.
The breeding system, possibly in conjunction with dormancy requirements, then drives
differentiation at the local scale. On Surtsey as well as on Heimaey the populations
remain significantly differentiated despite their close proximity.
The significant genetic distance and differentiation found among the Surtsey
populations indicates that the influx of genetic material is likely attributable to multiple
introductions from various source locations. Multiple introductions increase both the
effective population size and the population growth rate, and have been shown to lead
to an increase in gene diversity in newly colonized populations (Dlugosch and Parker,
2008). Further increases in gene diversity may have also been promoted due to
differentiation across the geographical distribution of populations in the source regions
(Ellstrand and Schierenbeck, 2000). Moreover, multiple introductions followed by
gene flow between formerly differentiated populations has been shown to result in
levels of diversity that exceed those in the source populations (Novak and Mack, 2005;
Dlugosch and Parker, 2008).
Therefore it seems probable that long distance dispersal from multiple locations is a
key driver of the current genetic structure on Surtsey and that H. peploides populations
in the Iceland region are highly genetically similar (panmictic). The negative genetic
consequences associated with colonization (e.g. the bottleneck effect or genetic drift)
are likely reduced through the constant influx of new genetic material (Nei et al., 1975;
Barrett and Husband, 1990). The maintenance and/or expansion of populations with
high gene diversity on the island is then most likely fostered by: a) the vast empty
niche space available there (Friðriksson and Johnsen, 1968); b) the sea barrier
inhibiting establishment of other (probably competing) dispersal groups; c) the
polyploid nature of the study species which is accompanied by fixed heterozygosity
that maintains gene diversity (Soltis and Soltis, 2000; Brochmann et al., 2004); and d)
the breeding system of the plant itself preventing successful self-fertilization (Tsukui
and Sugawara, 1992).
2.5.2
Genetic Differentiation Among
Populations and Regions
At the level of intra-island genetic diversity, genetic differentiation (FST) on both the
island of Surtsey as well as on Heimaey is only moderate but highly significant. This
indicates some population genetic structure with strong gene flow likely preventing
43
divergence within the islands (see Wright 1978). However, genetic differentiation on
mainland Iceland is great and indicates that there is strong genetic structuring on the
island and that limited gene flow between the two populations could promote
divergence (Wright 1978). This strong differentiation is likely due to environmental
heterogeniety and differential selection pressures (Audigeos et al., 2013; Pannell and
Fields, 2014) as sampling locations in mainland Iceland are much older and seperated
by roughly 85 km of highly variable, mountainous topography. On the other hand,
sampling locations within Surtsey and Heimaey are relatively recent and quite
proximal (~1 & 5 km apart respectively), with limited topography seperating them.
At the global scale, the greater FST between sampling locations leads us to believe that
the dispersal of H. peploides over its entire range is similar to the propagule model in
which the founding event is severe with few colonists coming from a single source
(see Slatkin, 1977). This is thought to result in increased differentiation between
locations. On the other hand, the FST between regional locations is far less. This paints
a picture which is more similar to the migrant model in which colonization is the result
of multiple immigration events from several populations (Slatkin, 1977), resulting in
greater population cohesion. This spatial variation in intensity of genetic differentiation
is once again most likely explained by the plants dispersal ability and breeding system.
Mantel tests of the full data set revealed a positive and highly significant correlation
between genetic distance/differentiation and physical distance. Furthermore, a Mantel
test that excluded only Denmark populations revealed a smaller and less significant
correlation than the mantel test which excluded only the Greenland population. This
indicates that gene flow between Denmark and all other sample locations is quite
limited and that there is significant isolation by distance at the global scale, similar to
the propagule model postulated by Slatkin (1977). Moreover, this also implies that
isolation by distance is affecting gene flow between the Greenland and Iceland
locations to a lesser degree than it is affecting gene flow between all the Iceland and
Denmark locations. Furthermore, similar to Slatkins' (1977) migrant model, the nonsignificant mantel tests of select regions and locations indicate that there is significant
gene flow between all locations except Denmark. These results parallel other studies
which suggest that the presence of IBD (isolation by distance) patterns depends greatly
on the spatial scale in question (Gaudeul et al., 2000; Stehlik et al., 2001; Ægisdóttir et
al., 2009; Eidesen et al., 2013).
One possible explanation for this is that, similar to other plants found in Iceland
(Thórsson et al., 2010), Western Europe is the source region from which the original
genetic material was obtained. The most probable means of dispersal likely being drift
via the North Atlantic current during the early Holocene (Buckland et al., 1986).
