Guidelines for the Reintroduction of Plant Species With

GUIDELINES FOR THE REINTRODUCTION OF PLANT SPECIES WITH

ISSUES RELATED TO LOW GENETIC DIVERSITY

Gary Shanks

A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Public Horticulture

Summer 2015

ProQuest Number: 1602359

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GUIDELINES FOR THE REINTRODUCTION OF PLANT SPECIES WITH

ISSUES RELATED TO LOW GENETIC DIVERSITY

Gary Shanks

Approved: ______Robert E. Lyons, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee

Approved: ______Blake C. Meyers, Ph.D. Chair of the Department of Plant and Soil Sciences

Approved: ______Mark Rieger, Ph.D. Dean of the College of Agriculture and Natural Resources

Approved: ______James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS

I would like to thank my thesis advisory committee, Dr. Robert Lyons, Dr. Matt Taylor, Abby Hird and Dr. Joyce Maschinski, for their guidance and support throughout this process. I would specially like to thank Dr. Lyons for remaining on my committee, while the program transitioned in its leadership. I would like to thank Longwood Gardens and the University of Delaware for making it possible for me to complete my Masters Degree in the United States. I also would like to thank my classmates for their support and making life that much more interesting, especially on our international and domestic trips. Finally, I would like to thank my friends and family for their support, entertainment and encouragement.

iii TABLE OF CONTENTS

LIST OF TABLES ...... vi LIST OF FIGURES ...... viii ABSTRACT ...... ix

Chapter

1 INTRODUCTION ...... 1

2 LITERATURE REVIEW ...... 3

Reintroduction ...... 10 Genetic Modification Techniques ...... 20

3 MATERIALS AND METHODS ...... 24

Surveys ...... 25 Case Studies and Interviews ...... 26

4 RESULTS ...... 29

Survey ...... 29 Expert Interviews ...... 43 Dr. Randall Wisser ...... 43

Case Studies ...... 47

Kirstenboch Botanical Gardens ...... 47

Erica verticillata ...... 48 Current Management ...... 49 Future Genetic Concerns ...... 53 Measuring Success and Future Plans ...... 56

Fairchild Tropical Botanic Garden ...... 59

Consolea corallicola ...... 59 Current Management ...... 61 Future Management ...... 63

iv Pseudophoenix sargentii ...... 65 Current Management ...... 66 Future Management ...... 67

Missouri Botanical Garden ...... 69

Astragalus bibulatus ...... 70 Current Management ...... 70 Future Management ...... 72

American Chestnut Research and Restoration Projet ...... 74

Current Management ...... 74 Future Management ...... 75

5 DISCUSSION ...... 78

Collection Management ...... 79 Seed Banking ...... 81 Pollination Experiments ...... 84 Monitoring ...... 87 Site Management ...... 88 Genetic Diversity ...... 90 Advanced Methods of Improving Genetic Diversity ...... 91

Hybridization ...... 91 Managed Relocation ...... 94 Polyploidization ...... 96 Mutagenesis ...... 97 Genome Editing ...... 98 Genetic Modification ...... 99

Conclusions and Recommendations ...... 102

REFERENCES ...... 109

Personal Communication ...... 109

Appendix

A SURVEY I ...... 122 B SURVEY II ...... 137 C HUMAN SUBJECTS REVIEW BOARD ...... 177

LIST OF TABLES

Table 1 Case study and individual interview participants, their institutions and the reasons for contact...... 28

Table 2 Responses to survey question 1, “Is your organization currently involved in plant reintroduction projects?” ...... 30

Table 3 Responses to survey question 4, “Are you currently reintroducing any species that is known to have low genetic diversity in the wild?” ...... 31

Table 4 Summarized responses to survey question 6, “Please briefly describe the number of populations and or individuals remaining alive in the wild?” ...... 32

Table 5 Responses to survey question 7, “Please indicate whether population numbers are currently increasing, stable or declining?” Respondents could select more than one choice...... 33

Table 6 Responses to survey question 8, “Keeping the species in mind that you selected, what threat(s) have led to its current low genetic diversity?” Respondents could select more than one choice...... 33

Table 7 Responses to question 11, “Do you receive outside funding to support species conservation?” ...... 34

Table 8 Responses to survey question 13, “Is there seed of this species currently stored in a seed bank?” ...... 35

Table 9 Responses to survey question 14, “Are there plans for any banked seed of this species to be used in future reintroduction efforts by your institution?” ...... 36

Table 10 Responses to survey question 20, “How long after reintroduction (out- planting) does your institution monitor this species?” ...... 37

