Arenberg Doctoraatsschool Wetenschap & Technologie Faculteit Wetenschappen Departement Biologie

2012

CONTRIBUTION TO THE CONSERVATION OF TEMPERATE ORCHID SPECIES: PATTERNS OF

HYBRIDIZATION, SEED ECOLOGY AND POTENTIAL FOR ECOLOGICAL RESTORATION

Koen DE HERT

Proefschrift voorgedragen tot het behalen van de graad van Doctor in de Wetenschappen (Biologie)

CONTRIBUTION TO THE CONSERVATION OF TEMPERATE ORCHID SPECIES: PATTERNS OF

HYBRIDIZATION, SEED ECOLOGY AND POTENTIAL FOR ECOLOGICAL RESTORATION

Koen DE HERT

2012

Supervisor: Prof. Dr. Ir. Olivier Honnay Co-supervisors: Prof. Dr. Ir. Hans Jacquemyn Prof. Dr. Isabel Roldán-Ruiz Members of the Examination Committee: Prof. Dr. Filip Volckaert, Chairman Prof. Dr. Martin Hermy Prof. Dr. Wannes Keulemans Dr. An Vanden Broeck (Inbo) Prof. Dr. Daniel Tyteca (UCL)

Proefschrift voorgedragen tot het behalen van de graad van Doctor in de Wetenschappen (Biologie)

© 2012 Katholieke Universiteit Leuven, Groep Wetenschap & Technologie, Arenberg Doctoraatsschool, W. de Croylaan 6, 3001 Heverlee, België Alle rechten voorbehouden. Niets uit deze uitgave mag worden vermenigvuldigd en/of openbaar gemaakt worden door middel van druk, fotokopie, microfilm, elektronisch of op welke andere wijze ook zonder voorafgaandelijke schriftelijke toestemming van de uitgever. All rights reserved. No part of the publication may be reproduced in any form by print, photoprint, microfilm, electronic or any other means without written permission from the publisher.

iii

WOORD VOORAF

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WOORD VOORAF

v

SAMENVATTING

vi

vii

SUMMARY

The Orchidaceae are the most diverse plant family and occur on all vegetated

continents. However, because of their often very specific habitat requirements and

species-specific biotic interactions (mycorrhizas and/or pollinators), many orchid

species are rare and often restricted to nature reserves where habitat conditions are

still intact. Major threatening processes include habitat destruction, fragmentation,

nitrogen pollution, invasive alien species and climate change. Especially in the highly

urbanized Flanders region orchid species are highly threatened. In order to conserve

the orchid species diversity, conservation and restoration plans are necessary.

Although the extraordinary floral diversity in orchids indicates the importance

of orchid-pollinator associations for orchid evolution, many terrestrial orchid species

are generalists with respect to the choice of their pollinators, which may facilitate

hybridization. Moreover, high levels of natural hybridization have been reported

among species and genera of terrestrial, food-deceptive orchids. Most prominent is a

guild of food-deceptive orchids in the genera Orchis, Dactylorhiza, Anacamptis and

Neotinea. Although natural hybridization may have important evolutionary

consequences, it can also result in reduced biological diversity through the potential

loss of rare taxa as a consequence of outbreeding depression and genetic

assimilation. Since many orchid species are rare and hybridization is common in

terrestrial orchids, potential negative effects of hybridization may have a large impact.

Many orchid populations occur in small habitat fragments, which increases the

possibility of gene mixing with more common orchids. Therefore, it is important to

examine the extent of hybridization in natural hybrid zones, when conservation

strategies are designed.

We investigated several natural hybrid zones of sympatric Dactylorhiza

species in Flanders. Species of this genus are known to hybridize frequently, even

between species of different ploidy levels. Our general aim was to study hybridization

patterns based on morphological and genetic marker data (AFLP). Furthermore, we

investigated whether ecological restoration of natural populations was possible from

seeds. To do so, seed introduction experiments were combined with seed viability

analyses to investigate the potential of orchid species to colonize unoccupied habitat

viii

patches and to assess the potential of orchid seed banks to contribute to population

viability.

Frequent hybridization was observed in two hybrid zones with three

Dactylorhiza species (D. incarnata, D. fuchsii and D. praetermissa), but no

backcrosses or F2 hybrids were observed as a result of strong post-zygotic barriers.

Hybrid sterility is the major cause hampering hybridization to extend beyond the F1

generation. Hybridization between all species pairs, and hence between different

ploidy levels, was possible. However, the frequency of hybridization between species

varied substantially between different sites. Significant spatial genetic structure was

observed and could be the result of limited dispersal of seed or pollen or of patchy

occurrence of suitable mycorrhizal fungi. The occurrence of putative triple hybrids

provide evidence for some secondary gene flow, but no risk of genetic assimilation

and breakdown of species boundaries seemed to be present. When no negative

effects of hybridization are present, as in our case, hybrid zones should be protected

together with the pure species since they may provide the stage for evolutionary

processes in orchids. When, however, serious threats for the pure species should be

observed, through genetic assimilation or replacement by better adapted hybrids,

removal of the hybrids can be considered in order to maintain species diversity.

The results of our seed baiting experiment show that seeds of all three study

species (D. fuchsii, D. praetermissa and H. monorchis) can grow into protocorms in

occupied and unoccupied habitats, showing that dispersal limitation plays a

significant role in the inability of orchids to colonize restored sites. The absence of

recruitment limitation suggests that manual introduction of orchid seeds in isolated

restored habitats can be a valuable and even necessary practice to restore orchid

populations.The seed burial experiment revealed that seeds of seven of the

investigated orchid species survived at least 2.5 years in the soil, while D. fuchsii

seeds survived only one year in the soil. These results suggest that all of our study

species can form a short-term persistent seed bank, which can have important

implications for their persistence.

ix

LIST OF ABBREVIATIONS AFLP amplified fragment length polymorphism FCM flow cytometry Ln natural logarithm PCA principal components analysis PCO principal co-ordinates analysis PLP percentage polymorphic loci RI reproductive isolation

x

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LIST OF SYMBOLS b regression slope of Fij

df degrees of freedom

F1 kinship coefficient of the first distance interval with the standard error

of the mean estimated by a jackknife procedure

Fij Hardy’s kinship coefficient

Fst estimate of genetic differentiation among populations

n number of individuals sampled

Pp percentage of polymorphic loci

Ps percentage of species-specific loci

q posterior probability

SD standard deviation

SE standard error

Sp Sp statistic

Tq Structure threshold

α significance level

ΦST analog for Fst

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TABLE OF CONTENTS Voorwoord .................................................................................................................. iii

Sammenvatting ............................................................................................................ v

Summary ................................................................................................................... vii

List of abbreviations .................................................................................................... ix

List of symbols ............................................................................................................ xi

Table of contents ...................................................................................................... xiii

Chapter 1. General introduction ................................................................................. 1 1.1 Orchid species ...................................................................................................... 2

1.2 Hybridization in Orchidaceae ................................................................................ 4

1.3 Hybridization and species conservation ................................................................ 7

1.4 Restoration of orchid populations ......................................................................... 8

1.5 Objectives and thesis outline ................................................................................ 9

Chapter 2. Patterns of hybridization between diploid and derived allotetraploid

species of Dactylorhiza (Orchidaceae) co-occurring in Belgium ................................ 13 2.1 Abstract .............................................................................................................. 14

2.2 Introduction ......................................................................................................... 14

2.3 Materials and Methods ....................................................................................... 17

2.4 Results ................................................................................................................ 23

2.5 Discussion .......................................................................................................... 31

2.6 Conclusions ........................................................................................................ 35

2.7 Acknowledgements............................................................................................. 36

Chapter 3. Reproductive isolation and hybridization in sympatric populations of three

Dactylorhiza species (Orchidaceae) with different ploidy levels ................................ 37

3.1 Abstract .............................................................................................................. 38

3.2 Introduction ......................................................................................................... 38

3.3 Materials and Methods ....................................................................................... 41

3.4 Results ................................................................................................................ 47

3.5 Discussion .......................................................................................................... 56

3.6 Conclusions ........................................................................................................ 61

xiv

3.7 Acknowledgements............................................................................................. 61

Chapter 4. Germination failure is not a critical stage of reproductive isolation

between three congeneric orchid species.................................................................. 63

4.1 Abstract .............................................................................................................. 64

4.2 Introduction ......................................................................................................... 64

4.3 Methods .............................................................................................................. 65

4.4 Results ................................................................................................................ 69

4.5 Discussion .......................................................................................................... 73

4.6 Conclusions ........................................................................................................ 74

4.7 Acknowledgements............................................................................................. 75

Chapter 5. Absence of recruitment limitation in restored dune slacks suggests that

manual seed introduction can be a successful practice for restoring orchid

populations ............................................................................................................... 77

5.1 Abstract .............................................................................................................. 78

5.2 Introduction ......................................................................................................... 78

5.3 Methods .............................................................................................................. 79

5.4 Results ................................................................................................................ 84

5.5 Discussion .......................................................................................................... 85

5.6 Implications for conservation .............................................................................. 87

5.7 Acknowledgements............................................................................................. 87

Chapter 6. Seed longevity of eight European orchid species –a burial experiment .. 89

6.1 Abstract .............................................................................................................. 90

6.2 Introduction ......................................................................................................... 90

6.3 Methods .............................................................................................................. 91

6.4 Results ................................................................................................................ 92

6.5 Discussion .......................................................................................................... 94

6.6 Conclusions ........................................................................................................ 96

Chapter 7. General discussion and conclusios ......................................................... 97

7.1 Outline of main results ........................................................................................ 98

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7.2 Implications for conservation ............................................................................ 100

7.3 Implications for restoration ................................................................................ 101

7.4 Research perspectives ..................................................................................... 102

Appendix .................................................................................................................. 105

References .............................................................................................................. 107

Chapter 1.

General introduction

Chapter 1

2

1.1 Orchid species

The Orchidaceae are the most diverse of all angiosperm families with estimates of

>25,000 species (Dressler 1993; Mabberley 1997; Cribb et al. 2003). Orchids

comprise five subfamilies and c. 870 genera, and are considered almost ubiquitous,

occurring on all vegetated continents and even on some Antarctic islands (Dressler

1993; Chase et al. 2003). Two-thirds of orchid species are epiphytes and lithophytes,

with terrestrial species comprising the remaining third. Orchid distribution and

abundance are distinctly skewed towards the tropics and vary between continents

and within regions, following hotspots of species richness and high angiosperm

endemism, as described by Myers et al. (2000). Orchid-rich areas include the

northern Andes of South America, Madagascar, Sumatra and Borneo for mostly

epiphytic species, Indochina for both epiphytic and terrestrial species, and

southwestern Australia as a centre of terrestrial orchid richness (Cribb et al. 2003).

Orchids often have very specific habitat requirements (physico-chemical

properties of soil (often calcium rich, high pH and low nutrient content), ground water

level, microclimate) (Bournérias and Prat 2005). Furthermore, orchids require the

presence of a compatible fungal partner (mycorrhiza) for seed germination and

continued growth (Rasmussen 1995; Peterson et al. 1998). This is because the

minuscule seeds lack nutritional resources and the necessary energy for germination

and seedling growth is provided by mycorrhizal fungi. Mycorrhizal specificity is highly

variable between different orchid species, ranging from a few genera to a single

fungal species (McCormick et al. 2004; Dearlaney 2007; Shefferson et al. 2008;

Jacquemyn 2011, 2012). Molecular studies have shown that fully myco-heterotrophic

orchid species have a high degree of mycorrhizal specificity (Taylor and Bruns 1997;

Selosse et al. 2002), while photosynthetic orchids can have both a high or low

degree of specificity. Several authors have found photosynthetic orchids associating

with a surprisingly narrow range of fungi over large geographic areas (Shefferson et

al. 2005, 2007; McCormick et al. 2006; Bonnardeaux et al. 2007; Irwin et al. 2007).

However, Jacquemyn et al. (2010, 2012) revealed a low specificity or preference for

a widespread fungal symbiont for several species from the genera Orchis and

Dactylorhiza, using DNA arrays. Also, Shefferson et al. (2008) showed evidence for

Introduction

3

widespread fungal symbionts for three orchid species.

In orchids, the extraordinary floral diversity indicates the importance of orchid-

pollinator associations for orchid evolution and orchid pollination biology is regarded

as a driving force in orchid diversification and speciation (Van der Pijl and Dodson

1966; Cozzolino and Widmer 2005). However, orchid pollination is not always

species specific. A compilation of pollinator information for European orchids by Van

der Cingel (1995) reveals that species-specific orchid-pollinator relationships have

evolved in only a few groups, such as the sexually deceptive genus Ophrys (Schiestl

et al. 1999) (Fig. 1.1), whereas many orchid species are generalists with respect to

the choice of their pollinators. Most prominent is a guild of food-deceptive orchids in

the genera Orchis, Dactylorhiza, Anacamptis and Neotinea, which share large

conspicuous flowers, offer no reward to pollinators and are thought to attract and

deceive mostly naïve pollinators (Dafni 1984, 1987).

Fig.1.1: Ophrys insectifera pollinated by a wasp (Argogorytes mystaceus)

Due to these often highly specialized interactions and the very specific habitat

requirements, many orchid species are rare and often restricted to nature reserves

where habitat conditions are still intact. As a result, the Orchidaceae, more than any

other plant family, has a high proportion of threatened species (Swarts and Dixon

2009). Although terrestrial orchid species comprise only one third of all orchid

species, almost half of the extinct species according to the World Conservation Union

(IUCN 2012) are terrestrial herbaceous perennials. Terrestrial orchids thus represent

Chapter 1

4

a life-form class likely to experience a greater extinction risk as a result of the

multiplicity of threatening processes, particularly under current climatic change

scenarios. Especially in the highly urbanized Flanders region orchid species are

under a lot of pressure. From the 38 orchid species that occurred in Flanders, already

six species (16%) are extinct and 18 species (47%) belong to one of the three

threatened categories (vulnerable, endangered and critically endangered) (Van

Landuyt et al. 2006; Fig. 1.2). Only two species (5%) have a low risk of extinction

(least concern).

Fig. 1.2: Percentage of the 38 Flemish orchid species in the different Red List Categories (based on data from Van Landuyt et al. 2006. Atlas van de Flora van Vlaanderen en het Brussels Gewest)

1.2 Hybridization in Orchidaceae

1.2.1 Hybridization and reproductive isolation

Hybridization is defined as interbreeding of individuals from different species or

genera. Natural hybridization is a common phenomenon and has long been

suspected to be a potent evolutionary force, in particular in the plant kingdom

(Anderson 1949; Stebbins 1959; Grant 1981). Indeed, introgression (the transfer of

genetic material from one species into another via hybridization) has been

documented in a wide variety of both plant and animal taxa (Burke and Arnold 2001),

and there is evidence that it may serve as a source of adaptive genetic variation

(Arnold 1997; Cozzolino et al. 2006). Moreover, it has been suggested that a

16%

26%

16%

5%

32%

5%

extinct

critically endangered

endangered

vulnerable

rare

least concern

Introduction

5

significant fraction of flowering plants may be of hybrid origin (Ellstrand et al. 1996;

Rieseberg 1997, Rieseberg et al.1999) and at least a quarter of plant species are

involved in hybridization and potential introgression with other species (Mallet 2005).

The most common mechanism of plant speciation through hybridization is

allopolyploidy (Soltis and Soltis 1999), however, there is strong empirical evidence

that hybridization can also give rise to new species without a change in ploidy level

(“homoploid hybrid speciation”) (Rieseberg et al. 1995; Arnold 1997; Wolfe et al

1998; Buerkle et al. 2000).

Reproductive isolation is of fundamental importance for maintaining species

boundaries in sympatry. Flowering plants posses various reproductive isolation

mechanisms that prevent or limit interspecific gene flow (Cozzolino et al. 2004;

Moccia et al. 2007). Therefore, the extent to which hybridization occurs depends on

the strength of different reproductive barriers and their potentially complex

interactions (Rieseberg and Carney 1998; Coyne and Orr 2004; Scopece et al.

2008). These barriers can act before or after pollination and are therefore

conveniently categorized as pre- and post-mating barriers. Pre-mating barriers

include geographic, temporal and pollinator isolation. Post-mating barriers are further

subdivided in pre-zygotic and post-zygotic mechanisms. Pre-zygotic barriers reduce

the likelihood that heterospecific gametes will combine to form a viable zygote and

include pollen–stigma and sperm cell–ovule interactions. Post-zygotic barriers reduce

the viability or reproductive potential of interspecific hybrids and include embryo

mortality, germination failure, hybrid inviability and sterility. Hybrid inviability and

sterility are also referred to as hybrid inferiority since the hybrids have a lower fitness

than their parental species. On the contrary, hybrids may have a higher fitness than

their parents. This hybrid superiority can have important evolutionary implications

(Burke and Arnold 2001).

1.2.2 Hybridization in Orchidaceae

Since orchid pollination is less species-specific than previously thought (Van der

Cingel 1995; Cozzolino et al. 2005), hybridization between different orchid species

may occur (Fig. 1.3). Moreover, high levels of natural hybridization have been

reported among species and genera of terrestrial, food-deceptive orchids (Willing and

Willing 1985; Hédren 1996a; Bournérias and Prat 2005; Kretzschmar et al. 2007).

Chapter 1

6

These species offer no reward to their pollinators and therefore often attract a diverse

range of rather unspecialized naïve pollinators (Cozzolino and Widmer 2005;

Cozzolino et al. 2006; Moccia et al. 2007). This frequent sharing of pollinators (Van

der Cingel 1995; Cozzolino et al. 2005) is believed to facilitate hybridization (Fig. 1.3).

Fig. 1.3: Hybrid between D. praetermissa and D. incarnata visited by a bumble bee

Even within the sexually deceptive genus Ophrys, known for its peculiar

pollinator specificity, interbreeding is frequent (Stebbins and Ferlan 1956; Danesh

and Danesh 1972; Delforge 2001; Soliva and Widmer 2003) and likely contributed to

the difficulties in the taxonomy of this genus. The number of species within the genus

Ophrys has been estimated at anything between 21 (Nelson 1962) and 215 (Delforge

1994). In some hybrid zones of sympatric orchid species even hybrid swarms are

found (Lord and Richards 1977; Harris and Abbott 1997; Kretzschmar et al. 2007). A

hybrid swarm is a population of individuals in which introgression has occurred to

various degrees by varying numbers of generations of backcrossing to one or both

parental taxa, in addition to mating among the hybrid individuals themselves (Rhymer

and Simberloff 1996).

The widespread occurrence of hybridization within the Orchidaceae suggests

that it may play a significant role in orchid speciation and evolution. Whether or not

interspecific hybridization is important as a mechanism generating biological diversity

in Orchidaceae is, however, a matter of controversy. Several orchid hybrid zones

Introduction

7

have recently been documented and investigated with molecular markers, providing

evidence either for (Aagaard et al 2005; Bateman et al 2008; Cortis et al 2009;

Bellusci et al 2010) or against (Cozzolino et al 2006; Moccia et al 2007; Scopece et

al. 2008) the potential role of hybridization in orchid evolution and diversification.

Especially in the food-deceptive orchid genus Dactylorhiza hybrid speciation via

allopolyploidy seems to have played an important role in the diversification in the

genus (Hédren 1996a). In the case of Neotropical orchids, only limited and indirect

evidence are available for the strength of reproductive isolation between related

species (Van der Pijl and Dodson 1966).

1.3 Hybridization and species conservation

Although natural hybridization may have important evolutionary consequences, as

mentioned above, it can also result in reduced biological diversity through the

potential loss of rare taxa as a consequence of outbreeding depression and genetic

assimilation (Mayr 1992; Rieseberg 1995; Arnold 1997; Allendorf et al. 2001). This

can happen when hybrids are fertile and have ecological requirements that are

similar to one or both parents (Wolf et al. 2001; Hedge et al. 2006). Hybrids may then

backcross with either parent resulting in introgression. If hybrids do not exhibit

reduced fitness relative to parental taxa, they may ultimately displace pure

populations of one or both parental taxa. This process may especially favour the

genetic assimilation of the rarer taxa due to a proportionally greater frequency of

interspecific pollination and recombination with hybrids. Even if hybrids have reduced

fitness relative to parental species and do not contribute to introgression, the rarer

taxon may yet decline because of pollen swamping from the more common taxon

(Ellstrand and Elam 1993; Arnold 1997).

Especially when hybridization is human mediated, it may have harmful effects.

The three interacting anthropogenic activities that contribute most to increased rates

of hybridization include introductions of plants and animals, fragmentation, and

habitat modifications (Rhymer and Simberloff 1996; Allendorf et al. 2001). As a

consequence of these human activities rare species may come in contact and

hybridize with more common and/or introduced taxa (Ellstrand and Schierenbeck

2000; Ferdy and Austerlitz 2002). Of particular concern for conservation is the

Chapter 1

8

possibility that novel genotypes created through hybridization may be well suited to

anthropogenically altered habitats (Rieseberg et al 1995; Vilà et al. 2000).

Since many orchid species are rare and hybridization is common in terrestrial

orchids, potential negative effects of hybridization may have a large impact. Many

orchid populations occur in small habitat fragments, which increases the possibility of

gene mixing with more common orchids. Since hybridization can have both positive

and negative consequences for orchid species, it is important to study natural

hybridization in the field, when conservation strategies are designed. So far, very few

studies investigated hybridization between a rare and common orchid in the field

(Chung et al. 2005a; Worley et al. 2009). They found no evidence for genetic

assimilation. However, the negative effects of pollen swamping can still play a role.