Through time and space, the Iceland/Greenland region has become genetically isolated
and is possibly still in the early stages of establishment compared to the Denmark
populations. Recent molecular studies also support this view, showing that circumpolar
species such as Juniperus (Adams et al., 2003), Saxifraga (Abbott, 2000) and
Vaccinium (Alsos et al., 2005) are of Eurasian (western Siberia) origin. These studies
show that circumpolar species tend to split into Eurasian and North American lineages.
The Eurasian lineages often have distributions spanning from Eurasia and northern
Scandinavia to Iceland and Greenland. However an analysis of the North American
biota revealed that the majority of endemic species are a result of extensive postglacial migration (Brochmann et al., 2003; Alsos et al., 2007).
44
Using only data from Surtsey, Mantel tests revealed a negative and non-significant
correlation between genetic distance/differentiation and physical distance. This
negative spatial correlation, although not significant, may suggest that at such a small
scale, dissimilar genotypes are occurring in close spatial association to one another,
like that found in other studies (Matesanz et al., 2011; Diniz-Filho and Bini, 2012;
Diniz-Filho et al., 2013). This is also supported by the genetic structure seen in the unrooted NJ tree, the moderate genetic differentiation (FST) found between locations on
Surtsey and the bayesian analysis. This is a further indication that the breeding system
and outcrossing nature of the plant might be keeping inbreeding depression in check
via reduced fitness of self-fertilized embryos.
In accordance with this, an analysis of molecular variance indicates that despite the
clonal capabilities of H. peploides, most of the genetic variance is within populations
(sampling locations). These results parallel similar studies on H. peploides conducted
in Spain and Portugal (Sánchez-Vilas et al., 2010). This high degree of within
population genetic variation is likely explained once again by the outcrossing nature of
the plant itself. Unpublished greenhouse experiments show that very few, small seeds
are produced via active self-fertilization by hand, no seeds are produced by autodeposition and active outcrossing produces a much higher seed set than open
pollination (see section 2.2.1). We therefore postulate that the variable success of long
distance dispersal of the species maintains great genetic differentiation at the global
scale while the higher success of dispersal at the regional scale maintains population
cohesion and negates IBD. The outcrossing nature of the plant then acts as a buffer to
maintain moderate genetic differentiation at the local scale.
2.5.3
Phylogeography of H. peploides in
the Iceland Region
There is a clear northwest-southeast split in the Surtsey populations. This clear split
found with the Bayesian analysis on the island of Surtsey and in the data set as a whole
is well supported by both the un-rooted NJ tree as well as the MDS analysis. Once
again, this, along with the moderate differentiation found, indicates multiple
colonization events to Surtsey from several sources as well as a clear genetic split
between the Iceland/Greenland and Denmark locations. This is evident from the fact
that populations from Garður, Greenland, Heimaey and the western coast of Surtsey
(SC & SD) are clearly differentiated from the Denmark populations. While Stokkseyri
(ST) and the locations on the eastern side of Surtsey (SK, SE & SF) show an
intermediate between the Denmark and Greenland locations, with greater membership
in the same genotype cluster as Denmark.
It is therefore most probable, as stated above, that the colonization of Iceland by H.
peploides took place from the east and that through time and space the
Iceland/Greenland populations have become isolated and diverged substantially from
the Denmark populations. The initial migration to the region could have taken place in
a stepping stone like fashion via drift on ice and wood during the early Holocene
(Johansen and Hytteborn, 2008). As suspected by Magnússon et al. (1996) and
revealed here by our genetic structure analysis, the colonization of Surtsey likely took
place from several source locations including Heimaey, the Reykjanes peninsula and
the southern coast of Iceland. Judging from the bayesian analysis, NJ tree, MDS plot
and FST, the populations closer to the Reykjanes peninsula and from Heimaey are the
45
most likely source of genetic material for the populations on the western side of the
island (SC & SD). The southern shore of mainland Iceland is the most probable source
of colonists for the eastern side of the island (SK, SE & SF).