Table 11 Responses to Survey question 23, “What is the main reproductive mechanism for this species?” ...... 38

Table 12 Responses to survey question 26, “What percentage of this seed is estimated to be viable?” ...... 38

vi Table 13 Responses to survey question 27, “Has hand pollination of this species resulted in successful seed set?” Describe these results ...... 39

Table 14 Responses to survey question 29, “Does your institution use any methods to increase or maximize the genetic diversity of this species?” ...... 40

Table 15 Summarized responses to survey question 30, “Please briefly explain the methods used by your institution to increase the genetic diversity of this species. For example conservation breeding, laboratory techniques, curatorial practices etc.” ...... 41

Table 16 Responses to survey question 31, “Do you think that your institution’s reintroduction project is successful or soon to be successful?” ...... 41

Table 17 Summarized responses to survey question 33, “How do you define the success of your institution’s reintroduction efforts for this species? Please list your own reasons for success.” ...... 42

vii LIST OF FIGURES

Figure 1 Erica verticillata flowering in midsummer at Tokai in the Table Mountain National Park, South Africa...... 48

Figure 2 The successful method of planting Erica verticillata in transects on the edge of the wetland at Tokai, South Africa, 2009. Photo credit Anthony Hitchcock...... 52

Figure 3 Erica verticillata thriving six years after being planted at Tokai using the transect method. South Africa, 2015...... 53

Figure 4 Specimens of Erica verticillata during an intense fire at Rondevlei Nature Reserve, South Africa, 2013. Photo credit Dalton Gibbs...... 58

Figure 5 Erica verticillata plants retaining leaves and flower color after a prescribed burn, Rondevlei Nature Reserve, South Africa, 2013. Photo credit Dalton Gibbs...... 58

Figure 6 A close-up of the flower of Consolea corallicola...... 60

Figure 7 Clones of different individuals of Consolea corallicola, identified from a RAPD analysis, maintained in the conservation nursery at the Fairchild Tropical Botanic Garden, Coral Gables, Florida, USA...... 62

Figure 8 Pseudophoenix sargentii growing in the nursery at Fairchild Tropical Botanic Garden, Coral Gables, Florida, USA...... 66

Figure 9 Mature flowering plant of Astragalus bibulatus reintroduced to the Cedar Glade Habitat in Tennessee, USA, 2014. Photo credit Ashley Morris...... 69

Figure 10 Astragalus bibulatus being reintroduced to the Cedar Glade habitat by Dr. Matthew Albrecht (right) and Dr. Quinn Long (Left) in Tennessee, USA, 2012. Photo credit David Kennedy...... 71

Figure 11 Astragalus bibulatus plants propagated from seed in a nursery setting at Missouri Botanical Garden, St. Louis, Missouri, USA, 2012. Photo credit Dr. Quinn Long...... 72

viii ABSTRACT

An increasing number of plant species are being threatened with population fragmentation and extinction, mainly due to anthropogenic actions. Populations with low numbers usually consist of low genetic diversity, which can result in deleterious symptoms that cause further species decline. As a result, numerous species currently exist only as a few genotypes in botanical collections around the world, with little hope of successful reintroduction back into the wild. This research will explore how species with low genetic diversity can best be managed to facilitate reintroduction back into a natural ecosystem, and what can be done to save those species that are on the verge of extinction. This research investigated the current management practices used for plants with a known low genetic diversity through a survey of botanic garden conservation programs, case studies and interviews. Alternate methods of increasing genetic diversity of plant material in other sectors were also explored as potential conservation measures.

Survey results showed most institutions utilize general management practices to facilitate species reintroduction, but few have specialized methods of maintaining or increasing genetic diversity apart from mixing of seed sources. Case studies and interviews with reintroduction experts and crop geneticists supported the development of a set of guidelines for best management practices pertaining to genetic diversity. These guidelines focus on knowledge of species demographics and distribution, stored

ix germplasm, species biology, reintroduction history and the potential for conservation breeding and managed relocation. Additionally, several methods of gene manipulation were identified that may be useful in creating diversity in genetically depauperate species. These include hybridization, genetic modification, mutagenesis, polyploidization and genome editing.

x Chapter 1

INTRODUCTION

Genetic diversity can be described as the total number of genetic characteristics in the genetic makeup of a particular species or population of that species (Falk et al. 2001). Having some measure of genetic diversity in a species is essential for the following: phenotypic and genotypic variation, coping with current environmental variability, reducing the deleterious effects of inbreeding among close relatives and providing the basis for adaptation (Falk et al. 2001). Of the estimated 400, 000 plants species on earth (BGCI 2014a); an increasing number is threatened with extinction, mainly as a result of widespread habitat degradation or conversion (Maunder et al. 1998). In 2014 the International Union for Conservation of Nature (IUCN) reported that of the 20,185 plants assessed, some 10,896 plant species are threatened with extinction, with 2,205 of those being critically rare. This equates to over 50% of evaluated plants being threatened (IUCN 2015). An additional 113 species are possibly extinct or extinct in the wild and 37 species are known to be extinct in the wild.