1.4 Restoration of orchid populations

Many plant species face the risk of extinction as result of a fast changing world.

Nearly 12.5% of the global vascular flora is threatened with extinction due to human

activity (IUCN 2012). Especially orchids, with their high degree of rarity and

ecological specialization, are extremely threatened (Cribb et al. 2003; Swarts and

Dixon 2009). Major threatening processes include habitat destruction, fragmentation,

nitrogen pollution, invasive alien species and climate change (Butchart et al. 2010;

Swarts and Dixon 2009). In combination with the often typical specificity of orchid

species for certain pollinators and mycorrhizal fungi, the adverse effects of these

threats can be expected to be even bigger. Habitat fragmentation results in small and

isolated orchid populations that are more prone to extinction due to the loss of

genetic variation (Wallace 2002). Random genetic drift and inbreeding are the main

causes of the decline in genetic variation. Also, the risk of genetic assimilation with

more common and widespread orchids is higher in small and isolated populations.

Although much effort is currently put into the conservation of remnant habitats,

passive protection of these remnants alone is likely not sufficient to guarantee long-

term persistence of many species, since these habitats have often become too

fragmented (Brudvig 2011). Therefore, ecological restoration, focusing on the

enlargement and defragmentation of the remaining habitats, is gaining importance

(Benayas et al. 2009). The success of habitat restoration depends on the ability of

target plant species to reach the restored patch (dispersal limitation) and on the

Introduction

9

ability to recruit after seed dispersal (recruitment limitation) (Rasmussen and

Whigham 1998; Verheyen and Hermy 2001). Knowledge of the relative importance of

dispersal vs. recruitment limitation is crucial for predicting the success of habitat

restoration measures. If the orchid species seem to be dispersal limited, artificial

introduction of seeds in the restored habitats can be considered when no seed bank

is present. Although little information on orchid seed banks is available (e.g.

Whigham et al. 2006), persistent seed banks can be very important for maintaining

and restoring population genetic diversity (Honnay et al. 2008), but also for restoring

orchid populations after habitat improvement in a natural way. Before restoration

measures are designed, also information on seed longevity should be obtained.

To increase the probability of successful restoration, it has recently been

suggested that more attention should be directed towards aboveground-belowground

linkages (Kardol and Wardle 2010). This is especially the case when restoring

populations of orchid species as the presence of mycorrhizal fungi at a restoration

site will not only be critical for orchid seed germination (Rasmussen 1995;

McCormick et al. 2012), but also for seedling establishment (Bidartondo and Read

2008).

1.5 Objectives and thesis outline

Hybridization is of major evolutionary importance in the plant kingdom and at least a

quarter of plant species are involved in hybridization and potential introgression with

other species (Mallet 2005). In addition to its potential evolutionary role, however,

hybridization can also have negative effects on rare species as a consequence of

outbreeding depression and genetic assimilation (Allendorf et al. 2001). Since

hybridization is common in terrestrial orchids and many orchid species are rare,

knowledge of the extent of hybridization in natural hybrid zones is important.

We investigated several natural hybrid zones of sympatric Dactylorhiza species in

Flanders. Species of this genus are known to hybridize frequently, even between

species of different ploidy levels. Our general aim was to study hybridization patterns

based on morphological and genetic marker data (Fig. 1.4). Furthermore, we

investigated whether ecological restoration of natural populations was possible from

seeds. To do so, seed introduction experiments were combined with seed viability

analyses to investigate the potential of orchid species to colonize unoccupied habitat

Chapter 1

10

patches and to assess the potential of orchid seed banks to contribute to population

viability (Fig. 1.4).

Fig 1.4: Scheme of research objectives

More specifically, the aims of this thesis are to answer the following questions:

• Do all three Dactylorhiza species (D. incarnata, D. praetermissa and D.

fuchsii) hybridize with each other in natural sympatric populations?

• If hybridization occurs, is there a difference in frequency of hybridization

between different species pairs? Which hybrid classes are found?

• How strong are post-mating barriers between the different species? Is

germination failure an important barrier?

• Do fine-scale patterns of genetic variations reveal different patterns of gene

flow for different species?

• What is the relative importance of dispersal vs. recruitment limitation in the

failure to colonize restored dune slacks

• What is the seed longevity of eight European orchid species? Are there

indications for a persistent seed bank?

Introduction

11

In Chapter 2 and 3, research on hybridization patterns in natural hybrid zones

is presented. In Chapter 2, a hybrid zone of D. incarnata and D. praetermissa in the

Vaarttaluds nature reserve was investigated. Chapter 3 presents the results of two

hybrid zones with three sympatric species; D. incarnata, D. praetermissa and D.

fuchsii. The locations here were the Ter Yde and Paelsteenpanne nature reserves.

The importance of germination failure as reproductive barrier was compared with

early acting barriers (fruit set and embryo mortality) in Chapter 4.

Chapter 5 and 6 report the results on seed ecology. In Chapter 5, the relative

importance of dispersal vs. recruitment limitation was investigated using seed baiting

methods for three orchid species (D. praetermissa and D. fuchsii and H. monorchis).

The seed longevity of eight orchid species (Dactylorhiza incarnata, D. fuchsii, D.

praetermissa, D. maculata, Orchis mascula, Gymnadenia conopsea, Epipactis

palustris and Herminium monorchis) was investigated using a seed burial experiment

in Chapter 6.

In Chapter 7 implications for conservation and restoration of orchid

populations are discussed, followed by general conclusions and research

perspectives.

Chapter 2.

Patterns of hybridization between diploid and derived allotetraploid

species of Dactylorhiza (Orchidaceae) co-occurring in Belgium

Adapted from:

De hert K, Jacquemyn H, Van Glabeke S, Roldán-Ruiz I, Vandepitte K, Leus L, Honnay O (2011) Patterns of hybridization between diploid and derived allotetraploid

species of Dactylorhiza (Orchidaceae) co-occurring in Belgium. American Journal of

Botany 98: 946–955.

Chapter 2

14

2.1 Abstract

• Premise of the study: Although the potential for gene flow between species with

large differences in chromosome numbers has long been recognized, only few

studies have thoroughly investigated in situ hybridization across taxa with different

ploidy levels. In this study, we combined morphological, cytological, and genetic

marker data with pollination experiments to investigate the degree, direction, and

spatial pattern of hybridization between the diploid Dactylorhiza incarnata and its

tetraploid derivative, D. praetermissa.

• Methods: To identify hybrids, 169 individuals were genotyped using AFLPs and

morphologically characterized. Individuals were clustered based on their AFLP profile

using the program Structure. To reduce the dimensionality of the plant trait matrix

PCA was applied. The origin of suspected hybrid individuals was verified using flow

cytometry. An AMOVA and spatial autocorrelation analysis were used to indirectly

infer the extent of gene flow.

• Key Results: Only five individuals were regarded as putative hybrids based on the

AFLP data; all had been assigned to the D. praetermissa morphotype. Only one had

a deviating DNA content and was presumably a triploid. High ΦST values between

different subpopulations and significant spatial genetic Structure were observed,

suggesting localized gene flow.

• Conclusions: Using combined data to study hybridization between D. incarnata and

D. praetermissa, very few unequivocal hybrids were observed. We propose several

non-mutually exclusive explanations. Localized pollen flow, in combination with

different microhabitat preferences, is probably one of the reasons for the low

frequency of hybrids. Also, the triploid first-generation hybrids may experience

difficulties in successful establishment, as a result of genic incompatibilities.

2.2 Introduction Interspecific hybridization is widespread among plants (Rieseberg and Carney 1998;

Burke and Arnold 2001; Baack and Rieseberg 2007). It has been suggested that it

occurs in 25% of plant species (Mallet 2005) and that a significant proportion (50–

70%) of flowering plant species may be of hybrid origin (Ellstrand et al. 1996;

Rieseberg 1997). Although hybridization is often considered a negative factor

Hybridization between two Dactylorhiza species

15

because of the potential loss of rare taxa due to outbreeding depression and the risk

of genetic assimilation (Mayr 1992; Allendorf et al. 2001), attention is increasingly

being paid to its evolutionary significance (Arnold 1997; Burke and Arnold 2001;

Cozzolino et al. 2006). Also, the development and increased availability of high-

resolution molecular markers has contributed to a renewed interest in interspecific

hybridization (Rieseberg and Ellstrand 1993; Martinsen et al. 2001).

Hybridization and introgression can have several evolutionary consequences.

First, an increase in within–species genetic diversity can impact on both ecological

and evolutionary processes (Anderson 1948; Martinsen et al. 2001). Second, the

transfer of adaptations, or even the production of novel adaptations, can result in new

colonization abilities (Arnold 1997; Rieseberg and Carney 1998). Third, reproductive

barriers can be broken-down or, on the contrary, be reinforced (Levin et al. 1996;

Rieseberg 1997). Finally, new ecotypes or even species may arise, often through

polyploidization (Barton and Hewit 1989; Rieseberg 1997; Soltis and Soltis 1999;

Coyne and Orr 2004).

Patterns and intensity of gene flow are essential aspects in this context, as

without gene flow, no interspecific hybridization is possible. For instance, spatially

restricted pollen flow in combination with a patchy occurrence of related species is

predicted to seriously hamper hybridization. Therefore, it is important to assess the

spatial extent of gene flow within pure species when examining the degree of

hybridization and underlying causes in situ (Valbuena-Carabaña et al. 2005; Friar et

al. 2007). Knowledge of the direction of gene flow between hybridizing species is also

imperative, as imbalances can lead to asymmetric patterns of introgression (Oddou-

Muratorio et al. 2001).

Hybridization patterns are of particular relevance for orchid species. On the

one side, it has traditionally been suggested that the extraordinary floral diversity in

the Orchidaceae is a consequence of highly specific plant–pollinator associations.

Consequently, premating reproductive barriers have been proposed as the main

reproductive isolation mechanism among orchid species (Van der Pijl and Dodson

1966). Contrary to expectations, however, high levels of natural hybridization have

been reported among species and genera of terrestrial, food-deceptive orchids

(Kretzschmar et al. 2007). These species offer no reward to their pollinators and

therefore often attract a diverse range of rather unspecialized naïve pollinators

(Cozzolino and Widmer 2005; Cozzolino et al. 2006; Moccia et al. 2007). This

Chapter 2

16

frequent sharing of pollinators (Van der Cingel 1995; Cozzolino et al. 2005) is

believed to facilitate hybridization.

Hybridization within the food-deceptive orchid genus Dactylorhiza has been well-

documented (Heslop-Harrison 1953, 1957; Lord and Richards 1977; Hedrén 1996a;

Shaw 1998; Lammi et al. 2003; Aagaard 2005; Ståhlberg and Hedrén 2009).

Different Dactylorhiza species often co-occur and hybrids are frequently formed.

Moreover, evidence from morphological and cytological studies indicates that some

Dactylorhiza species have a recent hybrid origin (Hedrén 1996a; Pillon et al. 2007).

Allotetraploids have evolved on several occasions due to repeated hybridization

(Hedrén 1996a, 2003) between two main groups of parental taxa,—one consisting of

the diploid marsh orchids, D. incarnata sensu lato, the other consisting of spotted

orchids—the diploid D. fuchsii and more rarely also the autotetraploid D. maculata

(Heslop-Harrison 1953, 1956; Hedrén 1996a, 2001; Devos et al. 2006; Pillon et al.

2007). Hedrén et al. (2001) suggested that allotetraploidization dominates over

introgression as a speciation mechanism in the genus.

The extent of introgression between individuals of different ploidy levels is

generally expected to be limited, due to fitness and fertility problems in the resulting

polyploids (Ramsey and Schemske 1998; Rieseberg and Carney 1998; Burton and

Husband 2000; Burke and Arnold 2001). On the other hand, evidence exists that

Dactylorhiza species of different ploidy levels frequently hybridize (Heslop-Harrison

1957; Lord and Richards 1977; Hedrén 1996a; Aagaard 2005; Ståhlberg and Hedrén

2009). The diploid D. incarnata and the allotetraploid D. praetermissa have yielded

several field observations of putative hybrids (Lambinon et al. 1998; Weeda et al.

2003; Bournérias et al. 2005; Van Landuyt et al. 2006) and there is one quantitative

study that suggested substantial hybridization based on morphological data (Shaw

1998). When examining hybridization, however, ideally morphological, cytological,

and molecular data should be combined (Pellegrino et al. 2000; Wallace 2006;

Bateman et al. 2008; Ståhlberg and Hedrén 2009). Here, we combined morphological

and genetic marker data with flow cytometry and evidence from a pollination

experiment to study the degree and spatial pattern of in situ hybridization between D.

incarnata (L.) Soó and D. praetermissa (Druce) Soó. We also assessed patterns of

gene flow in both species by investigating fine-scale patterns of genetic variation

using spatial autocorrelation analyses.

Hybridization between two Dactylorhiza species

17

2.3 Materials and Methods

2.3.1 Study species and study site

Dactylorhiza incarnata and D. praetermissa are herbaceous, perennial, food-

deceptive orchid species. Dactylorhiza praetermissa is an allotetraploid (2n = 4x =

80), whereas D. incarnata is diploid (2n = 2x = 40) and one of the parental species of

D. praetermissa (Hedrén 1996a,b). The descriptions of morphology and ecology of

our study species are based on their occurrence in Belgium. Dactylorhiza incarnata

has a Eurosiberian distribution and is predominantly found in wet meadows, dune

slacks, fens, and marshes, where it often occurs in somewhat calcareous, nutrient-

poor, humid soils. Dactylorhiza incarnata is pollinated by bumblebees (Bombus) and

other relatively large insects (Hymenoptera, Coleoptera) (Lammi et al. 2003; Vallius

et al. 2004). Dactylorhiza praetermissa prefers calcareous soils, and sites with some

disturbance. It occurs in periodically flooded dune slacks, roadsides, canal verges,

raised terrains, grazed or mown fens. Dactylorhiza praetermissa has an Atlantic

distribution, ranging from Brittany in France to Great Britain and Denmark (Van

Landuyt et al. 2006). Similarly to D. incarnata, D. praetermissa is pollinated primarily

by bumblebees (Ferdy et al. 2001).

Dactylorhiza incarnata has a dense inflorescence with pink-purplish, purplish or

occasionally almost white flowers. The flowers are relatively small and the labellum

has reflexed lateral lobes. The labellum markings consist of loops which are

somewhat lung-shaped. Leaves of D. incarnata are lanceolate, and usually have a

hooded leaf tip. Some subspecies of D. incarnata can have yellow flowers (ssp.

ochroleuca) or spots on both sides of the leaves (ssp. cruenta) (Bateman and

Denholm 1985), but these subspecies—some authors (e.g. Bournérias et al. 2005)

treat them as distinct species—do not occur in Belgium. Inflorescences of D.

praetermissa are dense and comprise bright pink to purplish flowers (Lambinon et al.

1998; Bournérias et al. 2005). The labellum is often rather flat and bears many small

spots, which may be accompanied by short lines or even loops. The lanceolate

leaves of D. praetermissa can be spotted, but if so only on the upper surface.

The study was conducted at Vaarttaluds in Moen (Zwevegem), Belgium. This

nature reserve originated when the Kortrijk-Bossuit canal was widened and banks of

calcareous clay were consequently formed. The two species studied occur where the

Chapter 2

18

infiltrated rainwater reaches the soil surface. Three distinct subpopulations were

found. According to our original visual determination, two of the subpopulations

contained both species, whereas one contained only D. praetermissa individuals (Fig.

2.1). All individuals were mapped using a high–precision GPS (Trimble GmbH).

Distance (m)

0 100 200 300 400 500 600 700

Dis

tanc

e (m

)

0

100

200

300

400

500

D. incarnataD. praetermissaputative hybrids

Fig. 2.1. Spatial distribution of Dactylorhiza incarnata (open circles), D. praetermissa (open triangles), and putative hybrid (filled squares) individuals in Vaarttaluds, Moen, Belgium. Subpopulations 1 and 3 contain both species, whereas subpopulation 2 contained D. praetermissa only.

2.3.2 Phenology, fruit set, and potential for autogamous pollination

Because the flowering phenology of the two species can have an influence on the

potential for hybridization, we counted the number of flowering individuals of the two

species every two or three days during the peak flowering period in 2008. For each

sampled individual, we determined the number of flowers (June) and fruits (August)

and calculated fruit set as the proportion of flowers that set fruit. To estimate the

1

2

3

Hybridization between two Dactylorhiza species

19

degree of autogamous pollination ten individuals of each species were bagged so

that pollinators could not reach the flowers. At the end of the flowering season we

counted the number of fruits carried by the bagged individuals. The percentage of

autogamy was calculated as the average percentage fruiting success of bagged

individuals.

2.3.3 Pollination experiment and viability of seeds

To assess the viability of hybrid seeds, we pollinated ten flowers of ten individuals of

each species with pollinia removed from the other species. We used several donors

per individual to negate differences in pollen quality between different plants. Pollinia

of the artificially pollinated flowers were removed to prevent self-pollination. To test

the viability of the seeds we used the tetrazolium method, according to the protocol of

Van Waes and Debergh (1986); only viable embryos are stained red after the

treatment. Because the tetrazolium method can overestimate the real germination

rate, the seed viability percentages should be treated with caution. However, for

comparative purposes this method is sufficient to investigate differences among

pollination treatments. We counted a subsample of 200 to 300 seeds from each

capsule. We calculated the percentage of viable seeds as the ratio of the number of

colored seeds over the total number of seeds. We also tested the seed viability of

open pollinated plants (ten fruits of ten plants for each species) as reference for

hybrid seed viability.

2.3.4 Morphological data

We sampled 169 flowering individuals in June 2008 (95 of D. incarnata and 74 of D.

praetermissa). Since no clearly intermediate forms occurred, putative hybrids were

included in these figures. To estimate differences in morphological trait expression

between parental species and putative hybrids, the morphological variation in flower

and vegetative traits was estimated. The following vegetative traits were measured in

the field: length and width of the three largest leaves, plant height, stem diameter,

inflorescence length, number of leaves, and number of flowers.

For the analysis of flower traits, two or three young flowers were sampled per

individual. The flowers were stored in a denatured ethanol preservative and

transported to the laboratory. Each flower was dissected and a digital image was

Chapter 2

20

taken. The following flower traits were measured using the image analysis software

IMAGEJ 1.41 (Rasband, 2011): spur length and width, labellum length and width,

length and width of the middle lobe of the labellum, length and width of the lateral

lobes of the labellum, length and width of the middle sepal, length and width of the

lateral sepals, length and width of the two lateral petals. The following labellum traits

were recorded in the field: ground color on a scale 0–3 (0 = white, 1 = pale pink, 2 =

deep pink, 3 = purple), type of markings on a scale 0–4 (0 = no markings, 1 = spots,

2 = spots and dashes, 3 = dashes and loops, 4 = loops). A list of all morphological

characters is given in Table 2.1.

Table 2.1. List of morphological characters Vegetative characters 1 Leaf length, average of the length of the three largest leaves (cm) 2 Leaf width, average of the width of the three largest leaves (cm) 3 Number of leaves 4 Plant height, measured from ground level to apex of inflorecence (cm) 5 Inflorescence length, measured from lowermost flower to apex of inflorescence (cm) 6 Stem diameter, measured below inflorescence (mm) 7 Number of flowers Floral characters 8 Spur length (mm) 9 Spur width (mm) 10 Lateral sepal length (mm) 11 Lateral sepal width (mm) 12 Middle sepal length (mm) 13 Middle sepal width (mm) 14 Lateral petal length (mm) 15 Lateral petal width (mm) 16 Labellum length (mm) 17 Labellum width (mm) 18 Labellum shape index 19 Middle lobe of labellum length (mm) 20 Middle lobe of labellum width (mm) 21 Lateral lobe of labellum length (mm) 22 Lateral lobe of labellum width (mm)

23 Labellum ground colour, on a scale 0-3 (0 = white, 1 = pale pink, 2 = deep pink, 3 = purple)

24 Labellum markings, type of markings on a scale 0–4 (0 = no markings, 1 = spots,

2 = spots and dashes, 3 = dashes and loops, 4 = loops)

Hybridization between two Dactylorhiza species

21

2.3.5 DNA extraction and AFLP analysis

For each individual, leaf samples were collected for DNA analysis. The materials

were immediately frozen in liquid nitrogen and stored at –80°C before freeze drying

for 48 h. Dried material was vacuum packed and stored at room temperature until

DNA extraction. Forty milligrams of the dried material were ground and DNA-isolation

was carried out according to the CTAB-extraction procedure of Lefort and Douglas

(1999). DNA concentrations were estimated using a NanoDrop ND-1000

spectrophotometer running software v3.0.1 (NanoDrop Technologies), following the

manufacturer’s instructions.

We used AFLP markers (Vos et al. 1995) to screen the general genetic diversity

of our samples. AFLP analysis was carried out according to the protocol described in

Vandepitte et al. (2009). Four primer combinations were used: EcoRI-AGG/MseI-

CTAG, EcoRI-AGG/MseI-CTGG, EcoRI-ACAG/MseI-CTA, and EcoRI-ACAG/MseI-

GGT (MWG biotech, Ebersberg, Germany). AFLP-fragments were separated on an

ABI3130 sequencer on 50 cm capillaries using the polymer Pop7 (Applied

Biosystems). GeneScan 500 Rox labelled size standard (Applied Biosystems) was

injected with each AFLP sample, to allow sizing of the DNA fragments. The

fluorescent AFLP patterns were scored using GeneMapper 3.7 (Applied Biosystems).