The full NJ tree shows two distinct clusters which indicate a similar pattern as was
found with the Bayesian analysis. However, the tree gives us clearer resolution on the
relationship that the Denmark populations have with the rest of the samples, indicating
a very clear separation from the Iceland/Greenland region. This relationship can most
likely be attributed to the time since colonization of Iceland by H. peploides from
Europe following the end of the last glacial maximum (LGM) roughly 10,000 years
ago (Hallsdóttir, 1995). As the plant colonized islands along the way from mainland
Europe to the Iceland region it likely encountered variable habitats which might have
favored survival of diverse genotypes not favored by mainland conditions. The
dispersal of these genotypes in a stepping stone fashion towards the Iceland region
could then have slowly increased divergence from the Denmark populations (Le Corre
and Kremer, 1998). This could also have contributed to the increased gene diversity as
the availability of nearby sources of variation following colonization has been shown
to mitigate founder effects in newly colonized populations (Helsen et al., 2013)
The Surtsey NJ tree also shows two distinct clusters which indicate that there is
significant genetic structure on the small island. This is likely explained by the
relatively young age of the island itself and by multiple colonization events combining
genotypes of differentiated populations from the source regions (Ellstrand and
Schierenbeck, 2000; Novak and Mack, 2005). The original reports of plant
colonization to the island indicate that H. peploides initially colonized the northeastern
shore (Friðriksson, 1970). This report indicates that during the summer of 1968 at
location SK (F15 in report) there were two plants (plants # 28 & 34) which had five
and two branches respectively. Likewise at site SD (B13 in report) there were two
plants (plants # 75 & 76); however these plants had only one stem each meaning that
they were younger. This indicates several colonization events, possibly at differing
times as none of the plants had produced seed yet. This could be a feasible explanation
for the significant genetic distance and differentiation found between sites on the
island.
Also, two sampling locations on the northwest side of the island of Surtsey (SC & SD),
which are separated by a distance of up to 800 m and a 100 m high volcanic crater,
have a genetic distance that is less than half the genetic distance between SD and the
closest sampling location, SK, located less than 150 m away. These sampling locations
were colonized at different times. The SD location was colonized first in 1968 and the
SC (I8 in report) location was not colonized until after 1978 (Friðriksson, 1982). The
genetic similarity of SC and SD could possbly indicate that seed movement and spread
of H. peploides within the island of Surtsey is being facilitated by seabirds or
migratory passerines such as the snow bunting (Plectrophenax nivalis). This is a
feasible scenario as previously Friðriksson (1975) reported finding seeds (not H.
peploides) in the digestive tracts of P. nivalis caught on the island, suggesting that
these birds are capable of distributing seeds long distances. These seeds were later
germinated and produced viable offspring (S. Friðriksson, pers. comm., 2013).
Moreover, tagged P. nivalis from as far away as Norfolk England have been sighted
near Stokkeyri in southern Iceland, Akureyri in the north and Seyðisfjörður in the east
46
(Middleton, 2012) and it is quite probable that these same birds use Surtsey as a
stopping point on their long journey from western Europe.
2.5.4
Present Research and Future Prospects
It is through the dynamic interplay of spatio-temporal factors, breeding systems, seed
dispersal and polyploidy that heterozygosity is maintained in colonizing arctic plant
populations and the recently colonized Honckenya peploides population on Surtsey is
no exception. Despite being faced with inbreeding and bottlenecks these populations
still have the ability to maintain relatively high heterozygosity. It is likly that
numerous, long distance seed dispersal events from multiple differentiated sources
have lead to high genetic diversity during colonization. Heterozygosity was then
maintained by the outcrossing nature of the plants dioecious breeding system and its
tetraploid genomic constitution. In polyploid plants, this guarantees genetic variation
through fixed heterozygosity in duplicated genomes (Brochmann et al., 2004; Soltis
and Soltis, 2000).
When such populations are confronted with various novel, heterogeneous
environments, the presence of duplicated genomes within each individual allows for
selection to act on a greater number of available traits (Soltis and Soltis, 2000). This
increases survival of diverse genotypes in variable niches. Such a statement has some
implications for conservation efforts as it implies that conservation strategies in the
Arctic should be focused on large numbers of small, spatially separated populations
and the migratory-dispersal routes separating them rather than a small number of large
populations. This will maintain pockets of diversity that can then be utilized in a
similar manner as seed banks (León-Lobos et al., 2012). Such an effort can assure
influxes of diverse genetic material for populations struggling to adapt to
anthropogenic environmental changes, helping to secure ecological diversity for
coming generations (Vitt et al., 2010).
The inferences drawn from the preceeding analysis should be further validated with a
more detailed investigation combining the methods used above with detailed
cytogenetic, physiological and morphometric analysis. Such studies should sample
populations from all shoreline areas of Iceland and compare them with populations
from Greenland, Denmark, Norway, Northern Siberia, Spain, Portugal as well as the
Shetland, Orkney and Faroe islands. If only sampling is considered, such an
investigation is quite realistic and rather cost effective. Collaborations with other
scientists can reduce travel expenses and consolidate time so that additional funding
can be utilized to improve analytical methods. A phylogeography study of this scale
will give a more comprehensive understanding of the dispersal history of H. peploides
in the north Atlantic region, provide valuable insight into the long standing glacial
“refugia” debate (reviewed by Habel et al., 2010) as well as perhaps most importantly
provide a greater and novel understanding of the processes which drive the evolution
of gene diversity.