Though beneficial, species conserved through ex situ collections and horticulture usually represent only a fragment of the genetic diversity once represented in wild populations (Maunder et al. 1998). Reintroduction attempts using this material will likely result in populations with low genetic diversity. Sexually propagated material may also suffer from genetic drift, where plant material becomes adapted or artificially selected for ex situ conditions and be less able to survive under wild

1 conditions. Genetically depauperate populations risk inbreeding depression due to a lack in variation of pollen partners (Falk et al. 2001). Inbreeding depression can compound the problem further by creating populations that are even more genetically depauperate. This can lead to individuals that are phenotypically and genotypically similar, which can prevent adaptation to changes in the environment, and an increase in disease and pest susceptibility (Falk et al. 2001). These threats compromise the long-term viability of populations of rare plants (Falk and Holsinger 1991). Though there are several ex situ methods for long-term preservation over time (tissue culture, living plant collections, cryopreservation, seed banking), when possible, the ideal conservation goal should be appropriate reintroduction back into the wild. The aim of this research is to assess and recommend methods for improving and increasing genetic diversity of species currently in extremely precarious situations that, without the aid of man, will face certain extinction. To this end, this research highlights innovative and sometimes controversial approaches to management and enhancement of genetic diversity that may increase in viability as more research enriches the body of scientific knowledge.

2 Chapter 2

LITERATURE REVIEW

This literature review summarizes the overarching theme of plant reintroduction as a conservation measure, its history and broad reasons for successes and failures. This review focuses on reintroduction of plant species with symptoms related to low genetic diversity, specifically targeting methods to conserve or improve remaining genetic diversity. Several species with low genetic diversity are exemplified and the limitations and current status of their existence are highlighted. Due to the effects of genetic diversity being very species specific, for the purposes of this research, low genetic diversity is defined as a species that is genetically depauperate and having one or more of the deleterious symptoms present in that species or population of that species. Plants are necessary for human life and nearly every animal species on this planet (Raven 2011; Kennedy et al. 2012). They give us food, shelter and medicines and have numerous other uses, providing the basis of life on this planet. Due to the impacts of humans and climate change much of this plant diversity is threatened (IUCN 2015). With time, these threats will only increase as humans put more pressure on the planet’s resources. There is an acute need for large-scale habitat restoration and species reintroductions, and botanic gardens and others play a fundamental role in these efforts. In addition to serving as resources for ex situ plant material, botanic

3 gardens also specialize in education and research, essential to successful integrated plant conservation (Kramer et al. 2011). The loss of plant diversity is a global issue, such that the United Nations’ Convention on Biological Diversity has adopted the Global Strategy for Plant Conservation Collections (GSPC). Its aim is to halt the loss of plant diversity, developing 16 outcome-orientated conservation targets with an updated deadline of 2020. Target 8 of the GSPC calls for, “At least 75% of plant species in ex situ collections, preferably within the country, and at least 20% available for recovery and restoration programs” (CBD 2014). The Botanic Gardens Conservation International’s 2013 review of plant collections in the United States showed that Target 8 is far from being reached, with some 3,000 additional species still needed in ex situ collections to meet the 2020 goal. Ex situ collections also need to be of consistently high quality, comprising well-documented material with suitable genetic diversity in order to provide seed and plant material needed for conservation purposes. Building ex situ collections to directly support conservation requires a significant investment in time, resources and expertise (BGCI 2013). Conservation can be achieved with two overarching concepts, namely in situ and ex situ conservation. In situ conservation consists of the conservation of wild plants in a natural setting. Species in this situation can be subject to some degree of species-specific horticultural and demographic management or only require general land management. The ultimate goal is populations conserved in situ that are self- sustaining with little or no management (Guerrant et al. 2004). Inter situ conservation consists of plants cultivated under near natural conditions, such as a managed population within restored semi-natural vegetation (Cochrane et al. 2010). Ex situ