Each marker was coded as 1 or 0 for presence or absence in an individual, forming a

binary data matrix. Each sample was thus represented by a vector of 1s and 0s. To

assess the reproducibility of the protocol, two independent DNA extractions were

carried out for nine samples. The overall error rate, i.e. the percentage of differently

scored loci between repeated samples, was low (3.4 %).

2.3.6 Flow cytometric analysis

We used flow cytometric analysis (FCM) to determine the ploidy level of our two

study species and putative hybrids. High-throughput ploidy analysis was performed

on a CyFlow Space (Partec, Münster, Germany) flow cytometer equipped with a

light–emitting diode (365 nm), using the protocol described by De Schepper et al.

(2001) with 4´,6-diamidino-2-phenylindole (DAPI) staining. Young leaf material (0.5

cm²) was co-chopped with leaf material of Lolium perenne cv. ‘Bellem’, which was

used as internal standard. Analyses were performed using Flomax software (Partec).

Chapter 2

22

Ploidy levels were inferred from the peak position ratios on the histograms. We

analyzed 20 D. incarnata and 67 D. praetermissa individuals, including all putative

hybrids. Ten samples were analyzed twice to verify the reproducibility of the protocol.

At least 2500 nuclei per sample were analyzed. Samples resulting in a coefficient of

variation higher than 4% were repeated.

2.3.7 Data analysis

2.3.7.1 Morphology

Morphological data were analyzed using a Principal Component Analysis (PCA) with

Varimax rotation in SPSS 16.0 for Windows (SPSS Inc., 2007). We used the scores

obtained for each individual on the first two PCA axes to interpret the main patterns

of morphological variation in the dataset. The traits used in the analysis are given in

Table 1.

2.3.7.2 Patterns of genetic differentiation

AFLP data were subjected to Bayesian analyses using methods implemented in the

program Structure version 2.2 (Pritchard et al. 2000). Structure uses a model-based

clustering method to assign individuals to groups in which deviations from Hardy–

Weinberg equilibrium and linkage equilibrium are minimized. Individuals assigned to

two sources with non-trivial probabilities are potential hybrids. In the Structure model,

the posterior probability (q) describes the proportion of an individual genotype

originating from each of K categories. In the present case, setting K = 2 corresponds

to the assumption of two species contributing to the gene pool of the sample. No prior

species information was used in any of the analyses. The AFLP data were entered as

Ploidy = 2 for all individuals, with the second line as missing data. Calculations

followed the admixture model of ancestry, assuming correlated allele frequencies.

We used a burn-in of 500,000 steps, followed by 1,000,000 iterations, after verifying

that the results did not vary significantly across multiple runs. We also tested whether

the results changed as we entered the data as only a single line for each individual

(Ploidy = 1) under the ‘no admixture’ model, but the results were the same. To

determine in which class an individual falls, we used as thresholds Tq = 0.90

(Burgarella et al. 2009). With Structure, individuals with q ≥ Tq are assigned to the

purebred category and indiviuals with q < Tq are assigned to the hybrid category.

Hybridization between two Dactylorhiza species

23

However, the Structure results should be treated with caution since the assumption of

Hardy-Weinberg equilibrium is not met because of the different ploidy level of the

parental species.

We also performed a Principal Coordinates analysis (PCO), based on Jaccard

distances (with squared lambda as vector scaling), using NTSYSpc2.1 (Rohlf 2000),

to visualize the genetic differentiation between the parental species and the putative

hybrids.

2.3.7.3 Spatial genetic Structure

Since three spatially distinct subpopulations could be distinguished in the field (Fig.

2.1), for each species total genetic diversity was partitioned among and within

subpopulations by carrying out a hierarchical analysis of molecular variance

(AMOVA) on Euclidean pairwise genetic distances, calculated according to Huff et al.

(1993), and using GenAlEx 6.2 (Peakall and Smouse 2006). The ΦST, an analog for

FST, was calculated based on Euclidean genetic distances, and significance testing

was performed using the permutational procedures offered in GenAlEx. We used spatial autocorrelation analysis to detect spatial genetic Structure at

AFLP loci within the two species. Putative hybrid individuals were not included in

these analyses. The spatial autocorrelation analyses were conducted using Hardy’s

kinship coefficient (Fij) for dominant markers (Hardy 2003) between individuals vs

distances in logarithmic scale using SPAGeDi version 1.2 (Hardy and Vekemans

2002). Spatial genetic Structure was quantified by the slope (b) of the regression line.

For estimation of kinship coefficients, we assumed no inbreeding. The number of

distance classes used for the analysis was 15 and 20 for D. incarnata and D.

praetermissa, respectively, so that at least 100 pairwise comparisons were included

in each distance interval. Standard errors of b for each distance class were obtained

by jackknifing across all loci (Vekemans and Hardy 2004).

To allow comparison of the SGS pattern in our two study species with the

strength of patterns observed in other species, the Sp-statistic was calculated. The

Sp-statistic is equal to –b/(1-F1) where F1 is the average Fij between individuals

belonging to the first distance class. We also calculated the Sp-statistic for each

subpopulation separately.

2.4 Results

Chapter 2

24

2.4.1 Phenology, fruit set, and potential for autogamous pollination

The results of the flowering phenology are shown in Fig. 2.2. The flowering peaks of

the two study species indicate that there is a substantial overlap in flowering period.

Dactylorhiza incarnata flowered from mid-May until the end of June and peaked at

the beginning of June. Dactylorhiza praetermissa flowered from the end of May until

the beginning of July and peaked in mid-June. The maximal overlap in flowering

curves was around 8th June. The results of our bagging experiment showed that if

solely relying on autogamy D. incarnata had a slightly higher percentage of fruit set

than D. praetermissa (5.45% vs 1.56% respectively, Table 2.2), but the difference

was not significant (Mann-Whitney U; z = 1.794; P = 0.073). Dactylorhiza

praetermissa had a significantly higher natural fruiting success than D. incarnata

(49.8% vs 25.6%, respectively; Mann-Whitney U; z = 7.837; P < 0.001).

Fig. 2.2. Flowering curves for Dactyorhiza incarnata and D. praetermissa in Belgium.

0

0.2

0.4

0.6

0.8

1

27-May 6-Jun 16-Jun 26-Jun 6-Jul

Flow

erin

g in

divi

dual

s (%

)

Date

D. incarnata

D. praetermissa

Hybridization between two Dactylorhiza species

25

Table 2.2. Measures of genetic variation and reproductive biology of D. incarnata and D.

praetermissa in Vaarttaluds, Moen, Belgium.

Statistic D. incarnata D. praetermissa

N 95 69 F1 (+ SE) 0.034 (+ 0.010) 0.113 (+ 0.012) B -0.009 -0.022 Sp 0.0092 0.0250 Fruiting success (%) 25.60 49.80 Autogamy (%) 5.45 1.56 Pp (%) 33.73 71.69 Ps (%) 6.63 40.96

Notes: N = the number of individuals sampled (without putative hybrids); F1 = the kinship coefficient of the first distance interval with the standard error of the mean estimated by a jackknife procedure; b = regression slope of Fij vs ln distance; Sp = Sp statistic; fruiting success = ratio number of fruits/number of flowers; autogamy = fruiting success of bagged individuals; Pp = percentage of polymorphic loci; Ps = percentage of species-specific loci.

2.4.2 Pollination experiment and viability of seeds

When plants were pollinated with pollinia from the other species, fruiting success was

almost 100% for both species. The average percentage of viable seeds of the 100

flowers of D. praetermissa pollinated with D. incarnata pollinia was 77.9%, whereas

that for D. incarnata receiving D. praetermissa pollinia was 51.1%. The average seed

viability of the cross-pollinated plants was very similar to those of the open-pollinated

pure species, which were 85.2% and 49.4% for D. praetermissa and D. incarnata,

respectively.

2.4.3 Identification of putative hybrids

The four primer combinations used for the AFLP analysis generated a total of 166

polymorphic loci. Using Structure, five putative hybrids were detected with a

threshold (Tq) of 0.90 (Fig. 2.3). The confidence intervals for the pure individuals

were very small, whereas these for the hybrid individuals were clearly larger. There

was some overlap between the hybrid and D. praetermissa individuals. Figure 2.4

shows the PCO plot of the two parental species and putative hybrids based on

individual genetic distances calculated with 166 polymorphic AFLP markers. The first

two axes explained 55.8% and 4.0% of the variation, respectively. The PCO (Fig. 2.4)

clearly shows that the parental species are strongly separated. Irrespective of their

population of origin, individuals clustered according to their morphological identities.

Chapter 2

26

The five putative hybrids did not take an intermediate position between the parental

species, but were genetically more similar to D. praetermissa than to D. incarnata.

The genetic variation in D. incarnata is much lower than in D. praetermissa. The

strong differences of polymorphic and species-specific loci between D. praetermissa

and D. incarnata, respectively 71.7% and 41.0% for D. praetermissa and 33.7% and

6.6% for D. incarnata (Table 2.2), are congruent with the low spread of D. incarnata

in the PCO plot, as would be expected given that the diploid D. incarnata is a

parental species of the tetraploid D. praetermissa.

0.0

0.2

0.4

0.6

0.8

1.0

q

H

D. incarnataD. praetermissa

D. praetermissa D. incarnata

Fig. 2.3. Clustering results for all individuals obtained for K = 2, using the program Structure. Each individual is represented by a vertical bar partitioned into K colored segments that correspond to the estimated membership proportions for each parental species (Dactylorhiza incarnata and D. praetermissa). Posterior probabilities (q) are given on the y-axis. The individuals identified as putative hybrids by Structure, at threshold Tq = 0.90, are indicated with “H”.

Individuals

Hybridization between two Dactylorhiza species

27

Fig. 2.4. Principal coordinates analysis (PCO) of parental species and putative hybrids based on individual genetic distances determined from 166 polymorphic AFLP markers. For Dactylorhiza praetermissa (left), and D. incarnata (right) different symbols indicate the subpopulation origin of the individuals (see Fig. 2.1). Filled symbols are putative hybrids. The first two axes described 55.8% and 4.0% of the variation, respectively.

Fig. 2.5. Principal component analysis (PCA) of parental species and putative hybrids based on morphological characters. The first two axes explained 29.1% and 16.8% of the variation, respectively.

Chapter 2

28

2.4.4 Ploidy level

The mean D. incarnata : Lolium ratio was 3.04 and the mean D. praetermissa :

Lolium ratio was 5.35 (SD among repeats = 0.047; SD among samples = 0.081). This

pattern corresponds with the diploid and allotetraploid nature of D. incarnata and D.

praetermissa, respectively (Hedrén 1996a,b). Four of the five putative hybrids

detected by Structure had the same ratio values as D. praetermissa individuals. One

individual had a value of 3.94, which is intermediate between values typical of D.

incarnata and D. praetermissa. This individual is presumably a triploid hybrid. One

putative pure D. praetermissa individual had a value of 8.11 and could therefore be

hexaploid.

2.4.5 Morphological analysis

Figure 2.5 shows the principal component analysis (PCA) plot of the two parental

species and putative hybrids based on morphological characters. The first two axes

explained 29.1% and 16.8% of the variation, respectively. The two parental species

are clearly separated, but there was no intermediate cloud. The five putative hybrids

identified using Structure all belong to the D. praetermissa group.

The main characters that were associated with the first component were type of

markings on labellum (0.948), labellum width (0.916), labellum ground color (0.883),

lateral lobe of the labellum length (0.876), lateral lobe of the labellum width (0.836),

middle lobe of the labellum width (0.767), height of plant (0.641), labellum length

(0.599), lateral sepal length (0.592). The main characters that were associated with

the second component were number of flowers (0.879), inflorescence length (0.837),

stem diameter (0.772), leaf width (0.728), and number of leaves (0.668). A complete

list of the vector loadings of all the different characters is given in Table 2.3.

Hybridization between two Dactylorhiza species

29

Table 2.3. Contributions of morphological characters to the first two multivariate axes of the principal component analysis, which explained 29.1% and 16.8% of the variation, respectively. Component 1 Component 2 1 Leaf length 0.221 0.589 2 Leaf width -0.040 0.728 3 Number of leaves 0.039 0.668 4 Plant height 0.641 0.560 5 Inflorescence length 0.181 0.837 6 Stem diameter -0.506 0.772 7 Number of flowers 0.055 0.879 8 Spur length 0.156 0.189 9 Spur width 0.120 -0.018 10 Lateral sepal length 0.592 0.004 11 Lateral sepal width 0.356 0.329 12 Middle sepal length 0.535 0.082 13 Middle sepal width 0.558 0.229 14 Lateral petal length 0.110 0.112 15 Lateral petal width 0.099 0.251 16 Labellum length 0.599 0.063 17 Labellum width 0.916 0.075 18 Labellum shape index -0.389 -0.090 19 Middle lobe of labellum length 0.092 0.065 20 Middle lobe of labellum width 0.767 0.071 21 Lateral lobe of labellum length 0.876 0.072 22 Lateral lobe of labellum width 0.836 0.047 23 Labellum ground colour 0.883 -0.033 24 Labellum type of markings 0.948 -0.056

2.4.6 Spatial genetic Structure

Analysis of Molecular Variance (AMOVA) indicated relatively high genetic

differentiation between the subpopulations. The ΦST value between subpopulations 1

and 3 of D. incarnata was 0.08 (df = 94). For D. praetermissa, ΦST values were 0.08

(subpopulations 1-2; df = 57), 0.11 (subpopulations 1-3; df = 48), and 0.15

(subpopulations 2-3; df = 40). All ΦST values were significantly different from zero (P

= 0.001).

The results from the spatial autocorrelation analysis indicated a highly

significant spatial genetic Structure for both species (Fig. 2.6). Mean values of the

regression slopes (b) were -0.009 and -0.022 for D. incarnata and D. praetermissa,

respectively (Table 2.2). The kinship coefficient for the first distance interval (F1) was

much higher for D. praetermissa (0.113 ± 0.012) than for D. incarnata (0.034 ±

0.010). For both species, positive values of Fij were found at short distances,

Chapter 2

30

whereas negative values of Fij occurred at larger distances, indicating that

neighboring individuals were more related than random pairs of individuals. The Sp

statistic was higher for D. praetermissa (0.025) than for D. incarnata (0.009) (Table

2.2), indicating a stronger genetic Structure within D. praetermissa. We also

conducted the autocorrelation analysis on individual subpopulations. For D. incarnata

the Sp values for subpopulations 1 and 3 were both 0.005. For D. praetermissa the

Sp values for subpopulations 1, 2 and 3 were respectively, -0.002, 0.035 and 0.127.

The Sp value is very variable between the different subpopulations of D.

praetermissa, but except for subpopulation 1, the values are still higher than the Sp

value of D. incarnata.

0 1 2 3 4 5 6 7Ln Distance (m)

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

F ij (m

ean ±

SE)

D. praetermissaD. incarnata

Fig. 2.6. Autocorrelogram of kinship coefficients vs. logarithmic distance

between Dactylorhiza incarnata and D. praetermissa individuals.

Hybridization between two Dactylorhiza species

31

2.5 Discussion

2.5.1 Hybridization

Although D. incarnata and D. praetermissa are thought to hybridize (Lambinon et al.

1998; Weeda et al. 2003; Bournérias et al. 2005; Van Landuyt et al. 2006), there is

no genetic evidence in the literature of ongoing hybridization between these two

species. Shaw (1998) suggested frequent hybridization between D. incarnata and D.

praetermissa, but only based on morphological characters. However, several studies

have shown that morphological characters are not always reliable for identifying

hybrids between similar parents (Wallace 2006; Bateman et al. 2008; Burgarella et al.

2009).

Contrary to our expectation of hybridization, only five out of 169 individuals were

classified as putative hybrids based on their AFLP profiles using Structure. Just one

of these individuals had a deviating DNA content and was probably a true primary

hybrid. Some pure D. praetermissa plants were perhaps misclassified as hybrids; the

HW equilibrium assumption in Structure may not have been met because of the

contrasting ploidy levels of the parental species. Despite the low number of hybrids,

our study provides evidence for hybridization between species with different ploidy

levels. Other studies have reported hybridization between polyploid derivatives and

their diploid progenitors. Ståhlberg and Hedrén (2009) found relatively few triploid

hybrids when they examined hybridization patterns between the diploid D. fuchsii and

the autotetraploid D. maculata. Their study also provided indications of introgressive

gene flow between ploidy levels. Heslop-Harrison (1953, 1957) studied morphological

intermediates between D. fuchsii and the tetraploids D. purpurella and D.

praetermissa. His main conclusions were that most hybrids were eutriploids and

showed a high level of seed-sterility. Lord and Richards (1977) reported a hybrid

swarm between the dipoid D. fuchsii and tetraploid D. purpurella based on

chromosome counts and floral characters. In addition to a large number of eutriploid

hybrids, they also found aneuploid individuals. Significant numbers of triploid hybrids

were also found by Aagaard et al. (2005), who examined a hybrid zone between

diploid D. incarnata ssp. cruenta and its putative allotetraploid derivative D.

lapponica. The results of their study suggest backcrossing between the triploid

hybrids and D. lapponica, and hence some hybrid fertility.

Chapter 2

32

A low number of hybrids has been reported before in other terrestrial orchid

species (Wallace 2006; Ståhlberg and Hedrén 2009). Different flowering phenologies,

different pollinators, and restricted gene flow have all been put forward as barriers

hindering the formation of hybrids (Scopece et al. 2007). Given the large overlap of

flowering curves of our study species, differences in flowering phenology can

probably be excluded. Since the main pollinators of both species are naïve

bumblebees (Bombus sp.) (Ferdy et al. 2001; Lammi et al. 2003; Vallius et al. 2004),

pollinator specificity is unlikely to play a role either. Spatial processes seem to play

an important role in the system studied. In the first place, the ΦST values between the

different subpopulations are significant, which may indicate spatially restricted gene

flow through pollen and/or seed. Spatially restricted gene flow is also suggested by

the results of the SGS analysis. In addition, the fine-scale spatial distribution of the

two species might also contribute to low rates of gene flow between them. Although

the two studied species occur in the same area, their occurrence is rather patchy

because of differences in microhabitat preference. Dactylorhiza incarnata is confined

to the most humid places, whereas D. praetermissa also occurs on slightly drier

places.

An additional factor contributing to low hybridization rates could be a lower

viability of the hybrid seeds through genic incompatibilities (Scopece et al. 2007).

However, the hybrid seeds generated by forced interspecific crosses displayed high

viability percentages. Interestingly, the interspecific crosses produced viable seeds in

both directions, which suggests that both species can be the maternal or paternal

plant. This is in contrast with the allopolyploid origin of many Dactylorhiza species

where D. incarnata can only act as pollen-parent and not as ovule-parent (Pillon et al.

2007).

On the other hand, the triploid nature of the hybrids may play an important role

in preventing the formation of successful hybrids in later life stages, through genic

incompatibilities (Ramsey and Schemske 1998; Rieseberg and Carney 1998; Burke

and Arnold 2001; Coyne and Orr 2004). Moreover, it is possible that the degree of

hybridization was overestimated in the past. Since D. praetermissa has a variable

morphology, like many other allopolyploid Dactylorhiza species, (Bateman and

Denholm 1983; Vanhecke 1989, 1993; Tyteca and Gathoye 1993; Shaw 1998), it is

likely that morphological variants were confused with interspecific hybrids in previous

studies, as was suggested by Wallace (2006) for Platanthera species.

Hybridization between two Dactylorhiza species

33

Most studies investigating hybridization in orchid species have reported hybrids

that grouped together and were intermediate between the parental species based on

either morphological (Caputo et al. 1997; Nielsen 2000; Cozzolino et al. 2006; Moccia

et al. 2007) or genetic data (e.g. Hedrén 1996a; Cozzolino et al. 2006; Moccia et al.

2007). In contrast, the five putative hybrids detected in our study area did not group

together and clearly belonged to the D. praetermissa morphotype (Fig. 2.5). From a

genetic viewpoint they were also more similar to D. praetermissa than to D.

incarnata. This finding is not unique. Wallace (2006), for example, who studied

hybridization between the North American orchids Platanthera aquilonis and P.

dilatata, concluded that morphology was not consistently reliable for determining

hybrid status even though the parental species differed significantly in several traits.

Bateman et al. (2008) concluded that one parent contributed substantially more to the

hybrid phenotype than did the other parent, based on morphometric and genetic

(AFLP) analyses on Orchis purpurea, O. simia and their interspecific hybrids.

Ståhlberg and Hedrén (2009), examining hybridization patterns between the diploid

D. fuchsii and the autotetraploid D. maculata, found that, based on morphology and

nuclear markers, hybrid triploids were more similar to tetraploids than to diploids. In a

hybrid zone between diploid D. incarnata ssp. cruenta and allotetraploid D. lapponica,

Aagaard et al. (2005) concluded that triploids were morphologically more similar to

tetraploids than to diploids. In the Asiatic orchids Liparis kumokiri and L. makinoana,

hybrid genotypes were also skewed towards one of the parental species (Chung et

al. 2005a).