47
3 Karyotype Variation of Honckenya
peploides Worldwide
S. H. Árnason and Kesara Anamthawat-Jónsson
Institute of Life and Environmental Sciences, University of Iceland, Askja Sturlugata
7, Reykjavík, IS-101, Iceland
3.1
Introduction
The global morphology of the nuclear genome displays great variation among plant
species (Johnson et al., 2003; Stuessy et al., 2004; Perný et al., 2005; Suda et al., 2007;
Sonnleitner et al., 2010; Travnicek et al., 2010; Spaniel et al., 2011;). As effective
plant cytological methods are being developed (e.g. Anamthawat-Jónsson, 2004) they
are increasingly being used to explore ploidy levels and chromosome morphology
within plant groups. The presence of widespread cytotypic and karyotypic variation
within plant species is causing some re-evaluation of previously held assumptions on
the degree of intra-specific karyotype variation found in plants (Delaney and Baack,
2012; Fehlberg and Ferguson, 2012; Kono et al., 2012). For some time the prevailing
paradigm has been that a species has only one chromosome number, ploidy level and
karyotype structure. This was thought to be due to the fact that variable chromosome
numbers usually bring about meiotic irregularities and/or sterility (Levin, 2002;
Stebbins, 1971). However, recent studies have identified plant species which have
more than one diploid somatic chromosome number, most frequently due to
intraspecific polyploidy (Frello and Heslop-Harrison, 2000; Stuessy et al., 2004; Suda
et al., 2007). Yet other species have shown complex paterns of chromosome numbers
thought to have arrisen through a dysploid series at the diploid level accompanied by
hybridization and polyploidy. These include Prospero autumnale (Hyacinthaceae)
which has 2n = 10, 12, 14, 25, 26, 27, 28, 42 (Ainsworth et al., 1983; Ebert et al.,
1996) and Scilla scilloides (Hyacinthaceae) with 2n = 16, 18, 26, 27, 34, 35, 36, 43
(Haga and Noda, 1976; Choi et al., 2008), to name a few. These intraspecific
karyotype variants have been termed cytotypes or cytoraces (Stuessy, 2009). Some
even suggest that various reproductively isolated cytotypes within species may indeed
represent cryptic species (Soltis et al., 2007) as various cytotypes might be found
allopatrically, parapatrically, or in mixed populations.
Slight morphological differences among cytotypes within species, particularly in floral
and fruit characteristics have been shown in several studies (Perný et al., 2005; Cires et
al., 2009; Spaniel et al., 2011). Investigations into the Crowberry, Empetrum, from
locations in the Czech Republic found that cytotype is strongly associated with flower
sex, with diploids having unisexual flowers and the tetraploids having exclusively
bisexual flowers (Suda et al., 2004). Also, in some instances cytotypic differences can
lead to changes in pollinator species, creating limitations on gene flow between
cytotypes. For instance, varying cytotypes of the rhizomatous herb, Alumroot,
Heuchera grossulariifolia, growing sympatrically in Northern Idaho have been shown
49
to attract differing pollinators. Diploid and autotetraploid H. grossulariifolia plants act
essentially as separate ecological species and may experience partial reproductive
isolation through differential visitation and pollination by their floral pollinators
(Thompson and Merg, 2008). Such morphological and physiological variation among
different cytotypes can lead to the use of diverse ecological niches (Johnson et al.,
2003), often leading to the reduction of one or more of the cytotypes or to further
genetic differentiation between them (Garcia et al., 2008). In other cases there may be
no detectable differences in appearance among cytotypes, but higher ploidy simply
provides the plants with the ability to cope with environmental stresses which is an
adaptive advantage (Leitch and Leitch, 2008). Numerous series of cytotypes and
polyploid complex via hybridization have been evident among alpine-arctic plants, e.g.
in Brassicaceae (Grundt et al., 2005).
Four subspecies of Honckenya peploides have been described. These include: subsp.
peploides which is found on the coasts of northern Norway down to northern Portugal;
subsp. diffusa which has a circumpolar distribution mainly in Arctic and northern
Boreal zones; subsp. robusta, found in Northeastern North America; subsp.
major/oblangifolia, found in the North West Pacific area and on the coasts of north
eastern Russia down to Japan (Houle, 1997; Jonsell, 2001; Probatova et al., 2004) (see
Section 1.6.1). Studies from several locations over the whole of the species range
report varying chromosome numbers (2n = 4x = 66, 68, 70) as well as possible
differing ploidy levels including 2n = 34 (Probatova et al., 2004) (see Table 6).