The fact that the putative hybrids were more similar to their allotetraploid

parent is not that surprising since triploid hybrids receive 2/3 of their genome from the

tetraploid parent and 1/3 from the diploid parent. Also, the large numbers of species-

specific markers that were found in the allotetraploid D. praetermissa compared with

the diploid D. incarnata may contribute to the similarity of the hybrids to D.

praetermissa. Because D. incarnata is the pollen–parent of D. praetermissa (Pillon et

al. 2007), this may further explain why the hybrids were skewed towards D.

praetermissa. If the genome constitution of D. incarnata is represented by II and of D.

praetermissa by FFII (FF coming from D. fuchsii as the ovule–parent of D.

praetermissa) (Hedrén 1996a; Pillon et al. 2007), the genome constitution of the F1

hybrids is FII. Whereas these hybrids differ markedly from D. incarnata in that they

Chapter 2

34

contain a copy of the F genome, they only differ quantitatively from the allotetraploid

FFII in numbers of F genome copies.

The low genetic variation of D. incarnata compared to that of D. praetermissa

(Fig. 2.4) is in accordance with the literature. Genetic variation has long been known

to be much lower in D. incarnata than in allotetraploid species (Hedrén 1996a;

Pedersen 1998; Hedrén et al. 2001; Pillon et al. 2007). However, genetic variation of

D. praetermissa in our study was not very high either, since the second axis of the

PCO is only covering 4% of the variation. We cannot explain this low genetic

variation in D. praetermissa, but historical bottlenecks or founder effects could be

possible explanations.

2.5.2 Spatial genetic Structure

In both species, spatial autocorrelation analysis demonstrated the presence of a

highly significant spatial genetic Structure. The positive values of Fij at short

distances indicate that neighboring individuals are genetically more closely related

than random pairs of individuals. The Sp statistic allows comparison of the spatial

genetic Structure pattern between studies and species (Vekemans and Hardy 2004).

The Sp value of D. praetermissa was more than twice the value of D. incarnata,

indicating a stronger genetic Structure within D. praetermissa. In comparison with

mean values for other species, the Sp statistic of D. incarnata (0.0092) is similar to

the value reported for outcrossing species (0.0126), whereas the value of D.

praetermissa (0.0250) is substantially higher. The Sp statistic of Orchis purpurea

(0.0222), also a food-deceptive orchid (Jacquemyn et al. 2006), is very similar as

those of the two species investigated here. Two other orchid species, Platanthera

aquilonis and P. dilatata, have higher Sp values (0.0790 and 0.0540, respectively;

Wallace 2006) than our study species. In the case of P. aquilonis this was likely due

to its status as a facultative autogamous plant. However, comparisons with other

species should be interpreted with caution, since our study was conducted at only a

single site.

Significant spatial genetic Structure can be the result of limited seed and/or

pollen dispersal. Although one would expect that limited seed dispersal does not play

an important role in the Orchidaceae because of their small, wind–dispersed seeds,

many studies have shown that seed dispersal and establishment most often happen

Hybridization between two Dactylorhiza species

35

over short distances (Murren and Ellison 1998; Chung et al. 2005b; Jacquemyn et al.

2007). In addition, pollen dispersal distances can be limited when plants occur in

clearly delimited subpopulations, and pollinators move around within patches before

flying to another subpopulation. The highly significant ΦST values indeed suggest

limited gene flow among subpopulations. Another possible cause for a significant

spatial genetic Structure could be a high degree of self-pollination. In our case this

would mean that D. praetermissa has a higher degree of self- pollination compared

with D. incarnata, but this was not supported by our bagging experiment. Therefore,

the higher Sp statistic of D. praetermissa is not caused by a higher degree of

autogamy. A possible explanation could be linked with the higher fruiting success of

D. praetermissa (49.8%) compared with D. incarnata (25.6%) due to higher pollinator

visitation rates. Possibly, due to higher intraspecific variation in flower morphology in

tetraploid species, naïve pollinators take longer to learn to avoid the flowers of

tetraploid D. praetermissa plants than those of diploid D. incarnata plants. As naïve

insects typically visit several flowers of the same plant before going to the next plant,

a higher pollination frequency of D. praetermissa may cause a higher degree of

inbreeding. Finally, it is also possible that the lower Sp value of D. incarnata is a

consequence of its lower genetic variation compared to D. praetermissa.

2.6 Conclusions

In this study we combined morphological, cytological, and molecular data to study

hybridization between D. incarnata and D. praetermissa. Although other Dactylorhiza

species with different ploidy levels may frequently form hybrids, we found very few

unequivocal hybrids in the chosen study area. Several non-mutually exclusive factors

can contribute to this outcome. Spatially restricted gene flow observed is probably

one of the reasons for the low number of hybrids detected. It is also possible that

triploid hybrids experience difficulties during establishment through genic

incompatibilities. Future research will assess the different reproductive barriers in

more detail.

2.7 Acknowledgements

Chapter 2

36

The authors thank K. Libaut, S. Provoost, and P. De Raeymaecker for assistance, M.

Leten for useful information, and W. Pauwels of Natuurpunt for permission to conduct

our research in the Vaarttaluds nature reserve. We also thank R. Bateman and an

anonymous reviewer for helpful comments. A grant from the Flemish fund for

scientific research (FWO) [G.0592.08] is gratefully acknowledged.

Chapter 3.

Reproductive isolation and hybridization in sympatric populations of three

Dactylorhiza species (Orchidaceae) with different ploidy levels

Adapted from:

De hert K, Jacquemyn H, Van Glabeke S, Roldán-Ruiz I, Vandepitte K, Leus L, Honnay O (2012). Reproductive isolation and hybridization in sympatric populations of

three Dactylorhiza species (Orchidaceae) with different ploidy levels. Annals of Botany

109: 709–720.

Chapter 3

38

3.1 Abstract

• Background and Aims The potential for gene exchange between species with different

ploidy levels has long been recognized, but only few studies have tested this hypothesis

in situ and most of them focused on not more than two co-occurring species. In this

study, we examined hybridization patterns in two sites containing three species of the

genus Dactylorhiza (diploid D. incarnata and D. fuchsii and their allotetraploid derivative

D. praetermissa).

• Methods To compare the strength of reproductive barriers between diploid species,

and between diploid and tetraploid species, crossing experiments were combined with

morphometric and molecular analyses using AFLP markers, whereas flow cytometric

analyses were used to verify the hybrid origin of putative hybrids.

• Key Results In both sites, extensive hybridization was observed indicating that gene

flow between species is possible within the investigated populations. Bayesian

assignment analyses indicated that the majority of hybrids were F1 hybrids, but in some

cases triple hybrids (hybrids with three species as parent) were observed, suggesting

secondary gene flow. Crossing experiments showed that only crosses between pure

species yielded a high percentage of viable seeds. When hybrids were involved as

either pollen-receptor or pollen-donor, almost no viable seeds were formed, indicating

strong post-zygotic reproductive isolation and high sterility.

• Conclusions Strong post-mating reproductive barriers prevent local breakdown of

species boundaries in Dactylorhiza despite frequent hybridization between parental

species. However, the presence of triple hybrids indicates that in some cases

hybridization may extend the F1 generation.

3.2 Introduction

Hybridization and introgression may increase genetic diversity within species, transfer

genetic adaptations between species, break down or reinforce reproductive barriers

between closely related groups, and lead to the emergence of new ecotypes or species

Hybridization between three Dactylorhiza species

39

(Barton and Hewitt 1989; Abbot 1992; Rieseberg 1997). Hybridization occurs either with

or without genome doubling. Hybridization with genome doubling (i.e.

allopolyploidisation) is one of the most common mechanisms of sympatric speciation

(Arnold 1997; Otto and Whitton 2000; Coyne and Orr 2004; Rieseberg and Willis 2007).

There is also strong empirical evidence that hybridization can give rise to new species

without a change in ploidy level (“homoploid hybrid speciation”) (Arnold 1997; Rieseberg

1997; Rieseberg and Willis 2007).

Hybrid zones between cytotypes with different ploidy levels are especially

interesting, as they allow studying the mechanisms involved in the early stages of

polyploid speciation and how reproductive isolation mechanisms may affect the

establishment of polyploids in diploid populations (Thompson and Lumaret 1992; Petit et

al. 1999; Coyne and Orr 2004). Although the potential for gene exchange between taxa

with different ploidy levels has long been recognized (Stebbins 1971), relatively few

studies have tested its frequency in the wild (Petit et al. 1999; Chapman and Abbott

2010; De hert et al. 2011). The reason for this is that gene flow between species with

different ploidy levels is thought to be unlikely, as they are assumed to be reproductively

isolated from each other due to strong post-zygotic barriers (Coyne and Orr 2004).

However, recent studies have suggested the evolution of reproductive barriers in contact

zones of diploid-polyploid species, including the possibility of introgression across

species boundaries (Aagaard et al. 2005; Shipunov et al. 2005; Pillon et al. 2007;

Ståhlberg and Hedrén 2009; Pinheiro et al. 2010).

The food-deceptive orchid genus Dactylorhiza is an excellent study group to test

the extent of gene flow between species of different ploidy levels. Polyploidisation within

Dactylorhiza has been relatively well-documented (Hedrén 1996a; Hedrén et al. 2001;

Devos et al. 2006; Pillon et al. 2007) and allotetraploids have evolved on several

occasions due to repeated hybridization (Hedrén 1996a, 2003) between two main

groups of parental taxa. These parental lineages include the diploid marsh orchids, D.

incarnata sensu lato, and the spotted orchids, the diploid D. fuchsii and more rarely also

the autotetraploid D. maculata (Heslop-Harrison 1953, 1956; Hedrén 1996a; Hedrén et

al. 2001; Devos et al. 2006; Pillon et al. 2007). Hedrén et al. (2001) suggested that

allotetraploidization dominates over introgression as speciation mechanism in this

Chapter 3

40

genus. Hybridization between species with different ploidy levels has been frequently

reported in the genus Dactylorhiza (Heslop-Harrison 1953, 1957; Lord and Richards

1977; Aagaard et al. 2005; Shipunov et al. 2005; Pillon et al. 2007; Ståhlberg and

Hedrén 2009). Hybridization and introgression between diploids and allotetraploids, and

between different independently derived allotetraploids may further have contributed to

genetic diversity at the tetraploid level (Hedrén 2003). Even the possibility exists that

some allotetraploid species have multiple origins (Hedrén 1996a).

Greater insights into the different pathways of the speciation processes in

Dactylorhiza requires studying in situ hybridization in natural hybrid zones and assessing

the strength of reproductive barriers. Pre-mating barriers are generally very weak in the

genus (Aagaard et al. 2005; Ståhlberg and Hedrén 2009), since Dactylorhiza species

are food-deceptive and depend on naïve pollinators (Mattila and Kuitunen 2000; Ferdy

et al 2001; Lammi et al. 2003; Vallius et al. 2004, 2007). Post-zygotic barriers, on the

other hand, are expected to be more important (Scopece et al. 2007, 2008), particularly

in species with differing ploidy levels. In this case, hybrid inviability and sterility are not

uncommon and may result from chromosomal rearrangements and/or genic

incompatibilities (Rieseberg and Carney 1998). However, relatively few studies have

examined in situ hybridization in Dactylorhiza and all of them focused on not more than

two co-occurring species. Some studies found indications of backcrossing (Aagaard et

al. 2005), whereas others found evidence for introgressive gene flow between ploidy

levels (Nordstrom and Hedrén 2008; Ståhlberg and Hedrén 2009), suggesting the

occurrence of introgression, and hence of some hybrid fertility. In hybrid zones

containing more than two species, patterns of hybridization may become more complex.

In this study, we examined hybridization patterns at two different sites containing

two diploid species (D. incarnata (L.) Soó and D. fuchsii (Druce) Soó) and one

allotetraploid species (D. praetermissa (Druce) Soó). To assess the strength of

reproductive barriers acting between the investigated species, molecular markers, flow

cytometry and morphological analyses were used. In addition, the genome compatibility

of parental species and the fertility of putative hybrids were assessed by manual

crossing experiments. More specifically, we addressed the following questions. (1) Do D.

incarnata, D. fuchsii and D. praetermissa hybridize with each other in natural sympatric

Hybridization between three Dactylorhiza species

41

populations? (2) If hybridization occurs, is there a difference in frequency of hybridization

between different parental species? (3) How strong are postmating barriers between

these taxa?

3.3 Materials and Methods

3.3.1 Study species

Dactylorhiza incarnata, D. fuchsii and D. praetermissa are herbaceous, perennial, food-

deceptive orchid species. D. praetermissa is an allotetraploid (2n = 4x = 80), whereas D.

incarnata and D. fuchsii are diploids (2n = 2x = 40) and likely the present-day

representatives of the progenitor lineages of D. praetermissa (Hedrén 1996a, b).

However, some authors (Devos et al. 2006; Pillon et al. 2007) have suggested that the

maternal parent of D. praetermissa may have been related to the southern D. saccifera

rather than D. fuchsii (or at least partly), as it contains some ITS variants typical of D.

saccifera. The descriptions of morphology and ecology of our study species are based

on their occurrence in Belgium. Dactylorhiza incarnata is predominantly found in wet

meadows, dune slacks, fens, and marshes, where it often occurs in somewhat

calcareous, nutrient-poor, humid soils. Dactylorhiza praetermissa prefers calcareous

soils, and sites with some disturbance. It occurs in periodically flooded dune slacks,

roadsides, canal verges, raised terrains, grazed or mown fens. Dactylorhiza fuchsii

occurs in humid forests, mesophilic grasslands and wet meadows, on nutrient-rich,

mostly neutral or alkaline substrate.

All three species are mainly pollinated by bumblebees (occasionally also by other

relatively large insects (Hymenoptera, Coleoptera)) (Ferdy et al. 2001; Lammi and

Kuitunen 1995; Lammi et al. 2003; Vallius et al. 2004). Dactylorhiza incarnata has a

dense inflorescence with pink-purplish, purplish or occasionally almost white flowers.

The flowers are relatively small and the labellum has reflexed lateral lobes. The labellum

markings consist of loops which are somewhat lung-shaped. Leaves of D. incarnata are

lanceolate, and usually have a hooded leaf tip. Some subspecies of D. incarnata can

have yellow flowers (ssp. ochroleuca) or spots on both sides of the leaves (ssp. cruenta)

(Bateman and Denholm 1985), but these subspecies—some authors (e.g. Bournérias

Chapter 3

42

and Prat 2005) treat them as distinct species—do not occur in Belgium. Inflorescences

of D. praetermissa are dense and comprise bright pink to purplish flowers (Lambinon et

al. 1998; Bournérias and Prat 2005). The labellum is often rather flat and bears many

small spots, which may be accompanied by short lines or even loops. The lanceolate

leaves of D. praetermissa can be spotted, but if so only on the upper surface. Both D.

incarnata and D. praetermissa have a hollow stem, in contrast to D. fuchsii which has a

solid stem. Inflorescences of D. fuchsii are dense and comprise small flowers which are

white or pink to pale purplish. The labellum of the flowers is very clearly trilobed; the

middle lobe is nearly as long as the lateral lobes. The labellum markings consist mainly

of loops which are somewhat ‘w’-shaped. The lanceolate leaves of D. fuchsii are nearly

always spotted on the upper surface of the leaf.

3.3.2 Study sites and data collection

The study was conducted in two dune slacks, one at a site named Ter Yde

(Oostduinkerke, Belgium, 51°08’03”N and 2°41’16”E) and one at a site named

Paelsteenpanne (Bredene, Belgium, 51°15’31”N and 2°59’04”E). The climate at both

sites is maritime temperate. Typical co-occuring species were Salix repens, Centaurium

littorale, Carex trinervis, and Epipactis palustris. Management of both areas consists of

mowing after the growing season. Both sites previously contained all three species and

putative hybrids, but at Ter Yde pure D. praetermissa individuals are no longer present

(W. Van den Bussche, National Botanic Garden of Belgium, pers. comm.). Within each

site, we first selected at random a set of apparently pure individuals, i.e. individuals that

clearly belonged to one of the three species based on the morphological characteristics

described above. Next, we selected a large set of putative hybrids throughout the site.

Putative hybrid individuals were selected to represent all morphological variation

present. At the same time, leaf samples were collected for DNA analysis. Leaf material

was immediately frozen in liquid nitrogen and stored at -80°C before freeze drying for 48

h. The dried material was vacuum packed and stored at room temperature until DNA

extraction. A total of 295 (91 D. incarnata; 46 D. fuchsii and 158 putative hybrids) and

163 (44 D. incarnata; 13 D. fuchsii; 52 D. praetermissa and 54 putative hybrids)

flowering individuals were mapped and sampled in Ter Yde (June 2008) and in

Hybridization between three Dactylorhiza species

43

Paelsteenpanne (June 2009), respectively. The estimated number of flowering

individuals was 9000 in Ter Yde (4000 D. incarnata; 1300 D. fuchsii and 3700 putative

hybrids) and 2500 in Paelsteenpanne (1500 D. incarnata; 20 D. fuchsii; 400 D.

praetermissa and 550 putative hybrids).

After fruit maturation, fruiting success was determined for each sampled plant as

the ratio of the number of fruits over the number of flowers. Fruits were harvested and

brought to the laboratory for further inspection. The proportion of viable seeds was

determined for five plants from each category (five fruits per plant) using the tetrazolium

method, according to the protocol of Van Waes and Debergh (1986); only viable

embryos are stained red after the treatment. Because the tetrazolium method can

overestimate the real germination rate, the seed viability percentages should be treated

with caution. However, for comparative purposes this method is sufficient to investigate

differences among pure species and hybrids and among pollination treatments. We

counted a subsample of 200 to 300 seeds from each capsule. We calculated the

percentage of viable seeds as the ratio of the number of colored seeds over the total

number of seeds.

3.3.3 DNA extraction and AFLP analysis

Forty milligram of the dried material was ground using a Retsch mill and DNA-isolation

was carried out according to the CTAB-extraction procedure of Lefort and Douglas

(1999). DNA concentrations were estimated using a NanoDrop ND-1000

spectrophotometer (NanoDrop Technologies). Amplified Fragment Length

Polymorphism (AFLP) markers (Vos et al. 1995) were used to screen the genetic

diversity of our samples. AFLP analysis was carried out according to the protocol

described in Vandepitte et al. (2009). Four primer combinations were used: EcoRI-

AGG/MseI-CTAG, EcoRI-AGG/MseI-CTGG, EcoRI-ACAG/MseI-CTA, and EcoRI-

ACAG/MseI-GGT (MWG biotech, Ebersberg, Germany). AFLP-fragments were

separated on an ABI3130xl sequencer on 50 cm capillaries using the polymer Pop7

(Applied Biosystems). GeneScan 500 Rox labelled size standard (Applied Biosystems)

was injected with each AFLP sample, to allow sizing of the DNA-fragments. The

fluorescent AFLP patterns were scored using GeneMapper 3.7 (Applied Biosystems).

Chapter 3

44

Each marker was coded as 1 or 0 for presence or absence in an individual, forming a

binary data matrix. Each sample was thus represented by a vector of 1s and 0s. To

assess the reproducibility of the protocol, two independent DNA extractions were carried

out for nine samples. The overall error rate, i.e. the percentage of differently scored loci

between repeated samples, was low (3.4%).

AFLP data were subjected to Bayesian analyses using methods implemented in

the program Structure version 2.2 (Pritchard et al. 2000). Structure uses a model-based

clustering method to assign individuals to groups in which deviations from Hardy–

Weinberg equilibrium and linkage equilibrium are minimized. Individuals assigned to two

sources with non-trivial probabilities are potential hybrids. In the Structure model, the

posterior probability (q) describes the proportion of an individual genotype originating

from each of K categories. In the present case, setting K = 3 corresponds to the

assumption of three species contributing to the gene pool of the sample. Preliminary

assignment analysis based on the criteria of Evanno et al. (2005) confirmed this

assumption. When we ran Structure on a dataset of only pure individuals, the Ln P(D)

values increased until K = 3 and then evened out. For K = 3 all pure individuals were

unambiguously assigned to their assumed species. Because we could not find clearly

pure D. praetermissa individuals in Ter Yde, we used pure D. praetermissa individuals

from the nature reserve Vaarttaluds (De hert et al. 2011), that were genotyped in the

same month as the samples from Ter Yde, as reference in the Structure analysis of this

site.

AFLP data were entered as Ploidy = 2 for all individuals, with the second line as

missing data. Calculations followed the admixture model of ancestry, assuming

correlated allele frequencies. We used prior population information: individuals of known

pure origin (Popflag=1) were used as learning samples to classify individuals of

unknown origin. We used a burn-in of 500 000 steps, followed by 1 000 000 iterations,

after verifying that the results did not vary significantly across multiple runs. We also

tested whether the results changed as we entered the data as only a single line for each

individual (Ploidy=1) under the ‘no admixture’ model, but the results were the same. To

determine in which class an individual falls, we used as thresholds Tq = 0.80. We used

this less strict threshold in stead of the more common used Tq = 0.90 (Burgarella et al.

Hybridization between three Dactylorhiza species

45

2009), because the assumption of Hardy–Weinberg equilibrium is probably violated due

to the different ploidy levels of the parental species and because D. praetermissa

originated from the two other species. Individuals with q ≥ Tq are assigned to the

purebred category and individuals with q < Tq are assigned to the hybrid category. We

also performed a principal coordinates analysis (PCO), based on Jaccard distances

(with squared lambda as vector scaling), using NTSYSpc2.1 (Rohlf 2000), to visualize

the genetic distances between the parental species and the putative hybrids.