Table 6: All published chromosome numbers for Honckenya peploides.
Location
Source
2n =
Svalbard
Flovik (1940)
66
Dragor, Denmark
Malling (1957)
68
Sakhalin Island, Russia
Sokolovskaya & Strelkova (1960)
66, 68, 70
Wrangle Island, Russia
Agapova (1993)
66, 68, 70
North Eastern Russia
Zhukova (1966)
68
Eastern Russia
Zhukova (1966)
66
Moneron Island, Russia
Probatova (2004)
34*
North Eastern Asia
Zhukova (1966)
68
Ogotoruk Creek, N.W. Alaska
Johnson & Packer (1968)
68
Meade River, N. Alaska
Packer & McPherson (1974)
68
Western Alaska
Murray & Kelso (1997)
68
Arctic Canada
Löve & Löve (1982)
68
Iceland
Löve & Löve (1950, 1956, 1970)
Sweden
* indicates diploid.
Lövkvist and Hultgård (1999)
66, 68, 70
68
The relationship between this cytotypic variation and variation in physiological,
morphological or life history characteristics remains largely unexplored. For example,
seed germination of the diploid (2n = 34) H. peploides subsp. oblongafolia/major occurred
50
within three weeks at temperatures of no less than 28°C without prior cold stratification
(Voronkova et al., 2011). However seeds of the tetraploid (2n = 68) H. peploides begain to
germinate only after cold stratification for up to 12 weeks at 2°C and germinated only at
temperatures of 5 - 15°C (Baskin and Baskin, 2001; S. H. Árnason, unpublished work).
Variation in morphology can also be seen between H. peploides in Greenland, Svalbard
and Norway, with the Svalbard ecotypes being generally smaller in all traits measured, as
well as having greater genetic diversity (M. Philipp, pers. comm.). Furthermore, on the
island of Iceland, three different chromosome numbers have been reported by Löve and
Löve (1950, 1956, 1970), yet to the authors knowledge, no investigation has been carried
out which attempts to explain these differences in karyotype using physiological,
morphological or life history variables.
In light of these apparent cytological uncertainties in the species as a whole,
cytogenetic methods used to characterize genome structure such as karyotyping and
fluorescence in situ hybridization (FISH) should prove to be valuable tools. Such
investigations can shed light on the roles that karyotype variation together with
environmental heterogeneity plays in the evolution of species (Lodh and Basu, 2013).
Differences in chromosome number, form and size within species can have substantial
phenotypic consequences (Balao et al., 2011) as the nuclear DNA content itself is
known to influence every stage of development, from the cellular to the somatic
(Richards, 1997). These differences often lead to differential survival of favorable
ecotypes, resulting in variable spatio-temporal distributions both globally and locally
(Ray, 2010; Bennett and Leitch, 2011). Therefore, karyotypes combined with
molecular phylogenetic analysis can provide information about taxonomic
relationships, ecotypic variation, genetic peculiarities and the evolutionary origins of
species (Watanabe et al., 1999; Ruffini Castiglione et al., 2010; Lysak and Koch,
2011). Furthermore, chromosome size differences, arm-length ratios and FISH can be
used to distinguish identifying features and loci between ecotypes (Truta et al., 2010;
Ebadi-Almas et al., 2012; Young et al., 2012), furthering our understanding of the
effects that karyotypic variation has on population genetic differentiation.
In order to obtain chromosome spreads suitable for karyotyping using the enzymatic
root tip squash method, the chromosomes must be very well spread apart and the
background clear of fragments of cytoplasm (Anamthawat-Jónsson, 2004). The cells
from which the chromosomes are obtained must be arrested in metaphase and be
appropriately condensed and free of damage. In order to obtain such chromosomes the
parts of the plant containing actively dividing cells such as the root tips, leaf
meristems, calli or protoplasts must be used. To obtain information regarding
chromosome number and karyotype variation between ecotypes, individuals of H.
peploides were chosen for chromosome isolation using the enzymatic root tip squash
method and stained using DAPI (4 ’,6-diamidino-2-phenylindole), a fluorescent stain
that binds to the A-T rich regions of DNA. The data obtained can provide a good basis
for distinguishing between subpopulations and will provide insight into the
mechanisms driving population differentiation.
The objectives of this study were as follows: (i) determine the chromosome number for
populations of H. peploides located on the Seltjarnarnes Peninsula, in Reykjavík Iceland;
(ii) obtain a collection of individual representatives for further studies intended to examine
karyotype variation in the worldwide distribution of the species; (iii) determine the proper
protocol for germination of H. peploides seed from the sample locations.
51
3.2
3.2.1
Materials and Methods
Plant Material
Samples for karyotyping were collected during September 2012 from Seltjarnarnes
peninsula (64° 09 N / 22° 01 W) in Reykjavík, Iceland. Live plants were placed into
plastic containers with the native substrate and kept at 15°C under a grow lamp with a
12/12 light/dark regime. In order to obtain plant material spanning the worldwide
distribution of the species, a correspondence with multiple research institutions around
the globe was initiated by the author SHÁ. Using this method, seeds of Honckenya
peploides were obtained from Keibu, Estonia (5 ° 4’ 4 .2 ” N, 23° 3 ’ 50. ” E),
Kolobrzeg, Poland (54° ’ 00.5 ” N, 6° 0 ’ 45. ” E), Cornwall, England (50° 32’
45.26”N, 5° 02’ 20.00W), Maryport, Scotland (54° 40’ 3.23” N, 4° 52’ 52.68” W),
Madeleine Island, Canada (4 ° 23’ 44. 4” N, 6 ° 50’ 58. 0” W), Miquelon Island,
Canada (4 ° 04’ 32” N, 56° 22’ 46.6 ” W) and Cold Bay, Alaska (55° 2’ 00.2 ” N,
62° 42’ 4 .25” W) (Figure 14). These seeds were treated to cold stratification,
germinated and grown under 12/12 light/dark regime. All plants were maintained and
grown in the Plant Genetics research laboratory growth room located on the second
floor of the Natural Sciences building at the University of Iceland, Reykjavík.
Figure 15: Collection locations worldwide. From east to west: Keibu, Estonia; Kolobrzeg,
Poland; Cornwall, England; Maryport, Scotland; Madeleine Island, Canada; Miquelon
Island, Canada; Cold Bay, Alaska. Map from Google Earth.
52
3.2.2
Seed Stratification, Sterilization and Germination
Seeds of H. peploides obtained from the various locations (Figure 14) were split into
two groups, those to undergo stratification and those to be germinated right away. For
each location, one half was stratified by placement into plastic bags with a moist,
sandy medium and refrigerated at 2 - 4°C for 12 weeks. The other half of the seeds
obtained from each location was sterilized right away using the procedure described
below and placed into germination directly after. Seeds that had been stratified for up
to 12 weeks as well as those that were germinated right away were sterilized by
placement in 5% CL solution for 2 minutes, then rinsed with distilled water and placed
in a Petri dish between two sterile filter papers. Petri dishes were then placed under a
12/12 light regime at 15°C during light hours and then moved into a dark refrigerator
and kept at 5°C during dark hours until germination. Germination success was
estimated visually and noted as % germination per plate. Once germinated, five
seedlings from each location were placed in plastic pots with a mixture of sand and
soil, labeled and separated according to location of collection. Root tips were then
collected from the Seltjarnarnes individuals in order to obtain chromosomes for
karyotyping using the enzymatic root tip squash and DAPI fluorochrome staining
method described below. Pending further funding for a more detailed analysis of
global karyotype variation using FISH, individuals from the other locations are being
stored at the Plant Genetics Laboratory growth room at the University of Iceland.
3.2.3 Enzymatic Root Tip Squash Method
Enzymatic root tip squash was performed according to Anamthawat-Jónsson (2010).
Young root tips were harvested from the live plants at mid-day. Root tips were cut to
approx. 1 - 3 cm. and placed in 15 mL tubes with ice water and kept at 4°C with ice for
24 - 27 h in order to synchronize mitosis and arrest as many cells as possible in
metaphase. Root tips were then placed in another 15 mL tube containing a fixative
solution of glacial acetic acid/96% ethanol (1:3) and allowed to sit at room temeprature
for about two hours before keeping them in a freezer at -28°C. After fixation, root tips
were submerged in an enzyme buffer twice in 20 minutes at room temperature and
then placed into an enzyme mixture at 37°C for 30 minutes. Roots were then
submerged again in the enzyme buffer and the solution changed after 5 minutes. Root
tips were then placed one at a time onto microscope slides that had been pre-cleaned
with chromic acid (chromium trioxide in 80% sulphuric acid) for 3 hours, rinsed
thoroughly in tap water and placed in 96% EtOH before use. The ends of the roots
were removed, leaving only the tip (1 - 2 mm) containing the meristematic tissue on
the slide. The slide was then placed under a stereo microscope at 20x magnification, a
drop of 45% acetic acid added and the meristem teased apart, allowing the mitotically
dividing cells to flow into the acetic acid. A cover slip was then placed on top and
pressed flat. The slides were analyzed for quality in a phase contrast microscope. Good
quality slides were then dipped in liquid nitrogen, the cover slip removed and slides
stored in an air tight storage box at 4°C until further use. Prepared slides were stained
with 20 μL of the fluorochrome DAPI (1 μL/ml) (4 ’,6-diamidino-2-phenylindole) for
1 minute and viewed under Nikon Eclipse 800 epifluorescent microscope using a UV
filter block with 340-380 excitation and 430-450 emission wavelengths (blue). The
images obtained with the 100x objective were captured with a CCD camera and used
for chromosome number determination and karyotyping.