3.3.4 Morphological data

For each plant sampled for DNA analysis, morphological characters from both

vegetative and floral parts of the plant were measured. The following vegetative

characters were recorded in the field: length and width of the three largest leaves, plant

height, stem diameter, inflorescence length, number of flowers, number of leaves,

presence of spots on the leaves and presence of a solid or hollow stem. In addition, a

large number of flower characters was recorded. For each plant, two to three young

flowers were sampled, stored in a denatured ethanol solution and transported to the

laboratory. Each flower was dissected and a digital image was taken. The following

flower characters were measured using the image analysis software IMAGEJ 1.41

(Rasband 2011): spur length and width, labellum length and width, length and width of

the three lobes of the labellum, length and width of the three sepals, length and width of

the two lateral petals. The following labellum characters were determined based on

digital images taken in the field: ground colour on a scale 0-3 (0 = white, 1 = pale pink, 2

= deep pink, 3 = purple), type of markings on a scale 0-5 (0 = no markings, 1 = spots, 2

= spots and dashes, 3 = dashes and loops, 4 = ‘lung’-shaped loops, 5 = ‘w’-shaped

loops).

Morphological data were analyzed using a principal component analysis (PCA)

with Varimax rotation in SPSS 16.0 for Windows (SPSS Inc. 2007). We used the scores

obtained for each individual on the first two PCA axes to interpret the main patterns of

morphological variation in the dataset. The characters used in the analysis are given in

Table 3.1.

Chapter 3

46

Table 3.1. List of morphological characters

Vegetative characters 1 Leaf length, average of the length of the three largest leaves (cm) 2 Leaf width, average of the width of the three largest leaves (cm) 3 Number of leaves 4 Plant height, measured from ground level to apex of inflorecence (cm) 5 Inflorescence length, measured from lowermost flower to apex of inflorescence (cm) 6 Stem diameter, measured below inflorescence (mm) 7 Number of flowers 8 Stem solid (1)/holow (0) 9 Spots on leaves (1/0) Floral characters 10 Spur length (mm)/Spur width (mm) 11 Lateral sepal length (mm) 12 Lateral sepal width (mm) 13 Middle sepal length (mm) 14 Middle sepal width (mm) 15 Lateral petal length (mm) 16 Lateral petal width (mm) 17 Labellum length (mm)/Labellum width (mm) 18 Labellum shape index (modified after Pedersen, 2006) 19 Middle lobe of labellum length (mm) 20 Middle lobe of labellum width (mm) 21 Lateral lobe of labellum length (mm) 22 Lateral lobe of labellum width (mm) 23 Labellum ground colour, on a scale 0-3 (0 = white, 1 = pale pink, 2 = deep pink, 3 = purple) 24 Labellum markings, type of markings on a scale 0-5 (0 = no markings, 1 = spots,

2 = spots and dashes, 3 = dashes and loops, 4 = 'lung'-shaped loops, 5 = 'w'-shaped loops)

3.3.5 Flow cytometric analysis

We used flow cytometry (FCM) to determine the ploidy of pure species and of putative

hybrid groups (based on Structure results). At each site, we analyzed 10 individuals of

each pure species and of the different putative hybrid group, unless less than 10

individuals per group were present. Ten randomly chosen samples were analyzed twice

to verify the reproducibility of the FCM protocol. High-throughput ploidy analysis was

performed on a CyFlow Space (Partec, Münster, Germany) flow cytometer equipped

with a light-emitting diode (365 nm), using the protocol described by De Schepper et al.

(2001) with 4´,6-diamidino-2-phenylindole (DAPI) staining. Young leaf material (0.5 cm²)

was co-chopped with leaf material of diploid Lolium perenne, which was used as internal

Hybridization between three Dactylorhiza species

47

standard. Analyses were performed using Flomax software (Partec). Ploidy levels were

inferred from the peak position ratios on the histograms. At least 2500 nuclei per sample

were analyzed. Samples resulting in a coefficient of variation higher than 4% were

reanalysed.

3.3.6 Experimental crosses

Experimental crosses were conducted to assess the strength of post-zygotic mating

barriers. The following treatments were applied: (i) intraspecific cross-pollination control;

(ii) interspecific cross-pollination; (iii) between hybrids (based on Structure results); (iv)

between pure species and hybrids. All crosses were conducted in both directions. For

treatments (i) and (ii), ten flowers from ten plants were pollinated for each cross. For

treatments (iii) and (iv) five plants (ten flowers per plant) were pollinated for each cross

(or less when not enough plants or flowers were available). Prior to pollination, pollinia of

the pollinated flowers were removed to prevent self-pollination. After fruit maturation,

fruit set and seed viability were determined for all pollinated plants using methods

outlined before.

For each different pollen recipient category, the difference in fruiting success

between pure species pollen donors and hybrid pollen donors was tested with a Mann-

Whitney U test. (the difference between all the separate pollen donor categories could

not be tested since for some crosses data from only one or two plants were available).

Also, the difference in seed viability between pure species pollen donors and hybrid

pollen donors was tested with a Mann-Whitney U test for each pollen recipient category.

An ANOVA (+ post hoc Tukey test) and a Kruskal-Wallis test (+ nonparametric multiple

comparisons test) were used to statistically compare fruiting success between pure

species and hybrids in the field, for respectively the Paelsteenpanne and Ter Yde. To

statistically compare seed viability between pure species and hybrids in the field an

ANOVA (+ post hoc Tukey test) was used.

3.4 Results

3.4.1 Molecular analyses

Chapter 3

48

The four AFLP primer combinations generated a total of 195 polymorphic markers for

the individuals sampled in Ter Yde, and 190 polymorphic markers for the individuals

sampled in Paelsteenpanne. Using Structure, different hybrid groups were detected with

a threshold (Tq) of 0.80 (Fig. 3.1 and 3.2). Fig. 3.3a and Fig. 3.3b show the PCO plots of

the parental species and these hybrid groups based on individual genetic distances. The

first two PCO axes described 27.84% and 18.52% of the variation in the case of

Paelsteenpanne, and 28.51% and 16.44% in the case of Ter Yde (Fig. 3.3a and 3.3b).

Parental species were clearly separated on the PCO plots. Individuals classified as

hybrids according to Structure had intermediate positions between their parental species

in the PCO plots. The mean species vs. standard sample ratio of the flow cytometric

analysis (FCM ratio) (SD among repeats = 0.047; SD among samples = 0.081) per

Structure group is given in Table 3.2. Specific and shared bands for the different groups

are given in Table 3.3 and the percentage of polymorphic loci is shown in Table 3.2.

Fig. 3.1. Structure clustering results for all individuals obtained for K = 3 in Paelsteenpanne. Each individual is represented by a vertical bar partitioned into K coloured segments, which correspond to the estimated membership proportions for each parental species (D. incarnata, D. fuchsii and D. praetermissa). Posterior probabilities (q) are given on the y-axis. Individuals were assigned to hybrid groups using q = 0.20 as a threshold (see text for details).

Individuals

Hybridization between three Dactylorhiza species

49

Fig.

3.2

. Stru

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Chapter 3

50

Fig. 3.3. Principal co-ordinates analysis (PCO) of parental species and putative hybrids based on individual genetic distances determined from 190 polymorphic AFLP markers in Paelsteenpanne (A) and 195 polymorphic AFLP markers in Ter Yde (B). The first two axes described 27.84% and 18.52% of the variation in (A) and 28.51% and 16.44% of the variation in (B).

A

B

Hybridization between three Dactylorhiza species

51

Table 3.2. Results of flow cytometric analysis and percentage of polymorphic loci (PLP) for the populations Ter Yde and Paelsteenpanne. Category Population

Ter Yde Paelsteenpanne

FCM ratio PLP (%) FCM ratio PLP (%)

D. incarnata (2n = 40) 3.14 (0.06) 54.36 3.05 (0.05) 45.26

D. fuchsii (2n = 40) 2.41 (0.03) 53.33 2.49 (0.06) 46.84

IF hybrids / / 2.77 (0.06) 54.74

D. praetermissa (2n = 80) / (62.05)* 5.48 (0.10) 13.68

PI hybrids 4.30 (0.05) 66.15 / /

PF hybrids 3.94 (0.07) 67.18 4.12 (32.11)†

Triple hybrids 2.74 (0.05) 62.56 4.39 (0.08) 42.63

The mean (± s.d.) FCM ratio of Dactylorhiza vs. Lolium of ten indivuals per group (or less when less than ten individuals were available) is given. Individuals were assigned to hybrid groups based on the Structure results. / = category not present. * Value of D. praetermissa individuals in Vaarttaluds. † Based on the only two individuals.

In Paelsteenpanne, by far the largest group of hybrids is that between the diploids D.

incarnata and D. fuchsii (Fig. 3.1). These IF hybrids have an intermediate position

between D. incarnata and D. fuchsii in the PCO plot (Fig. 3.3a) and their FCM ratio

(2.77), is between that of D. incarnata (3.05) and D. fuchsii (2.49) (Table 3.2). Two

hybrids between D. fuchsii and D. praetermissa were detected by Structure. The FCM

ratio of these PF individuals (4.12) was between those of D. praetermissa (5.48) and D.

fuchsii (2.49). According to Structure, a third group of hybrids had a part of all three

species in their genome (q > 0.2) (Fig.3.1). On the PCO these individuals lie in a central

position, among all three pure species (Fig. 3.3a). Their FCM ratio was 4.39.

In Ter Yde, different hybrid groups were detected with Structure (Fig. 3.2), which

were also visible in the PCO plot (Fig. 3.3b). Hybrid individuals between D. incarnata

and D. praetermissa were abundant (Fig. 3.2). These PI hybrids had a ploidy level ratio

(4.30) that was between that of D. incarnata (3.14) and D. praetermissa (5.48; value of

Chapter 3

52

Paelsteenpanne). Hybrids between D. fuchsii and D. praetermissa were also detected

by Structure (Fig. 3.2). The ploidy level ratio of these PF hybrids (3.94) was intermediate

between its parental species (D. praetermissa: 5.48 and D. fuchsii: 2.41), which is in

accordance with the Structure results. Both PI and PF hybrids have a larger proportion

in their genome coming from D. praetermissa than from the other parental species (Fig.

3.2) and are also lying closer to D. praetermissa on the PCO. A third group of hybrids

had contributions of all three species to their genome (q > 0.2), according to Structure

(Fig. 3.2), and had a more or less central position on the PCO (Fig. 3.3b). The FCM ratio

of these individuals is 2.74.

Tabel 3.3. Number of specific AFLP bands for each species and number of shared bands between all different groups for each site

D. incarnata D. fuchsii D. praetermissa IF hybrids PF hybrids Triple hybrids

(a) Paelsteenpanne (total number of bands = 190) D. incarnata 40

D. fuchsii 44 32 D. praetermissa 56 56 8

IF hybrids 81 80 72 PF hybrids 55 75 76 81

Triple hybrids 66 77 77 93 90

(b) Ter Yde (total numer of bands = 195) D. incarnata 14

D. fuchsii 73 9 (D. praetermissa) 117 104 9

PI hybrids 126 106 153 PF hybrids 124 115 156 158

Triple hybrids 124 109 147 148 158

3.4.2 Morphological analyses

Results of the molecular analyses were largely confirmed by the morphological

analyses. The first two axes of the PCA on morphological data for Paelsteenpanne

described 30.62% and 22.51% of the variation, respectively (Fig. 3.4a). A complete list

of the vector loadings of all the different characters for both sites is given in Table I

(Appendix). The main characters are also indicated. The three parental species are

clearly separated on the PCA. Dactylorhiza praetermissa is intermediate between D.

Hybridization between three Dactylorhiza species

53

incarnata and D. fuchsii, although its position is closer to D. incarnata. The largest group

of hybrids, the IF hybrids, is intermediate between its parental species D. incarnata and

D. fuchsii, but closer to D. fuchsii. The putative triple hybrids have no distinct position,

but are mainly situated near the central part of the plot.

For Ter Yde, the first two axes of the PCA described 27.10% and 20.45% of the

variation, respectively (Fig. 3.4b). The two pure species D. incarnata and D. fuchsii are

clearly separated on the PCA. The three different hybrid groups tended to cluster

separately. The putative triple hybrids lie between D. incarnata and D. fuchsii, mainly

near the center of the PCA.

A

B

Chapter 3

54

Fig. 3.4. Principal component analysis (PCA) of parental species and putative hybrids based on morphological characters. (A) Individuals sampled in Paelsteenpanne. The first two axes described 30.62% and 22.51% of the variation, respectively. (B) Individuals sampled in the Ter Yde. The first two axes described 27.10% and 20.45% of the variation.

3.4.3 Fitness of natural hybrids and artificial crosses

In naturally pollinated plants, there was no clear difference in fruiting success between

parental species and hybrid groups at both sites (Table 3.4). Seed viability, on the other

hand, showed very clear differences. In Ter Yde, seed viability of both pure species (D.

incarnata: 68.87% and D. fuchsii: 49.80%) was significantly higher than that of the three

hybrid groups (ranging from 0.07% to 14.23%, Table 3.4; post hoc Tukey, p < 0.001 (for

D. fuchsii - PF: p = 0.008)). In Paelsteenpanne, seed viability of both pure species (D.

incarnata: 57.16%, D. praetermissa: 67.46%, D. fuchsii produced no fruits in the year of

collection) was significantly higher than that of all the hybrid classes (ranging from

0.11% to 0.69%, Table 3.4; post hoc Tukey, p < 0.001). Only one hybrid class, the PF

hybrids in Ter Yde, had a relatively high percentage of viable seeds (14.23%), compared

with the other hybrid classes.

The results of the experimental crosses confirm pollination in natural conditions

(Table 3.5). When pure species were used as pollen donor, fruit set was high (average:

89.85%), but when hybrids served as pollen donor, fruit set was very low (average:

3.13%; Table 3.5A). This difference was significant for all six pollen recipient categories

(Mann-Whitney U, p < 0.01; p < 0.05 for PI hybrids). When pure species were pollinated

with pure species pollen, a high percentage of viable seeds was formed, but when

hybrid pollen were used almost no viable seeds were formed. This difference was

significant for all three pure species (Mann-Whitney U, p < 0.01). When hybrids were

pollinated with pure species pollen or hybrid pollen, seed viability was very low in both

cases. No significant differences were observed for all three hybrid classes.

Only when intra- and interspecific crosses between pure species were conducted,

there was a high percentage of viable seeds (average: 62.31%) (Table 3.5B). When

hybrids were involved as either pollen-receptor or pollen-donor, almost no viable seeds

were formed (average: 0.26%). For some crosses there are little or no data of fruit/seed

set because of herbivore damage or because some plants suffered from drought.

Hybridization between three Dactylorhiza species

55

Table 3.4. Fruit set and seed viability in natural populations. Fruit set (%) Seed viability (%)

Ter Yde Paelsteenpanne Ter Yde Paelsteenpanne

D. incarnata 50.27 a (91) 53.92 ac ( 44) 68.87 a 57.16 a

D. fuchsii 14.17 b (46) 24.05 b (11) 49.80 a –

D. praetermissa / 63.06 c (52) / 67.46 a

PI hybrids 30.40 c (104) / 4.67 b /

PF hybrids 20.62 b (39) 61.10 (2) 14.23 b 0.48

IF hybrids / 48.54 a (45) / 0.11 b

Putative triple hybrids 26.90 bc (15) 41.66 ab (7) 0.07 b 0.69 b

Values with different superscripts indicate that they are significantly different at the α = 0.05 level (fruit set; Ter Yde: nonparametric multiple comparisons, Paelsteenpanne: post hoc Tukey) or α = 0.01 level (seed viability; post hoc Tukey). The number of sampled individuals in each group is given in parentheses. Notes: / = category not present; – = fruits not present for this category Tabel 3.5. (A) Fruit-set (%) and (B) seed viability (%) produced from experimental crosses

Pollen donor

Pollen recipient D. fuchsii D. incarnata D. praetermissa IF hybrids PF hybrids PI hybrids (A) Fruit-set

D. fuchsii 99 (0.82) 96.7 (7.07) 96 (6.2) 0 0 (0) 0 (0) D. incarnata 95 (5.27) 100 (0) 98 (6.32) 0 (0) 5.56 (9.82) –

D. praetermissa 99 (3.16) 96 (5.16) 100 (0) – – –

IF hybrids 80 (28.28) 50.9 (33.36) – 0 (0) 0 5.56 (9.82) PF hybrids 78.06 (10.6) 77.78 (38.68) – 0 0 (0) 12.5 (15) PI hybrids 100 (0) – – 0 (0) 20 (28.28) –

(B) Seed viability D. fuchsii 58.31 (15.45) 43.44 (24.39) 74.05 (10.1) 0 0 (0) 0 (0)

D. incarnata 35.23 (23.94) 49.42 (15.29) 51.08 (25.84) 0 (0) 0 (0) –

D. praetermissa 86.19 (5.77) 77.85 (7.02) 85.18 (3.77) – – –

IF hybrids 0 (0) 0.23 (0.29) – 0 (0) 0 0 (0) PF hybrids 1.95 (0.57) 0.43 (0.75) – 0 0 (0) 0.55 (1.09) PI hybrids 0.7 (0.53) – – 0 (0) 0.06 (0.09) –

– = no data available because of drought or herbivore damage. Standard deviations are given in parentheses.

Chapter 3

56

3.5 Discussion

3.5.1 Hybridization

The three studied Dactylorhiza species are often found growing together in dune

habitats, which may provide ideal conditions for hybridization. In this study, we

characterized the genetic structure of two natural hybrid zones, where the three species

co-occurred. Both Structure and PCO results provided evidence for frequent

hybridization between Dactylorhiza species at both study sites, although the extent of

hybridization between species varied substantially between the two sites. These results

confirm earlier findings that the genetic structure of hybrid zones can differ (Burke and

Arnold 2001).

The AFLP markers combined with detailed cytometric analyses enabled us to

assign individuals to specific hybrid classes. In Paelsteenpanne, by far the largest group

of hybrids was between the diploids D. incarnata and D. fuchsii. Triploid hybrids between

D. incarnata and D. praetermissa, and between D. fuchsii and D. praetermissa were

abundant in Ter Yde, whereas these hybrids were rather rare or even absent in

Paelsteenpanne. This is remarkable, since no pure D. praetermissa individuals were

present anymore at Ter Yde. Perhaps the triploid hybrids were better adapted to

(changing) dune slack conditions than D. praetermissa and have outcompeted their

tetraploid parent.

Despite the marked differences between sites, our results provide clear evidence

that hybridization between all species pairs is possible. This is in accordance with

previous work on Dactylorhiza species. Triploid hybrids between D. fuchsii and D.

praetermissa were revealed through chromosome counts in a study of Heslop-Harrison

(1953). Shaw (1998) detected hybrids between D. incarnata and D. praetermissa, and

between D. fuchsii and D. praetermissa, based on morphology. Shipunov et al. (2005)

argued that some D. baltica individuals were possibly primary, diploid hybrids between

D. incarnata and D. fuchsii. Hedren et al. (1996a) found a few putative hybrids between

D. incarnata and D. fuchsii in some sympatric populations.

Several scenarios could be responsible for the difference in frequency of the

hybrid groups at the two sites. Different colonization histories or differences in phenology

Hybridization between three Dactylorhiza species

57

between the sites may have caused the observed difference in hybridization frequency.

In Paelsteenpanne D. praetermissa individuals lie mainly in one corner of the dune

slack. This somewhat isolated position could explain the very few hybrids with D.

praetermissa as a parent. The order of the flowering peaks of the three species is D.

incarnata, D. praetermissa and D. fuchsii. The absence of IF hybrids in Ter Yde may

therefore result from the small overlap in flowering curves between D. incarnata and D.

fuchsii (results not shown). The high abundance of PI and PF hybrids in Ter Yde is

probably the result from the intermediate flowering time of D. praetermissa. Another

explanation for the absence of certain hybrid groups, could be that hybrids may

disappear after a number of years since they produce almost no viable seeds.

3.5.2 Hybrid origin

Most hybrids in our two study areas are probably F1 hybrids, according to the Structure

(Fig. 3.1 and 3.2) and flow cytometric results (Table 3.2). The FCM ratio of the IF hybrids

(2.77) is between that of D. incarnata (3.05) and D. fuchsii (2.49) (Table 3.2), and these

hybrids have also an intermediate position between these two species (Fig. 3.3a). The

PI hybrids had a FCM ratio (4.30) that was between that of D. incarnata (3.14) and D.

praetermissa (5.48; value of Paelsteenpanne). The FCM ratio of the PF hybrids (3.94)

was intermediate between its parental species (D. praetermissa: 5.48 and D. fuchsii:

2.41). Moreover, most hybrid groups showed more or less intermediate morphological

positions between their parental species, indicating that most hybrids combine

characters of both parents. This is in agreement with several other studies that have

reported orchid hybrids that were morphologically intermediate between their parental

species (e.g. Caputo et al. 1997; Nielsen 2000; Cozzolino et al. 2006; Moccia et al.

2007). However, in the case of hybridization involving D. praetermissa, it was shown that

both the PI and PF hybrid groups in Ter Yde had a larger proportion coming from D.

praetermissa than from the other parental species (Fig. 3.2), and were skewed towards

D. praetermissa on the PCO. In this case, higher similarity towards the allotetraploid D.

praetermissa was expected a priori, as this species has the largest genome of the three

pure species considered here and hence contributes 2/3 of its genome to the triploid

hybrids. The fact that most sampled hybrids were probably F1 hybrids is in accordance

Chapter 3

58

with many other Dactylorhiza studies that reported predominantly F1 hybrids (e.g.

Heslop-Harrison 1953; Rossi et al. 1995; Hedrén et al. 2001; Aagaard et al. 2005).

3.5.3 Triple hybrids

At both sites a central group of hybrids with a part of all three species in their genome (q

> 0.2) was present, suggesting the presence of triple hybrids and thus providing

evidence that hybrids between two species can cross with a third species. The

occurrence of natural triple hybrids in the field has rarely been reported (Kaplan and

Fehrer 2007) and to our knowledge this is the first time that putative triple hybrids are

reported in Dactylorhiza species. Especially molecular evidence for three different

species contributing to natural hybrid individuals is scarce, although some exceptions

can be found, including Aesculus (dePamphilis and Wyatt 1990), Iris (Arnold 1993),

Quercus (Dodd and Afzal-Rafii 2004) and Potamogeton (Kaplan and Fehrer 2007).

According to their FCM ratio (4.39), the individuals in Paelsteenpanne could be

backcrosses of IF hybrids (2.77) with D. praetermissa (5.48). The triple hybrids in Ter

Yde could be backcrosses of PI hybrids with D. fuchsii or backcrosses of PF hybrids with

D. incarnata. The expected FCM ratio of these individuals should be around 3.5, which

is higher than the actual FCM ratio (2.74). However, it is possible that hybrids have a

lower genome size than expected because of chromosome rearrangements in the

genome of the hybrids (Van Laere et al. 2009). Nonetheless, clearly different genome

sizes between the triple hybrid groups can suggest a different hybridization history.

Given that D. praetermissa has not been detected in the current survey, another

possibility would be that the triple hybrids from Ter Yde are in fact hybrids between D.

incarnata and D. fuchsii, which could explain their low FCM ratio. However, this is not in

accordance with the Structure results, which clearly show influence of D. praetermissa in

these hybrids.

3.5.4 Hybridization across different ploidy levels

Hybridization was observed both between species with the same and with different

ploidy levels. The occurrence of putative triple hybrids suggests the possibility of

backcrossing. Our crossing experiment, which showed some hybrid fertility, also

Hybridization between three Dactylorhiza species

59

suggests the possibility of gene flow between different ploidy levels. These results

confirm previous studies that have reported hybridization between polyploid derivatives

and their diploid progenitors (Aagaard et al. 2005; Ståhlberg and Hedrén 2009; Pinheiro

et al. 2010). Ståhlberg and Hedrén (2009) found relatively few triploid hybrids in a hybrid

zone of the diploid D. fuchsii and the autotetraploid D. maculata. Their data also gave

indications that some gene flow between ploidy levels is occurring. In his study on

morphological intermediates between D. fuchsii and the tetraploids D. purpurella and D.

praetermissa, Heslop-Harrison (1953) concluded that most hybrids were eutriploids and

showed a high level of seed-sterility. Similarly, Lord and Richards (1977) found both

eutriploid hybrids and aneuploid individuals in a hybrid swarm of the diploid D. fuchsii

and tetraploid D. purpurella. The appearance of aneuploids was consistent with F2 and

backcrossed individuals, which suggests that the triploids were by no means totally

sterile. Significant numbers of triploid hybrids were also found by Aagaard et al. (2005),

who examined a hybrid zone between diploid D. incarnata ssp. cruenta and its putative

allotetraploid derivative D. lapponica. The results of their study suggested backcrossing

between the triploid hybrids and D. lapponica, and hence some hybrid fertility. Several

studies found indications of introgression between Dactylorhiza species (Hedrén 2003;

Shipunov et al. 2004, 2005; Devos et al. 2005; Pillon et al. 2007; Pedersen 2006).

3.5.5 Postmating isolation

Since both intra- en interspecific crosses yielded a high fruit set, no postmating pre-

zygotic isolation was present. Viable seeds were obtained in both directions for all

interspecific crosses, indicating that early postmating post-zygotic barriers were weak.

This is in agreement with Ståhlberg and Hedren (2009) who found that both diploids and

tetraploids may act as the maternal parent. Experimental crosses performed by Pinheiro

et al. (2010) revealed that hybridization occurred in both directions in the species

Epidendrum fulgens and E. puniceoluteum. The results of the genetic analyses and flow

cytometry clearly show the presence of F1 hybrids between all species pairs, indicating

that hybrid inviability is not a reproductive barrier either.

The experimental crosses further showed that only when the pollen donor is a pure

species, fruit set is high (Tabel 3.4A) and that only crosses between pure species

Chapter 3

60

yielded a high percentage of viable seeds (Table 3.4B). When hybrids were involved as

either pollen-receptor or pollen-donor, almost no viable seeds were formed. These

results thus suggest that hybrid sterility is the major cause hampering hybridization to

extend beyond the F1 generation, and confirms previous studies in Dactylorhiza. For

example, Heslop-Harrison (1953) found highly sterile triploid hybrids between D. fuchsii

and D. praetermissa. Rossi et al. (1995) suggested hybrid sterility as the cause of the

lack of recombinant hybrid classes between D. romana and D. saccifera.

However, some authors found viable seeds of certain hybrid classes when they

performed experimental crosses between Dactylorhiza species (Malmgren 1992;

Bateman and Haggar 2008). We found also some viable seeds in our experimental

crosses. Moreover, in some naturally pollinated groups, also relatively high seed viability

percentages were observed. The PI hybrids, and especially, the PF hybrids of Ter Yde

showed a remarkably higher seed viability than the IF hybrids of Paelsteenpanne. This

could indicate that triploid hybrids between one of the diploid species and the tetraploid

species have a higher fertility than hybrids between the two diploid species, which could

suggest a stronger reproductive isolation between the two diploid species than between

a diploid and the tetraploid species. Some hybrid viability can be expected since triploids

are known to produce fertile hyperdiploid (2x + 1) and hypotetraploid (4x – 1) offspring

occasionally (Ramsey and Schemske 1998).

3.5.6 Allopolyploid formation

Allotetraploid members of Dactylorhiza have evolved on multiple occasions (Hedrén

1996a, 2003). However, little is known about how new polyploids arise. One possible

genetic pathway is that fertile triploid hybrids act as a bridge in the formation of new

polyploid species (Ramsey and Schemske 1998; Coyne and Orr 2004). Our findings

lend some support to this hypothesis. Since some seed viability was found in our hybrids

and also hybrids with three different parents were found, there is strong evidence for the

presence of secondary gene flow. Through this secondary gene flow the present diploid

or triploid hybrids can further lead to the formation of new allopolyploids.

Hybridization between three Dactylorhiza species

61

3.6 Conclusions

Frequent hybridization was observed at both study sites and hybridization was possible

between all species pairs. Our molecular and flow cytometric data suggest that most

hybrids are F1 hybrids. However, the presence of putative triple hybrids indicates that

hybrids between two species can cross with a third species, which suggests secondary

gene flow and the possibility of backcrossing. Crossing experiments and data from the

field revealed low seed viability percentages of most hybrids, which indicates strong

post-zygotic barriers. These reproductive barriers probably prevent the local breakdown

of species boundaries in Dactylorhiza despite frequent hybridization between parental

species.

3.7 Acknowledgements

The authors thank K. Libaut, S. Provoost, and P. De Raeymaecker for assistance, M.

Leten for useful information, and ANB and Natuurpunt for permission to conduct our

research in the nature reserves, Ter Yde and Paelsteenpanne. A grant from the Flemish

fund for scientific research (FWO) [G.0592.08] is gratefully acknowledged.

Chapter 3

62

Chapter 4.

Germination failure is not a critical stage of reproductive isolation

between three congeneric orchid species

Unpublished manuscript:

De hert K, Jacquemyn H, and Honnay O (2012). Germination failure is not a

critical stage of reproductive isolation between three congeneric orchid species.

American Journal of Botany, under review.

Chapter 4

64

4.1 Abstract • Premise of the study: In food-deceptive orchid species, post-mating reproductive

barriers (fruit set and embryo mortality) have been shown to be more important for

reproductive isolation than pre-mating barriers (pollinator isolation). However,

currently there is very little knowledge about germination failure acting as a

reproductive barrier in hybridizing orchid species.

• Methods: In this study, we investigated germination and protocorm development of

pure and hybrid seeds of three species of the genus Dactylorhiza. To test the

hypothesis that germination failure contributed to total reproductive isolation,

reproductive barriers based on germination were combined with already available

data on early acting barriers (fruit set and embryo mortality) to calculate the relative

and absolute contributions of these barriers to reproductive isolation.

• Key Results: Protocorms were formed in all different crosses, indicating that both

hybrid and pure seeds were able to germinate and grow into protocorms. Also, the

number of protocorms per seed packet was not different between hybrid and pure

seeds. High fruit set, high seed viability, and substantial seed germination resulted in

very low reproductive isolation (average RI = 0.05). In two of six interspecific crosses,

hybrids were also fitter than the intraspecific crosses.

• Conclusions: Very weak post-mating reproductive barriers were observed between

our study species and may explain the frequent occurrence of first-generation hybrids

in mixed Dactylorhiza populations. Germination failure, which is regarded as one of

the most important bottlenecks in the orchid life cycle, was not important for

reproductive isolation.

4.2 Introduction

Hybridization between species and genera of European food-deceptive orchids has

been repeatedly reported in the literature (Willing and Willing 1985; Kretzschmar et

al. 2007). The extent to which natural hybridization occurs depends on the strength of

different reproductive barriers and differs significantly from one species pair to the

next (Scopece et al. 2008). These barriers are conveniently categorized as pre- and

post-mating barriers. Pre-mating barriers include geographic, temporal and pollinator

Germination failure as reproductive barrier

65

isolation, whereas post-mating barriers may result from pre-zygotic (pollen–stigma

and sperm cell–ovule interactions) and post-zygotic (embryo mortality, germination

failure, hybrid inviability and sterility) mechanisms.

In many food-deceptive orchid species pre-mating barriers are weak whereas

post-mating reproductive barriers can be very strong (Cozzolino et al. 2004, 2006;

Cozzolino and Scopece 2008). Scopece et al. (2007), for example, investigated the

relative importance of pre- and post-mating barriers in a large set of food-deceptive

and sexually deceptive orchids and found that post-mating barriers were most

important for reproductive isolation in food-deceptive orchids. However, currently

there is little knowledge about germination failure acting as a reproductive barrier in

hybridizing orchid species (but see Hollick et al. 2005; Jacquemyn et al. 2011).

Nonetheless, it was predicted that germination failure is an important barrier in

orchids, since germination is dependent on the presence of compatible mycorrhizal

fungi and different orchid species associate with different sets of mycorrhizal fungi

(Rasmussen 1995; Brundrett et al. 2003).

In this study, we tested the hypothesis that germination failure contributed to

total reproductive isolation in three species of the genus Dactylorhiza. Similar to other

food-deceptive orchid species, pre-mating barriers have been shown to be weak in

this genus, since Dactylorhiza species do not offer any reward to their pollinators and

rely on a wide range of inexperienced pollinators for sexual reproduction (Hedrén

1996a; Aagaard et al. 2005; Pillon et al. 2007; Ståhlberg and Hedrén 2009). Post-

zygotic barriers, on the other hand, are expected to be more important. To test this

hypothesis, experimental crosses were conducted between Dactylorhiza incarnata,

D. fuchsii and D. praetermissa and germination experiments were used to compare

the germination and protocorm development of hybrid and pure seeds. Finally, to

assess the role of germination failure acting as a post-mating barrier, data on seed

germination and protocorm development were combined with already available data

on fruit set and seed viability from De hert et al. (2012) to calculate the relative and

absolute contributions of the different mating barriers.

4.3 Methods

4.3.1 Study species

Chapter 4

66

Three species of the orchid genus Dactylorhiza were taken as study species:

Dactylorhiza incarnata (L.) Soó, D. fuchsii (Druce) Soó, and D. praetermissa (Druce)

Soó. These orchid species are herbaceous, perennial, and food-deceptive. D.

praetermissa is an allotetraploid (2n = 4x = 80), whereas D. incarnata and D. fuchsii

are diploids (2n = 2x = 40). The distribution and morphology of the species are

described in De hert et al. (2012). Dactylorhiza species mainly associate with fungi

from the genus Tulasnella, but also with members of the genera Thanatephorus and

Ceratobasidium (Rasmussen 1995; Shefferson et al. 2008; Jacquemyn et al. 2012).

Fig. 4.1. Design of our seed germination experiment. At each site, only seeds from crosses with the residing pure species were introduced. The first species of each cross is the ovule parent and the second one is the seed parent. F: D. fuchsii, P: D. praetermissa, and I: D. incarnata

Germination failure as reproductive barrier

67

4.3.2 Experimental crosses

To estimate the importance of germination failure as a post-mating reproductive

barrier, germination experiments were conducted using seeds from experimental

crosses between and within species (De hert et al. 2012). All experimental crosses

were performed on plants from single-species populations (Fig. 4.1). Both the D.

incarnata and D. praetermissa population were located in dune slacks, respectively at

the Westhoek (De Panne, Belgium) and Warandeduinen (Middelkerke, Belgium)

nature reserves. The D. fuchsii population was located in a calcareous marsh in

Torfbroek nature reserve (Kampenhout, Belgium). In each population, intra- and

interspecific crosses were performed on 30 randomly selected plants (sets of ten

plants were pollinated with pollen of one of the three species). Interspecific crosses

were conducted in both directions because reproductive barriers can be asymmetric,

depending on which species is the ovule parent and which species is the pollen

parent. Ten flowers from each plant were pollinated for each cross, resulting in a total

of 900 experimental pollinations. Prior to pollination, pollinia of the pollinated flowers

were carefully removed to prevent self-pollination.

In November 2008, seeds were introduced in the three study sites. At each

site, only seeds from crosses with the residing pure species were introduced (see

Fig. 4.1 for crossing design). Approximately 150 seeds were placed within a square

of 50-µm mesh phytoplankton netting, enclosed within a Polaroid slide mount

(Rasmussen and Whigham 1993). The pore size of the mesh was chosen to retain

the seeds without impeding fungal growth. Seed packets were placed vertically just

under the topsoil layer and were left in the ground for about 2.5 years. To cover as

much variation as possible, seeds were buried at 15 different places in each

population. Ten seed packets (two per treatment) were placed around a metal pin to

which they were attached with fishing wire. A total of 450 seed packets were buried

(150 per location). Seed packages were retrieved in summer 2011 using a metal

detector. After retrieval, they were maintained moist in cotton wool until examination.

For each slide mount the number of developed protocorms (see Fig. 4.2 for

photographs) and seedlings was determined by visual inspection under a dissection

microscope (magnification ×12). The germination rate of each category was

determined as the proportion of seed packets in which at least one protocorm was

observed.

Chapter 4

68

Fig. 4.2. Pictures of hybrid protocorms: (a) IP cross; (b) and (d) PF cross; (c) IF cross.

4.3.3 Data analysis

The possible effects of mother (‘home’) species and pollination treatment on seed

germination (the number of protocorms) were tested using a Generalized Mixed

Model with a Poisson loglinear link function. The number of protocorms was the

response variable and mother species and pollination treatment were the predictors.

A binary logistic regression was used to test the effects of ‘home’ species and

crossing on absence/presence of protocorms with absence/presence of protocorms

as response variable and mother species and pollination treatment as predictor

variables. For all seed packets in which protocorms were found, the number of

protocorms between the different groups was tested using a Kruskall-Wallis test.

To estimate the absolute and relative contribution of germination failure to total

reproductive isolation, components of reproductive isolation (RI) were determined

using methods outlined in Ramsey et al. (2003). The post-mating barriers used for

this analysis were fruit set, seed viability, and seed germination. Fruit set was

determined as the ratio of the number of fruits over the number of pollinated flowers

(data from De hert et al. 2012). For determination of seed viability the tetrazolium

Germination failure as reproductive barrier

69

method of Van Waes and Debergh (1986) was used (for details see De hert et al.

2012). Germination rate was determined as outlined above. We first tested whether

asymmetries in reproductive barriers occurred between the different species pair

combinations. We then computed for each component RIx as 1 minus the ratio of the

fitness of F1 hybrids to the fitness of the parents. This measure of hybrid fitness

varies between 0 and 1, except for comparisons in which the hybrids are fitter than

the respective parents (Ramsey et al. 2003). Because crosses were performed

reciprocally, for each species combination, two measures for each component of RI

were obtained. Finally, for each cross, total post-mating RI was calculated as

(Ramsey et al. 2003):

RItot = AC1 + AC2 + AC3 (1)

where

AC1 = RI1 (2)

AC2 = RI2(1 – AC1) (3)

AC3 = RI3[1 – (AC1 + AC2)] (4)

AC is the absolute contribution of each of the different post-mating barriers. The

subscripts in eqns (1)–(4) refer to RI due to fruit set (1), seed viability (2), and seed

germination (3). The relative contribution of each of these barriers was calculated as

RCn = ACn/RItot.

4.4 Results

In all nine crosses, seed packets with developed protocorms were found (Table 4.1).

Different crosses are represented by two letters; the first letter refers to the receptor

species and the second letter to the donor species (D. fuchsii = F, D. incarnata = I

and D. praetermissa = P). The proportion of seed packets in which at least one

protocorm was observed varied between 0.027 and 0.2 (Table 4.1). The mean

number of protocorms per seed packet varied between 1.5 and 7.5 (Table 4.1). Also,

a few seedlings were observed (in one seed packet of D. praetermissa, D. fuchsii,

and an IF cross). The results from the Poisson regression revealed a significant

effect of ‘home’ species (Wald Chi-Square = 8.39, df = 2, P < 0.015) on the number

of protocorms, but no effect of crossing. However, the interaction between species

Chapter 4

70

and crossing was significant (Wald Chi-Square = 22.86, df = 3, P < 0.001). For FP

and IF there seemed to be a negative home-site advantage for Torfbroek

(respectively, Wald Chi-Square = 5.45, df = 1, P = 0.02 and Wald Chi-Square =

20.69, df = 1, P < 0.001). The binary logistic regression showed no significant effects

of ‘home’ species and crossing on absence/presence of protocorms. Kruskall-Wallis

and Mann Whitney U tests revealed no significant differences in the number of

protocorms between the different groups.

Table 4.1. Fruit set, seed viability, and germination rate of nine experimental crosses between D. fuchsii (F), D. incarnata (I), and D. praetermissa (P) (first letter: receptor species; second letter: donor species). Cross Fruit set (%) Seed viability (%) Germination* # protocorms**

FF 99.0 ± 0.82a 58.31 ± 15.45bc 0.033 3.0

FI 96.7 ± 7.07a 43.44 ± 24.39bd 0.033 4.5

FP 96.0 ± 6.2a 74.05 ± 10.1ac 0.027 2.5

IF 95.0 ± 5.27a 35.23 ± 23.94d 0.085 4.2

II 100.0 ± 0a 49.42 ± 15.29bd 0.200 2.5

IP 98.0 ± 6.32a 51.08 ± 25.84bd 0.133 2.5

PF 99.0 ± 3.16a 86.19 ± 5.77a 0.097 7.5

PI 96.0 ± 5.16a 77.85 ± 7.02ac 0.121 2.4

PP 100.0 ± 0a 85.18 ± 3.77a 0.067 1.5

Notes: Values (± s.d.) represent the average of 100 crosses conducted on ten individual plants. Values with different superscript letters indicate that they are significantly different at the α = 0.05 level. * Proportion of seed packets in which at least one protocorm was observed. ** Mean number of protocorms/seed packet for all seed packets containing protocorms.

Our crossing experiments yielded very high levels of fruit set, both for inter-

and intraspecific pollinations (>95%; Table 4.1). No significant differences between

crosses were present for fruit set, indicating that pre-zygotic post-mating isolation

was very weak. The proportion of viable seeds, however, differed significantly

Germination failure as reproductive barrier

71

between crosses (F8,77 = 13.59, P < 0.001), varying between 35.23 % (IF) and 86.19

% (PF) (Table 4.1). The t-test results revealed asymmetries in seed viability for two

species pairs (FP-PF: t-4.461,18, P < 0.001; IP-PI: t-3.156,16, P = 0.006). For crosses with

D. incarnata as mother plant, the lowest seed viability percentages were observed

(from 35.23 % to 51.08 %), whereas crosses with D. praetermissa as mother plant

yielded seeds with the highest viability percentages (from 77.85 % to 86.19 %) (Table

4.1).

As a result, significant asymmetries in reproductive isolation were observed

(Fig. 4.3). For three interspecific crosses, reproductive isolation was positive (ranging

from 0.27 to 0.72) and for two interspecific crosses this value was negative (ranging

from -0.45 to -0.58). For FP this value approximated zero. For each cross, the

relative contribution of the different post-mating barriers was also calculated (Fig.

4.4). Seed inviability and germination failure contributed the most to the total

reproductive isolation. For FP and IF crosses both barriers were equally important,

whereas for FI only seed inviability was important. For IP, PF, and PI crosses

germination failure was the most important barrier. For PF and PI this was due to the

fact that germination for both crosses was better than for the pure seeds, which

resulted in a negative value for germination failure and hence in a negative RItot

value. For IP the values of germination failure and RItot were positive, resulting from

their lower germination rate compared with pure seeds.

Chapter 4

72

Fig. 4.3. Total reproductive isolation incorporating both pre- and post-zygotic post-mating barriers up to seed germination of reciprocal crosses between D. incarnata (I), D. fuchsii (F), and D. praetermissa (P). For each cross, the first species is the receptor species and the second the donor species.

Fig. 4.4. Relative contributions of reproductive barriers of reciprocal crosses between D. incarnata (I), D. fuchsii (F), and D. praetermissa (P). For each cross, the first species is the receptor species and the second the donor species.