53
3.3
3.3.1
Results
Seed Stratification, Sterilization and Germination
The sterilization procedure proved effective at removing most bacterial, mold or fungal
contaminants from the seed coat as only 7.1% (22 of 311) of Petri dishes containing
germinating seeds were contaminated. Of the unstratified seeds, only the samples
obtained from Cold Bay, Alaska, germinated under the 12/12, 5°C/15°C conditions,
with most germination occurring within five to seven days. None of the seeds obtained
from other locations germinated without prior cold stratification. Germination rates
following cold stratification for 12 weeks were quite high, with roughly an 80%
germination rate in all the non-contaminated plates at 12/12, 5°C/15°C.
3.3.2
Karyotype
After root tip samples had been prepared and stained with DAPI the slides were
scanned at 200-400x magnification to fine metaphases and then at 1000x magnification
to obtain the chromosome number. Microscope pictures indicate the presence of 68
chromosomes, such as the metaphase shown here (Figure 16a). In this cell, two
chromosomes with satellites are indicated and this is clearly the pair of homologous
chromosomes carrying active ribosomal loci NOR, Nucleolar Organizing Region
(Figure 16b). Chromosome sizes ranged from 1.36 μm – 4.66 μm and most seem to be
metacentric and sub-metacentric. A karyotype constructed shows chromosomes lined
up by size and paired with possible homologs (Figure 16c).
Figure 14: Honckenya peploides chromosomes stained with DAPI taken at 1000x
magnification (a), with drawn outlines (b) and karyotypically arranged. Arrows indicate
locations of satellites
54
3.4
3.4.1
Discussion
Significance of Chromosome Numbers
A total of 68 chromosomes were counted for H. peploides from Seltjarnarnes
peninsula, Iceland. This is the same number as Malling (1957) reported for plants from
N. America, Germany and Denmark. Furthermore it is also one of the three values that
Löve & Löve (1956) found for locations in Iceland. These results, lead us to believe
that 2n (4x) = 68 is an intermediate cytotype in Iceland and that this is one of three
tetraploid cytotypes found on the island (i.e. 2n = 66, 68, 70). Such thinking is not far
fetched as a survey of a wide range of species reports that 12-13% of all angiosperms
display some cytotypic variability (Wood et al., 2009). Furthermore, a litterature
review of cytotype comparisons from the sub-species H. oblongafolia (Probatova,
2004) from the western Pacific region revealed a possible genome duplication event.
Populations on the southern shores of the island of Moneron (~46°N, 141°E) were
found to be diploid (2n = 34), however populations from the nearby island of Sakhalin
(~50°N, 142°E) were found to be tetraploid with two cytotypes (2n = 4x = 68, 70)
(Sokolovskaya & Strelkova, 1960). Moreover, populations from the more northerly
Wrangle island (~71°N, 179°E) were found to be tetraploid and contained three
cytotypes (2n = 4x = 66, 68, 70) (Agapova, 1993; Goldblatt, 1985).
The co-existance of such variable cytotypes could be facilitated by ecological barriers
to gene flow or by numerous biological reproductive barriers which prevent
hybridization between proximal cytotypes (Husband and Sabara, 2003). Such barriers
can be present at various steps of reproduction. For instance, differences in both insect
pollinator composition (Seagraves and Thompson, 1999) and foraging patterns
(Kennedy et al., 2006) was found to reduce pollen exchange between cytotypes of
Heuchera grossulariifoli and Chamerion angustifolium respectively. Moreover,
cytotype hybridization is also thought to be prevented by gametophytic selection via
polen/ovule non-conformity (Howard et al., 1998). Also, spatial division of cytotypes
via ecological segregation is often observed (Bretagnolle and Thompson, 1995; FelberGirard et al., 1996; Duchoslav et al., 2010; Sonnleitner et al., 2010) and is thought to
have a large influence on ploidy coexistence.