-0.80 -0.60 -0.40 -0.20 0.00 0.20 0.40 0.60 0.80

PI

PF

IP

IF

FP

FI

RItot

Cro

ss

-100% -50% 0% 50% 100%

PI

PF

IP

IF

FP

FI

Relative contribution (RC)

Cros

s

Pre-zygotic barrier (fruit set)

Post-zygotic barrier I (seed viability)

Post-zygotic barrier II (germination)

Germination failure as reproductive barrier

73

4.5 Discussion

Our study revealed that protocorms were formed in all different crosses, indicating

that both hybrid and pure seeds were able to germinate and develop into protocorms.

Although we have no data on the specificity of fungi for hybrid and pure protocorms,

recent analyses have shown that the three investigated Dactylorhiza species are

generalist species with regard to mycorrhizal fungi (Jacquemyn et al. 2012). All

species were found to associate with at least six different Tulasnella fungi, some of

which were common and widespread. Within a single population, at least two

different fungal OTUs were observed, and in two populations six different OTUs were

found, which also indicates broad mycorrhizal specificity. Moreover, these species

shared several of their mycorrhizal fungi (Jacquemyn et al. 2012), which may explain

why no substantial differences in seed germination were observed between hybrid

and pure seeds. Similar results have been reported in hybridizing species of the

genus Orchis (Jacquemyn et al. 2011). Since developing protocorms were observed

in all crosses, this suggests that pure and hybrid seeds probably use the same set of

mycorrhizal fungi. Moreover, in some crosses the germination rate was higher for

hybrid seeds compared with pure seeds, suggesting that hybrid seeds may be less

limited by mycorrhizal fungi than seeds from pure crosses. This implies that

germination failure is not a critical stage of reproductive isolation between the three

studied Dactylorhiza species.

High fruit set, high seed viability, and substantial seed germination resulted in

low reproductive isolation. Compared with reproductive isolation data presented in

Scopece et al. (2007) the average reproductive isolation reported here was very low

(mean RI = 0.90 and 0.05, respectively). However, our results are consistent with

results of crosses conducted between Dactylorhiza saccifera and D. sambucina (RI =

0.43) and D. saccifera and D. romana (RI = 0.30) (Scopece et al. 2007). These

results suggest that reproductive barriers in the genus Dactylorhiza are in general

weak. They are also in agreement with the observation that hybrids can be found

frequently at sites where different Dactylorhiza species co-occur (Heslop-Harrison

1957; Ståhlberg and Hedrén 2009; De hert et al. 2011, 2012). Crosses with D.

incarnata and D. fuchsii as parental species had the highest RItot values. This is also

in agreement with the fact that hybrids between these two species are less frequently

Chapter 4

74

found in nature than hybrids between the other two species pairs (Hedrén 1996a;

Shaw 1998; De hert et al. 2012). In some cases, we even found negative RItot values,

indicating that interspecific crosses have a higher fitness than intraspecific crosses.

This is in contrast with many other studies that have shown that interspecific

pollinations generated fewer or less viable seeds than intraspecific controls (e.g.

Ramsey et al. 2003; Scopece et al. 2008) or that F1 hybrids exhibited reduced

germination rates (Ramsey et al. 2003).

Results from our crossing experiments further showed that post-zygotic factors

(seed inviability and germination failure) were more important for reproductive

isolation than pre-zygotic factors (fruit set). These results accord with findings of

Scopece et al. (2007), who showed similar patterns for food-deceptive orchids

growing in the Mediterranean (Orchis, Anacamptis, and Neotinea). For two species

pairs (D. incarnata and D. praetermissa; D. fuchsii and D. praetermissa) reproductive

isolation appeared to be asymmetric, which has also been found in other plant

species (Tiffin et al. 2001; Jacquemyn et al. 2011). The highest seed viability

percentages were observed for crosses with D. praetermissa as mother plant. Also,

Scopece et al. (2007) found asymmetries in seed viability for 26% of the investigated

food-deceptive species pairs. For the germination rate, the lowest values were

observed with D. fuchsii as mother plant and the highest values with D. praetermissa

or D. incarnata as mother plant (Table 4.1). Figure 4.3 clearly shows that the two

crosses with D. praetermissa as mother plant were the only two crosses that were

fitter compared with their parental species. These results indicate that the pollen

receptor mainly determines the fitness of the crosses.

4.6 Conclusions

Very weak post-mating reproductive barriers were observed between our study

species. Two of the six interspecific crosses were even fitter than the intraspecific

crosses. Seed germination, the most critical stage in orchid development, does not

appear to be an important reproductive barrier.

Germination failure as reproductive barrier

75

4.7 Acknowledgements

The authors thank M. Leten for useful information, and ANB for permission to

conduct our research in the Westhoek nature reserve. H. Desmet and J. Wouters of

Natuurpunt are acknowledged for their cooperation in respectively, the

Warandeduinen and Torfbroek nature reserves. A grant from the Flemish fund for

scientific research (FWO) [G.0592.08] is gratefully acknowledged.

Chapter 4

76

Chapter 5

Absence of recruitment limitation in restored dune slacks suggests that

manual seed introduction can be a successful practice for restoring orchid

populations

Adapted from:

De hert K, Jacquemyn H, Provoost S, Honnay O (2012). Absence of recruitment

limitation in restored dune slacks suggests that manual seed introduction can be a

successful practice for restoring orchid populations. Restoration Ecology, in press.

Chapter 5

78

5.1 Abstract Answering the question whether or not to introduce seeds of target species in restored

habitats depends on the relative importance of dispersal and recruitment limitation.

Especially in orchid species, recruitment limitation is expected to be important because

of their dependence on mycorrhiza for germination. Using a large seed introduction

experiment we investigated the relative importance of dispersal vs. recruitment limitation

in the failure of three rare orchid species to colonize restored habitat patches. For all

species, seeds developed successfully into protocorms in both occupied and

unoccupied habitats, and no significant differences in the number of protocorms per

seed packet were found between occupied and unoccupied habitats. These results

show that orchid species do not necessarily suffer from recruitment limitation when

introduced in restored habitats, and that manual introduction of orchid seeds can be a

successful and necessary ecological restoration practice.

5.2 Introduction

The success of habitat restoration depends on the ability of target plant species to reach

the restored patch (dispersal limitation) and on the ability to recruit after seed dispersal

(recruitment limitation) (Rasmussen & Whigham 1998; Verheyen & Hermy 2001).

Knowledge of the relative importance of dispersal vs. recruitment limitation is crucial for

predicting the success of habitat restoration measures, and for considering the artificial

introduction of seeds of the target species in the restored habitats. The most

straightforward way to examine the importance of recruitment limitation is to elevate

dispersal limitation through manually introducing seeds in both occupied and

unoccupied habitat patches (Ehrlén & Eriksson 2000).

To increase the probability of successful restoration, it has recently been

suggested that more attention should be directed towards aboveground-belowground

linkages (Kardol & Wardle 2010). This is especially the case when restoring populations

of orchid species as the presence of mycorrhizal fungi at a restoration site will not only

be critical for orchid seed germination (Rasmussen 1995; McCormick et al. 2012), but

Seed introduction as restoration practice in orchids

79

also for seedling establishment (Bidartondo & Read 2008). Together with the presence

of pollinators, predation and grazing these fungi are crucial in mediating orchid

demography (Brundrett 2007; McCormick et al. 2009).

Here, we investigated the relative importance of dispersal vs. recruitment

limitation in the failure of three rare and threatened orchid species to colonize restored

dune slacks. In our study area at the Belgian coast, these species are currently confined

to relatively young (<100 y) wet dune slacks, scattered in a matrix of largely fixed dry

sand dunes, covered by scrub and herbaceous vegetation. After a long history of dune

slack degradation because of scrub encroachment, many dune slacks have been

restored through mechanical scrub removal, followed by mowing or grazing (Bossuyt

2007). However, recolonization of these restored dune slacks from old source patches

does only sporadically occur for the three investigated species. To identify the cause of

this failure, and to answer the question whether seeds should be introduced or not, we

locally established a large seed introduction experiment.

5.3 Methods

5.3.1 Species

Dactylorhiza fuchsii and D. praetermissa are herbaceous, perennial, food-deceptive

orchid species. Herminium monorchis is a small, herbaceous, perennial, nectar-

producing orchid. Dactylorhiza species mainly associate with fungi from the genus

Tulasnella, but also with members of the genera Thanatephorus and Ceratobasidium

(Rasmussen 1995; Shefferson et al. 2008; Jacquemyn et al. 2012). For H. monorchis no

field data on fungal associates are available to our knowledge.

5.3.2 Study sites and data collection

The study was conducted at two dune sites [Ter Yde (Oostduinkerke) and Westhoek

(De Panne)] along the Belgian coast. All dune slacks in this area were surveyed and for

each of the three study species, occupied and unoccupied, but suitable patches were

identified. The latter were identified based on the vegetation composition, which was

Chapter 5

80

required to be identical to the occupied patches. We used the co-occurrence of all

following typical dune slack species as indicator of habitat suitability: Dactylorhiza

incarnata, Carex trinervis, Carex viridula var. pulchella, Centaurium littorale, Epipactis

palustris, Parnassia palustris, Pyrola rotundifolia, and Sagina nodosa. This procedure

resulted in nine suitable sites where at least one of the three study species was absent

(Fig. 5.1).

In November 2009, we introduced seeds of all three species in at least two

occupied, and at least four unoccupied dune slacks (Table 5.1). Approximately 150

seeds were placed within a square of 50-µm mesh phytoplankton netting, enclosed

within a Polaroid slide mount (Rasmussen & Whigham 1993). The pore size of the mesh

was chosen to retain the seeds without impeding fungal growth. For each species, 150

seed packets were buried in occupied dune slacks and 150 in unoccupied dune slacks.

Seed packets were placed vertically just under the topsoil layer and were left in the

ground for 21 months. When seed packets were retrieved in summer 2011, they were

maintained moist in cotton wool until examination. For each slide mount, the number of

(i) germinating (swollen) seeds (Fig. 5.2a), (ii) developed protocorms (Fig. 5.2b-e) and

(iii) seedlings (Fig. 2f-g) was determined by visual inspection under a dissection

microscope (magnification x 12).

Seed introduction as restoration practice in orchids

81

Fig. 5.1. Map of dune slacks in the two study areas (A: Westhoek; B: Ter Yde). The presence/absence of our study species is shown. Dune slacks where seed packets were buried are numbered.

A

B

Chapter 5

82

Fig. 5.2. Developmental stages of germinating seeds and protocorms. (a) Normal and germinating seed; (b-e) different stadia of protocorms; and (f-g) seedlings.

Seed introduction as restoration practice in orchids

83

Chapter 5

84

5.3.3 Data analysis

For each site we determined the frequency of seed packets in which germination,

development of protocorms, or development of seedlings took place. An

association between the presence/absence of a species and the frequency of

seed packets in which germinating seeds, protocorms or seedlings were found,

was examined using a χ² test. For all seed packets with protocorms, the mean

number of protocorms per seed packet was calculated for each site. A Mann-

Whitney U test was used to test the difference in the mean number of protocorms

per seed packet between occupied and unoccupied patches for each species.

5.4 Results

Whereas in most seed packets germinating seeds were found, protocorms were

less frequent and only six seed packets contained seedlings (four of D. fuchsii,

two of H. monorchis) (Table 5.1). For D. fuchsii, there were significant differences

between occupied and unoccupied dune slacks for the frequency of seed

packets with germinating seeds (χ² = 25.7, p < 0.001), protocorms (χ² = 4.2, p =

0.04) and seedlings (χ² = 4.1, p = 0.04). For H. monorchis, there was only a

significant difference for germinating seeds (χ² = 9.5, p = 0.002), but not for

protocorms (χ² = 0.49, p = 0.48). No significant differences were observed for D.

praetermissa (protocorms: χ² = 1.4, p = 0.24; germinating seeds were found in all

seed packets; no seedlings were found).

The mean number of protocorms per seed packet for occupied and

unoccupied sites for each species is visualized in Fig. 5.3. For all three species

the mean number of protocorms per seed packet was not significantly different

between dune slacks where the species was present and dune slacks where the

species was absent (D. fuchsii: Z = 1.82, p = 0.07; D. praetermissa: Z = 1.50, p =

0.14; H. monorchis: Z = 1.10, p = 0.27).

Seed introduction as restoration practice in orchids

85

Fig. 5.3. Mean number of protocorms per seed packet for patches where the species are absent and present. Standard deviation is shown.

5.5 Discussion

Our results show that seeds of all three study species can form protocorms in

occupied and unoccupied habitats, showing that dispersal limitation plays a

significant role in the inability of orchids to colonize restored sites. Also other

studies already observed some degree of dispersal limitation in orchids (Machon

et al. 2003; Jersakova & Malinova 2007; Jacquemyn et al. 2009), despite the

huge amounts of minute, dust-like seeds that are produced (Arditti & Ghani

2000).

Germination of orchid seeds in habitat patches where the species was

absent has been reported earlier. McKendrick et al. (2000) detected the best

germination of the orchid Corallorhiza trifida in a Salix repens community located

at a considerable distance from any extant C. trifida population. Although

McKendrick et al. (2002) found that germination of Neottia nidus-avis seeds

Chapter 5

86

occurred most frequently on sites containing adults, germination was also

observed on a site without adults. Phillips et al. (2011) used the seed baiting

technique for Drakaea species and found that both rare and common Drakaea

species germinated in suitable habitats that were not occupied.

The easy germination and protocorm formation in unoccupied habitats

strongly suggest the ubiquitous occurrence of mycorrhizal fungi, relatively low

specialization of orchid species for fungal species, or a combination of both. The

little data available indicate that species in the genus Dactylorhiza associate with

a diverse suite of mycorrhizal fungi (Shefferson et al. 2008; Jacquemyn et al.

2012), suggesting low specialization. In addition, Shefferson et al. (2008) found

that the mycorrhizal fungi associated with orchids that colonized mine tailing hills

were the same as those in the pristine habitats, suggesting that mycorrhizal

symbionts of Dactylorhiza may indeed be widespread. Also, Tĕšitelová et al.

(2012) found evidence for low fungal specificity in four Epipactis species with

divergent ecological preferences.

However, notwithstanding germination was frequently observed in

unoccupied habitats, germination appeared to be more frequent in occupied

dune slacks for D. fuchsii and H. monorchis. These results may suggest a higher

abundance of mycorrhiza near adult plants and are in agreement with other

experimental studies of seed germination that found the highest seed

germination near parent plants (Perkins & McGee 1995; McKendrick et al. 2000,

2002; Batty et al. 2001; Diez 2007; McCormick et al. 2009; Jacquemyn et al.

2007, 2011). Both the number of seed packets in which germination occurred

and the mean number of protocorms per seed packet were lower in H. monorchis

than in the two Dactylorhiza species. Differences in germination capacity

between species were also found in other studies. Brundrett et al. (2003), for

example, showed that common orchids germinated more frequently than those

which were uncommon at the field sites.

To conclude, our results indicate that orchid species are not necessarily

recruitment limited due to their mycorrhiza dependence. We therefore suggest

Seed introduction as restoration practice in orchids

87

that manual introduction of orchid seeds in isolated restored habitats can be a

valuable and even necessary practice to restore orchid populations.

5.6 Implications for Practice • Seed baiting methods can provide valuable information on whether the

colonization of orchid species after habitat restoration is dispersal or recruitment

limited.

• Although orchids are often thought to be recruitment limited due to their

mycorrhizal dependence, manual introduction of orchid seeds in isolated restored

habitats should be more frequently considered as a simple, but successful

restoration practice.

5.7 Acknowledgments

The authors thank K. Helsen for help with QGis, M. Leten for useful information,

and ANB for permission to conduct our research in the nature reserves,

Westhoek and Ter Yde. We also thank two anonymous reviewers for helpful

comments. A grant from the Flemish fund for scientific research (FWO)

[G.0592.08] is gratefully acknowledged.

Chapter 5

88

CHAPTER 6.

Seed longevity of eight European orchid species –a burial experiment

Unpublished manuscript:

De hert K., Jacquemyn H., and Honnay O. (2012). Seed longevity of eight

European orchid species –a burial experiment

Chapter 6

90

6.1 Abstract Persistent seed banks may have important implications for the persistence of plant

populations by conserving the genetic variation present in the population and

providing opportunities for recolonization. Many orchid species are currently

threatened and could benefit from the positive effects of a persistent seed bank.

However, very few studies have investigated the potential for persistent orchid seed

banks in the field so far. We established a seed burial experiment to examine the

seed longevity of eight European terrestrial orchid species, belonging to the genera

Dactylorhiza, Orchis, Gymnadenia, Epipactis and Herminium. The results of our

burial experiment show that seeds of seven of the investigated orchid species

survived at least 2.5 years in the soil, while seeds of one species survived only one

year in the soil. This suggests that all of our study species can form a short-term

persistent seed bank, which can have important implications for their persistence.

6.2 Introduction

Seed banks are an important part of the plant community because they allow species

to bridge temporally unsuitable habitat conditions for germination and establishment,

spread germination risk in time, and conserve population genetic diversity by

increasing the effective population size in small fragmented populations (Thompson

et al. 1997; Honnay et al. 2008). Next to their importance in mediating population

dynamics through time, the production of persistent seeds may also be a key factor

determining the success of ecological restoration, especially when newly established

habitats occur isolated from potential source habitats (Bakker and Berendse, 1999;

Bossuyt and Honnay 2008; Plassmann et al. 2009).

Many orchid species are currently threatened and their populations face the

risk of extinction due to human activity. Major threatening processes include land

clearance, habitat fragmentation, salinity, climate change, weed encroachment,

disease and pests (Swarts and Dixon, 2009). In combination with the often typical

specificity of orchid species for certain pollinators and mycorrhizal fungi, the adverse

effects of these threats can be expected to be even bigger. The characteristics of

orchid seeds are relatively well known. They are very small, extremely light and

produced in great numbers (Arditti and Ghani 2000). When the seeds are shed, the

Seed longevity of orchid species

91

embryos are morphologically immature and morphophysiological dormancy is likely

to be common in orchids (Baskin and Baskin, 1998). There is evidence of dormancy-

breaking mechanisms in temperate orchid seeds, such as specific temperature

regimes, lengthy periods of imbibitions, chemical or physical breakdown of the testa,

and chemical signals from fungi (Rasmussen 1995). Yet, the seed banking potential

is less well known, and in many demographic studies on orchid species, the seed

bank stage is simply ignored (e.g. Zotz 1998; Brzosko 2002; but see Nicolè et al.

2005).

Because of their threatened status, seed banks in orchid species can be very

important for maintaining and restoring population genetic diversity, but also for

restoring orchid populations after habitat improvement. Despite the traditional view of

easily dispersed minute orchid seeds (Arditi & Ghani 2000), many orchid species

seem to be dispersal limited (Jersakova & Malinova 2007; Jacquemyn et al. 2009; De

hert et al. 2012), and their recolonization is therefore often dependent on a persistent

seed bank. However, very few studies have investigated the potential for persistent

orchid seed banks in the field so far (van der Kinderen, 1995; Zelmer and Currah,

1997; Batty et al. 2000; McKendrick et al. 2000; Whigham et al., 2002, 2006).

Furthermore, only two of these studies examined European orchids (van der

Kinderen, 1995; McKendrick et al. 2000). The observed seed longevity values in

these studies were highly variable, and ranged from less than one year (Caladenia

arenicola and Pterostylis sanguine) to seven years (e.g. Corallorhiza odontorhiza and

Liparis liliifolia).

Here, we aimed at enlarging the available data set of orchid species where

seed longevity data are available. We established a seed burial experiment to

examine the seed longevity of eight European terrestrial orchid species, occurring in

Belgium. Four species belonged to the genus Dactylorhiza, while the other four

species each represented one different genus: Orchis, Gymnadenia, Epipactis and

Herminium.

6.3 Methods 6.3.1 Study species and data collection

Chapter 6

92

Dactylorhiza incarnata (L.) Soó, D. fuchsii (Druce) Soó, D. praetermissa (Druce) Soó,

D. maculata (L.) Soó and Orchis mascula (L.) L. are herbaceous, perennial, food-

deceptive orchid species. Gymnadenia conopsea (L.) R. Brown, Epipactis palustris

(L.) Crantz and Herminium monorchis (L.) R. Brown are herbaceous, perennial,

nectar-producing orchids. Seeds of D. incarnata, D. fuchsii, D. praetermissa and G.

conopsea were collected in 2008 in the nature reserves Westhoek (De Panne,

Belgium), Torfbroek (Kampenhout, Belgium), Warandeduinen (Middelkerke, Belgium)

and Viroin valley (province Namen, Belgium), respectively. Seeds of D. maculata, O.

mascula, E. palustris and H. monorchis were collected in 2009. D. maculata and O.

mascula seeds were collected in Vorsdonkbos (Aarschot, Belgium) and Viroin valley,

respectively. E. palustris and H. monorchis seeds were collected in Ter Yde

(Oostduinkerke, Belgium).

In November 2008, we introduced seeds of D. incarnata, D. fuchsii, D.

praetermissa and G. conopsea in the botanical garden of the Institute of Botany and

Microbiology at the University of Leuven (50°51'53"N, 4°41'20"O). In november 2009,

also seeds of D. maculata, O. mascula, E. palustris and H. monorchis were

introduced. Between 200 and 300 seeds were placed within a square of 50-µm mesh

phytoplankton netting, enclosed within a Polaroid slide mount (Rasmussen &

Whigham 1993). The exact number of seeds was determined under a dissection

microscope (magnification x 12). For each species, 50 seed packets were buried.