The spatial relationship between ploidy states within this study species can be
characterized as either sympatric, parapatric or allopatric. A sympatric and/or parapatric
cytotype distribution is likely found on the island of Iceland, resulting in several cytotypes
on the island. This is possibly the result of some yet unknown physiological or
morphological reproductive barriers. Changes in morphological or physiological
characteristics are frequently accompanied by changes in cytotype, often resulting in
ecological differentiation between cytotypes (Levin, 2002). An allopatric cytotype
distribution is likely found in the south-western pacific region, resulting from geographical
barrier between ploidy groups that maintains the diploid and tetraploid cytotypes.
Examples of all three forms of cytotype distribution have previously been described in the
literature. An extreme case of allopatric distribution can be found with one pantropical
species, Nymphoides indica. This aquatic plant of the Menyanthaceae family is diploid in
the Old World but tetraploid in the New World (Ornduff, 1970). In comparison, a
sympatric distribution was found throughout the overlapping range of the diploid and
tetraploid cytotypes of the Blue Ridge Mountain endemic Galax ureolata (Burton and
Husband, 1999). Lastly, an example of parapatry can be found in the Central European
55
distribution of Vicia cracca in which tetraploid cytotypes are found in the western limits of
the species range and diploids in the east (Travnicek et al., 2010).
The abundance of polyploidy within plants has led some to speculate as to the effects
of genome duplication on species diversification (Otto and Whitton, 2000; Leitch and
Leitch, 2008). Some argue that polyploidy simply accumulates as a unidirectional
mutation, having little influence on the degree or direction of evolutionary divergence
(Stebbins, 1971; Meyers and Levin, 2006). Proponents of this view hypothesize that
the evolution of polyploidy is a one-way process in which chromosome number can
increase but not decrease. If reversals do take place, the haploids derived are quite rare,
have reduced fitness and high sterility (Stebbins, 1980; Ramsey and Schemske, 2002).
Others propose that evolutionary diversification may be enhanced by polyploidy and
that it could lead to elevated levels of divergence and species richness (Otto and
Whitton, 2000). Elevated diversification could take place if polyploidy increases the
rate of adaptive divergence and the evolution of reproductive isolation or decreases
species extinctions (Otto and Whitton, 2000). Furthermore, the capacity to differentiate
extra gene copies (sub/neo-functionalization) or the effects of genomic restructuring on
current genetic and phenotypic variation could enhance such divergence (Schranz and
Osborn, 2004; Tate et al., 2005). Polyploidy is widespread in the flowering plants
(Bennett, 2004) and the success of polyploids is known to be attributable to their
highly plastic genome structure and the ability to restructure in response to external
changes (Leitch and Leitch, 2008).
Variation in the cytotype of Honckenya peploides likely contributes in some way to the
morphological variation found within the species. Slight morphological variability has
led to the classification of the four recognized sub-species (Jonsell, 2001). However,
since morphological divergence is often used as a criterion for sub-species delineation,
some of the reported polyploid lineages may not be recognized at the sub-species level.
This is especially true for autopolyploids such as this as they have a close resemblance
to their diploid progenitors (Soltis et al., 2007). A more indepth cytogenetic study
conducted throughout the species range is likely to confirm the existing cytotypes and
may reveal more cytotypes than have been reported so far.
3.4.2
Future Prospects
Further studies must be conducted on the individuals grown from seed from all
locations and new locations must be sampled throughout the species range. The use of
chromosome banding should assist in the evaluation of the global variation in
karyotype for the species (Guerra, 2008; Jang et al., 2013). Furthermore, correlating
this variation with individual morphological features as well as environmental aspects
from the plants with different origins will further our understanding of the mechanisms
influencing changes in karyotype morphology, in turn shedding further light on the
factors driving population differentiation and speciation similar to that found in other
plant groups ( Li et al., 2010; Balao et al., 2011; Schmickl and Koch, 2011). In the
future we would like to use Fluorescent in situ hybridization (FISH), specifically
targeting the highly conserved 45S and 5S ribosomal regions in order to study H.
peploides karyotype evolution. In numerous plant groups the FISH mapping of these
ribosomal genes has been shown to have a very high physical resolution for
cytotaxonomic purposes (e.g. Taketa et al., 2005; Chokchaichamnankit et al., 2008;
Barros e Silva et al., 2013). Morphometric data on Honckenya peploides attained from
56
this pilot study and environmental data from the original habitats would then be
correlated with cytotype variation in order to paint a clearer picture of what is driving
diversity within this system. Combining these data with similar analysis of Schiedea, a
sister genus of herbs, vines, and small shrubs endemic to the Hawaiian Islands,
consisting of 34 known species which are all thought to have arisen from a single
colonization event (Wagner et al., 2005), will also pose some interesting questions and
hopefully answers in future studies.
57
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