Each seed packet contained seeds of a single individual. All sampled individuals

originated from a large population, to avoid problems with seed viability. Seed

packets were placed vertically just under the topsoil layer and every one or two

months one seed packet of each species was retrieved.

For each slide mount, the proportion of viable seeds was determined using the

tetrazolium method, according to the protocol of Van Waes and Debergh (1986); only

viable embryos are stained red after this treatment. We calculated the percentage of

viable seeds as the ratio of the number of colored seeds over the original number of

seeds with embryo. The seeds were counted under a dissection microscope

(magnification x 12).

Seed longevity of orchid species

93

6.4 Results Our results clearly show decreasing seed viability percentages with time, although a

lot of variation between different counts within one species was present. Seed

viability of the four orchid species that were buried in 2008 varied between 35 and

50% in the first months (Fig. 6.1). After 11 months, most seeds of D. fuchsii had lost

their viability, while for D. praetermissa and G. conopsea viable seeds were still

observed after 34 and 38 months, respectively. D. incarnata retained a seed viability

percentage of 14.4% after 42 months.

Seed viability of the four orchid species that were buried in 2009 was between

20 and 50% at the start of the experiment (Fig. 6.2.). Seeds of E. palustris and D.

maculata lost most of their viability after 30 and 31 months, respectively. Surprisingly,

in five seed packets of E. palustris protocorms were found (number of protocorms per

packet were: 7, 1, 2, 1 and 3 after 13, 15, 23, 27 and 31 months, respectively). The

seed viability percentages of H. monorchis and O. mascula after 31 months were still

9.4 and 8.5%, respectively.

Fig. 6.1. Percentage of viable seeds of four orchid species as a function of the number of months of burial in the soil. The seed packets were buried in 2008.

0

10

20

30

40

50

60

70

80

90

0 10 20 30 40 50

Perc

enta

ge o

f via

ble

seed

s

Number of months

D. fuchsii

D. praetermissa

G. conopsea

D. incarnata

Chapter 6

94

Fig. 6.2. Percentage of viable seeds of four orchid species as a function of the number of months of burial in the soil. The seed packets were buried in 2009. 6.5 Discussion

The results of our burial experiment show that seeds of seven of the

investigated orchid species survived at least 2.5 years in the soil, while seeds of one

species survived only one year in the soil. Orchid species with short lived seeds

might be expected to be rarer than species with persistent seeds, due to their more

limited recolonization possibilities. When the rarity of our study species is considered

(based on the rarity classes in Van Landuyt et al. 2006) no relationship between rarity

and seed longevity seems present. The seed longevity of the two least rare species

D. maculata (relatively rare, class 4) and D. fuchsii (rare, class 3) was 2.5 and 1 year,

respectively. E. palustris, D. praetermissa and D. incarnata are categorized as very

rare (class 2) and their seeds remained viable between 2.5 and 3.5 years. Seeds of

the three extremely rare species (class 1), O. mascula, H. monorchis and G.

conopsea, remained viable during at least 2.5 years. Some of our study species have

more specific habitat requirements compared with other species. For example, H.

monorchis is restricted to dune slacks whereas less rare species as D. maculata and

D. praetermissa have much larger ecological amplitudes. However, these differences

are not reflected in different seed longevity values. The fact that protocorms of E.

palustris were found in several seed packets, is very interesting because this species

is normally restricted to dune slacks and calcareous marshes. However, this species

can sometimes also be found on humid raised terrains. The fact that seeds of E.

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30 35

Perc

enta

ge o

f via

ble

seed

s

Number of months

E. palustris

D. maculata

O. mascula

H. monorchis

Seed longevity of orchid species

95

palustris could grow into protocorms in our botanical garden suggests that it

associates with widespread mycorrhizal fungi.

Different types of seed banks have been distinguished, according to the

longevity of seeds in the soil: transient, short-term persistent and long-term persistent

(Thompson et al. 1997). Transient seed banks refer to seeds that persist in the soil

for less than one year; short-term persistent seed-banks refer to seeds that persist in

the soil for at least one year, but less than five years; while seeds that persist in the

soil for at least five years are considered as long-term persistent. Our results suggest

that all of our study species can form a short-term persistent seed bank. Van der

Kinderen (1995) found similar results for two related European orchid species,

Dactylorhiza maculata and Epipactis helleborine. Very few viable seeds of D.

maculata were reported to survive after a 15 months period in the soil. E. helleborine

on the other hand had still a high percentage of swollen embryos after 15 months of

burial (between 13 and 60%), suggesting some seed viability. Zelmer and Currah

(1997) found that most seeds of the North American orchid Spiranthes lacera had

germinated within one year. Batty et al. (2000) reported evidence for a transient seed

bank in two terrestrial Australian orchids. They studied the soil seed bank dynamics

of Caladenia arenicola and Pterostylis sanguine, and found that no viable seeds

remained after one growing season. Other studies have found a relatively high seed

longevity in certain orchid species. Whigham et al. (2006) investigated seed viability

of seven North American orchid species and found that seeds of four species

(Corallorhiza odontorhiza, Aplectrum hyemale, Liparis liliifolia and Tipularia discolor)

were still viable after almost seven years, indicating the presence of a long-term

persistent seed bank (Thompson et al. 1997). Seeds of two species (Platanthera

lacera and Galearis spectabilis) remained viable after four years of burial whereas

seeds of one species (Goodyera pubescens) remained viable during only one year.

Seeds of the European orchid Corallorhiza trifida remained viable after 31 months of

burial (Mckendrick et al. 2000).

The fact that all of our study species seem to form a short-term persistent

seed bank can have important implications for their persistence. When a population

should go extinct due to extreme environmental conditions, recolonization from the

seed bank could be possible, at least when the extinction event did not deteriorate

habitat quality. Also in less extreme situations where only a part of the individuals die

or when reproduction is suppressed for a short period of time, a seed bank can be

Chapter 6

96

important by conserving the genetic variation present in the orchid population. The

mitigating effect of the seed bank will, however, only remain for two or three years. It

is important to note that the buried seeds originated from one population only, and

that the seeds were not buried in their natural habitat. This may make our estimates

conservative. On the other hand, the tetrazolium method may overestimate the real

germination rate, although some studies have reported higher germination

percentages than predicted through biochemical viability testing (van der Kinderen

1995). A lot of variation in the seed viability percentages was present between

different measurements within one species. This is likely due to variation in seed

quality between different fruits and individuals in one population, which may be

caused by differences in pollen quality (e.g. Meekers & Honnay 2011).

6.6 Conclusions The results of our burial experiment show that seeds of seven of the investigated

orchid species survived at least 2.5 years in the soil, while seeds of one species

survived only one year in the soil. This suggests that all of our study species can form

a short-term persistent seed bank, which can have important implications for their

persistence.

Chapter 7.

General discussion and conclusions

Chapter 7

98

7.1 Outline of main results

7.1.1 Hybridization and reproductive isolation

We have characterized the genetic structure of three natural hybrid zones containing

different Dactylorhiza species, on the basis of morphological measurements, their

AFLP profiles and flow cytometry. Our results provided clear evidence that

hybridization between all species pairs, and hence between different ploidy levels, is

possible. However, the frequency of hybridization between species varied

substantially between different sites. While frequent hybridization was observed at

the two sites with three sympatric species, very few unequivocal hybrids were found

where only D. incarnata and D. praetermissa co-occurred.

Spatial processes seem to play an important role in shaping the hybrid zone

with the two species. In the first place, the ΦST values between the different

subpopulations were significant, which may indicate spatially restricted gene flow

through pollen and/or seed. In addition, in both species, spatial autocorrelation

analyses demonstrated the presence of a highly significant spatial genetic structure.

The positive values of Fij at short distances indicate that neighboring individuals were

genetically more closely related than random pairs of individuals. This significant

spatial genetic structure could be the result of limited dispersal of seed or pollen

(Chung et al. 2005b; Jacquemyn et al. 2006, 2007) or of patchy occurrence of

suitable mycorrhizal fungi (Jacquemyn et al. 2012b). Pollen dispersal distances can

be limited when plants occur in clearly delimited subpopulations and pollinators move

around within patches before flying to another subpopulation. Limited seed dispersal

may be important in causing the spatial genetic structure, since there is growing

evidence that seed dispersal tends to be limited in many orchid species despite their

huge amounts of dust-like seeds (Machon et al. 2003; Jersakova & Malinova 2007;

Jacquemyn et al. 2009; De hert et al. 2012b) Finally, patchy mycorrhizal distributions

may also affect the spatial distribution of orchid species. In some orchid species,

succesfull seed germination was shown to be directly related to the distance from the

maternal plant (McKendrick et al. 2000, 2002; Batty et al. 2001; Diez 2007),

suggesting a declining abundance of essential mycorrhizal partners further away

from adult plants. The spatially restricted gene flow observed is probably one of the

main reasons for the lack of certain hybrid classes.

General discussion and conclusions

99

According to the molecular data most hybrids were probably F1 hybrids. Strong

post-zygotic barriers (hybrid sterility) prevent further backcrossing. Most hybrid

groups also showed more or less intermediate morphological positions between their

parental species, indicating that most hybrids combine characters of both parents.

Diploid hybrids between D. incarnata and D. fuchsii were intermediate between their

parental species on the PCO, whereas triploid hybrids between the allotetraploid D.

praetermissa and one of the two diploid species were skewed towards the

allotetraploid parent on the PCO and had a larger proportion coming from D.

praetermissa than from the other parental species. The fact that the triploid hybrids

were more similar to their allotetraploid parent is not very surprising, given that

triploid hybrids receive two-thirds of their genome from the tetraploid parent and one-

third from the diploid parent.

As both intra- and interspecific crosses yielded a high fruit set, no post-mating

pre-zygotic isolation was present. Viable seeds were obtained in both directions for

all interspecific crosses, indicating that early post-mating post-zygotic barriers

(embryo mortality) were weak. Also, germination failure is not an important post-

zygotic barrier since our seed baiting experiment revealed that both hybrid and pure

seeds were able to germinate and grow into protocorms. Moreover, in two of six

interspecific crosses hybrids were fitter than the intraspecific crosses. The

experimental crosses further showed that only when the pollen donor is a pure

species fruit set is high and that only crosses between pure species yielded a high

percentage of viable seeds. When hybrids were involved as either pollen-receptor or

pollen-donor, almost no viable seeds were formed. These results thus suggest that

hybrid sterility is the major cause hampering hybridization to extend beyond the F1

generation, and confirm previous studies in Dactylorhiza (e.g. Heslop-Harrison 1953,

Rossi et al. 1995). However, some seed viability was found in the studied hybrids.

Together with the occurrence of putative triple hybrids, this suggests the presence of

some secondary gene flow.

7.1.2 Seed ecology and germination

Orchids are dependent on mycorrhiza for successful germination and

protocorm formation because their minuscule seeds lack nutritional resources and

the necessary energy for germination is provided by mycorrhizal fungi (Rasmussen

Chapter 7

100

1995; Peterson et al. 1998). Therefore, successful (re)colonization depends on the

ability of orchid species to reach a suitable habitat patch and the presence of suitable

fungi. Seeds of all three study species (D. fuchsii, D. praetermissa and H. monorchis)

can form protocorms in occupied and unoccupied habitats, showing that dispersal

limitation plays a significant role in the inability of orchids to colonize restored sites

and no recruitment limitation is present. However, notwithstanding germination was

frequently observed in unoccupied habitats, germination appeared to be more

frequent in occupied dune slacks for D. fuchsii and H. monorchis. These results may

suggest a higher abundance of mycorrhiza near adult plants, which is also found by

other authors (McKendrick et al. 2000, 2002; Batty et al. 2001; Diez 2007).The easy

germination and protocorm formation in unoccupied habitats strongly suggest the

ubiquitous occurrence of mycorrhizal fungi, relatively low specialization of orchid

species for fungal species, or a combination of both. The little data available indicate

that species in the genus Dactylorhiza associate with a diverse suite of mycorrhizal

fungi (Shefferson et al. 2008; Jacquemyn et al. 2012), suggesting low specialization.

Evidence for low fungal specificity was also found by Tĕšitelová et al. (2012) for

Epipactis species and by Jacquemyn et al. (2010) for Orchis species.

To obtain data on the seed longevity of eight terrestrial orchid species a burial

experiment was established. The results of this experiment show that seeds of seven

of the investigated orchid species survived at least 2.5 years in the soil, while D.

fuchsii seeds survived only one year in the soil. Seeds of E. palustris and D.

maculata remained viable during 2.5 years. Seeds of D. praetermissa and G.

conopsea remained viable for three years, while seeds of D. incarnata were still

viable after 3.5 years. H. monorchis and O. mascula had still viable seeds after 2.5

years. These results suggest that all of our study species can form a short-term

persistent seed bank, which can have important implications for their persistence.

7.2 Implications for conservation

Orchid species in the highly urbanized Flanders region are almost entirely restricted

to nature reserves because of their very specific habitat requirements. Since many

General discussion and conclusions

101

nature reserves are small and fragmented, orchid populations are also fragmented

and different species are forced to grow sympatrically in the few remaining intact

habitat fragments. This may favour hybridization between related species and in the

worst case, cause genetic assimilation with more common orchids (Mayr 1992;

Rieseberg 1995; Arnold 1997; Allendorf et al. 2001). Jacquemyn et al. (2012b)

reported very frequent hybridization and partial genetic assimilation between the rare

orchids Orchis purpurea and O. militaris. Complete assimilation of one of the species

was not likely to occur because of the different microhabitats of the species and the

patchy distribution of their mycorrhizal fungi. Even in the absence of genetic

assimilation, the rarer taxon may yet decline because of pollen swamping from the

more common taxon (Ellstrand and Elam 1993; Arnold 1997). For example, Buggs

and Pannell (2006) reported the rapid displacement of monoecious Mercurialis annua

due to pollen swamping by a dioecious relative. So far, no studies investigated the

effect of pollen swamping in orchid species.

Dactylorhiza species often occur sympatrically in Flemish nature reserves and

hybrids are frequently found. We observed frequent hybridization in two hybrid zones

with three Dactylorhiza species, but the F1 hybrids were almost sterile. Some putative

triple hybrids provide evidence for some secondary gene flow, but no risk of genetic

assimilation and breakdown of species boundaries seemed to be present. However,

at one study site pure D. praetermissa individuals were not present anymore,

whereas its hybrids were still very abundant. We don’t know the cause for this, but

one possibility is that the hybrids were better adapted to changed environmental

conditions than their parental species. This might also suggest the possibility that

hybrids may be better suited to anthropogenically altered habitats (Rieseberg et al

1995; Vilà et al. 2000). In this case the hybrids could replace their parental species

and hence reduce species diversity. Therefore, when natural habitats that contain

related rare species are modified by human activity, the ongoing gene flow processes

should be examined in detail. When serious threats for the pure species should be

observed, removal of the hybrids can be considered. On the other hand, natural

hybrid zones may provide the stage for evolutionary processes in orchids (e.g.

Hedrén 1996a). When no potential negative effects are observed, the hybrid zones

should be protected together with the pure species.

Chapter 7

102

7.3 Implications for restoration

Since our results indicate that orchid species are not necessarily recruitment limited

due to their mycorrhiza dependence, we suggest that manual introduction of orchid

seeds in isolated restored habitats can be a valuable and even necessary practice to

restore orchid populations. However, we must mention that our experiment only

considered the protocorm stage. As a result, we cannot make any predictions about

the establishment success of adult individuals. Especially in the context of climate

change and habitat destruction, assisted translocation/migration will be necessary for

the conservation of many orchid species (Swarts and Dixon 2009). Before restoration

measures are designed, knowledge of the relative importance of dispersal vs.

recruitment limitation is crucial. Seed baiting methods can provide valuable

information on whether the colonization of orchid species after habitat restoration is

dispersal or recruitment limited. The fact that several studies found low mycorrhizal

specificity (Shefferson et al. 2008; Jacquemyn et al. 2010, 2012; Tĕšitelová et al.

2012), which is also suggested by our results, is interesting in a restoration context

since it may indicate a potentially successful translocation of orchid species.

Our seed longevity data show that all of our study species are able to form a short-

term persistent seed bank. This can have important consequences in a restoration

context, if the seeds should be able to germinate in their natural habitat. When an

orchid population should go extinct due to extreme environmental conditions,

recolonization from the seed bank could be possible, at least when the extinction

event did not deteriorate habitat quality.

7.4 Research perspectives

Hybrids between all species pairs were found, but certain hybrid groups were absent

in some sites, whereas they were present in other sites (Chapter 2 and 3). Although

we suggested that spatially restricted gene flow is probably causing the absence of

some hybrid classes, it cannot explain all the differences between sites. To obtain

more insights into general hybridization patterns in the field more hybrid zones should

be investigated. Also, more different habitat types should be considered since

General discussion and conclusions

103

different environmental conditions (biotic and abiotic) may influence hybridization

patterns. The fact that triple hybrids were found is surprising and to examine the

frequency of triple hybrids in general other hybrid zones with three (or more)

sympatric species should be examined.

Pollination experiments showed that viable seeds were formed in all different

interspecific crosses and could develop into protocorms (Chapter 4). However,

differences between species pairs were observed; hybrids with D. praetermissa as

mother plant performed better than hybrids with D. praetermissa as father plant. It

would be interesting to know if these asymmetries between species pairs are also

reflected in the field. However, data on the maternal and paternal species of the

observed F1 hybrids are lacking. Therefore, further research should use maternally

inherited markers (chloroplast DNA) to determine the mother species of the observed

hybrids. In combination with paternity analysis more insights into detailed

hybridization patterns could be obtained.

The pollination experiments involving crosses between hybrids and between

pure species and hybrids were performed on a limited number of individuals per

category due to practical constraints (Chapter 3). External factors (drought and

herbivore damage) reduced the number of individuals of some crosses and caused a

lack or underrepresentation of these crosses. Ideally, these pollination experiments

should be performed on a larger scale, involving more individuals and different sites.

Seed burial experiments yielded some interesting insights in the seed

longevity of eight orchid species (Chapter 6). Due to practical constraints the seeds

were buried in a botanical garden. However, to maximize the ecological relevance

the seeds should be buried in their natural habitats and also germination should be

observed. More studies should investigate the potential for persistent seed banks for

orchid species since seed banks can be very important for natural recolonization and

conservation of the genetic variation present in orchid populations (Thompson et al.

1997; Honnay et al. 2008). However, very little data are available on seed longevity

of orchids (see Whigham et al. 2006 and references therein).

Although recent analyses have shown that the three investigated Dactylorhiza

species are generalist species with regard to mycorrhizal fungi (Jacquemyn et al.

2012), it would still be interesting to examine the specificity of fungi for hybrid and

pure protocorms using DNA arrays (Chapter 4). It would also be interesting to

Chapter 7

104

compare the present mycorrhizas between occupied and unoccupied habitats

(Chapter 5).

Natural hybridization can have important evolutionary implications, both

positive and negative. Therefore, research on natural hybrid zones should be

stimulated. Especially when a rare orchid species should come into contact with a

more common one, the potential negative effects of hybridization should be

investigated. In the case of Dactylorhiza, sites where the very rare D. spagnicola co-

occurs with other Dactylorhiza species deserve special attention, given the high

potential of hybridization between Dactylorhiza species. Also, sites with more than

one allotetraploid species (e.g. D. praetermissa and D. majalis) or with an

autotetraploid species (D. maculata) and diploids or allotetraploids would be

interesting to examine in an evolutionary context, because different species

combinations from varying ploidy levels might generate different hybridization

patterns.

105

Appendix Table I Contributions of morphological characters to the first two multivariate axes of the principal component analysis, which described 30.62 and 22.51% of the variation, respectively, for Paelsteenpanne and 27.10 and 20.45% of the variation, respectively, for Ter Yde. The most important characters are in bold type. Paelsteenpanne Ter Yde Component 1 Component 2 Component 1 Component 2 1 Leaf length -0.360 0.180 0.158 0.072 2 Leaf width -0.031 0.137 0.086 -0.087 3 Number of leaves 0.716 0.215 0.012 0.607 4 Plant height 0.117 0.139 0.247 0.423 5 Inflorescence length -0.079 0.180 0.097 0.037 6 Stem diameter -0.343 0.212 0.003 -0.435 7 Number of flowers 0.159 0.231 0.064 0.290 8 Stem solid/hollow 0.555 -0.039 -0.188 0.805 9 Leafspots 0.722 0.219 0.055 0.876 10 Spur length/width 0.599 -0.148 -0.073 0.461 11 Lateral sepal length -0.279 0.313 0.867 -0.007 12 Lateral sepal width 0.163 0.694 0.736 -0.197 13 Middle sepal length -0.346 0.344 0.822 0.103 14 Middle sepal width 0.116 0.651 0.759 -0.043 15 Lateral petal length -0.492 0.533 0.869 -0.037 16 Lateral petal width 0.199 0.761 0.751 -0.046 17 Labellum length/width -0.565 -0.304 -0.315 -0.584 18 Labellum shape index -0.934 0.050 -0.043 -0.911 19 Middle lobe of labellum length 0.848 0.040 0.202 0.869 20 Middle lobe of labellum width 0.587 0.288 0.535 0.271 21 Lateral lobe of labellum length 0.486 0.712 0.806 0.141 22 Lateral lobe of labellum width -0.363 0.815 0.762 -0.121 23 Labellum ground colour -0.360 0.103 0.242 0.845 24 Labellum type of markings 0.128 -0.097 0.255 0.131

106

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