4 conservation simply means maintenance of living plant material off site, away from a species’ natural habitat (Guerrant et al. 2004). Ex situ conservation can take the form of living plants grown and propagated in a plant collection, or as stored and preserved material in special storage facilities such as seed banks. Ex situ conservation can be divided into two groups based on their applications to conservation. Community gardens, commercial production, small collections of plants in living/reference collections usually serve as poor resources for conservation, unless multiple genotypes are grown and adequate provenance documentation is available. Plants grown in special conservation collections in large field orchards can preserve high genetic diversity and serve as a better resource for conservation (Guerrant et al. 2014). High genetic diversity can also be stored in seed banks or preserved as vegetative material in cryopreservation and tissue culture facilities (Guerrant et al. 2004). Long-term ex situ storage can provide a long measure against extinction, but does not provide a solution to the extinction crisis, only a means to buy time, as seed or vegetative cannot be stored forever. Seed banks are also still prone to manmade and natural disasters (BGCI 2014b). If the global goal for plant conservation is to minimize or prevent extinctions, then building these ex situ collections needs to be a priority. Emphasis needs to be placed on assessing and ensuring their conservation value. Genetically representative collections are essential if they are to be used for reintroduction work (Pritchard et al. 2012). With removal of threats, reintroduction of rare species back into the wild is an essential measure to conserve those threatened species (Akeroyd and Wyse Jackson 1995). Living collections in botanic gardens are also prone to threats, such as disease and are not “safe” without the management of man (Rae 2011).

5 On a global scale, the ability of botanical institutions to sample and store a wide variety of the world’s diversity has been proven (CBD 2014). Gardens and seed banks are retaining germplasm that has been extirpated on a local and global scale, as well as germplasm that may have disappeared entirely from the wild (Guerrant et al. 2014; Maunder et al. 2000; Hitchcock 2007; Maunder et al. 2001). An example is Chamaerops humilis in France, which was nearly extirpated from the wild if it weren’t for the efforts of botanical collections in France. The same can be said for a population of Leucojum aestivum collected in 1909 and preserved at the Royal Botanic Gardens at Kew, but completely lost from its original locality (Maunder et al. 2001). In North America, Franklinia alatamaha has been completely extirpated from the wild. It was first collected by William Bartram in the mid to late 1700s, and had completely disappeared from the wild by the 1800s (Owens and Rix 2007). Recent expeditions have failed to find any individuals alive in the wild (Bronaugh 2008). Mr. Bartram distributed cuttings and seed, which have resulted in more than 2000 trees being cultivated in botanic gardens worldwide (Owens and Rix 2007). The genetic diversity of the cultivated genotypes is low, since they all originated from the one collection made by Mr. Bartram (Missouri Botanical Garden 2014). Without banked seed, living plant collections are increasingly becoming some of the last options left to prevent a species’ extinction. A case in point is Erica verticillata, from South Africa, which was thought to be extinct in the wild by the first half of the 20th Century. It is currently ranked as “extinct-in-the-wild” by the Red List of South African Plants in 2012 (South African National Biodiversity Institute 2012), and previously listed as extinct by the IUCN (IUCN 1997). The Erica was rediscovered in plant collections far from the marshlands where it once prospered. It

6 was subsequently sent back to Kirstenbosch National Botanical Garden in Cape Town, propagated by horticulturists, and reintroduced to seasonal marshlands south of the city. Due to the persistence of this species in botanical collections around the world, eight different genotypes have been discovered and returned to South Africa. Plants were propagated and reintroduced into areas thought to be native habitat, and they have subsequently produced flowers and seed (Hitchcock 2007). Although the limited number of genotypes used for reintroduction might not constitute a genetically diverse population, the production of seed in the wild is a positive step toward the species’ recovery. Another example comes from Easter Island where Sophora toromiro was completely extinct in the wild by 1960 (Maunder et al. 2000). As with Erica verticillata, several specimens were preserved in botanic gardens around the world (Alden 1991; Godley 1992). Even though trees maintained at the National Botanic Garden at Vina del Mar in Chile represent somewhat higher genetic diversity than collections in Europe, some important genotypes have already been lost due to poor management of cultivated stocks (Ricci and Eaton 1997; Maunder et al. 1999). The process of genetic analysis and preservation is the first step in the conservation process so that total remaining genetic variability can be accurately characterized, as many of the trees may be duplicates from a single initial collection. Ideally propagation facilities need to be set up on Easter Island and suitable habitat restoration needs to take place if this species has any chance of being successfully reintroduced back to its historic range (Maunder et al. 2000). A similar example involving a species that has found a home in the horticultural industry comes from the Canary Islands off the coast of Africa (Ojeda

7 and Santos-Guerra 2011). Lotus berthelotii is widely cultivated in many regions, especially in Mediterranean climates, but has exceptionally low genetic diversity (Owens 1985). The range of genotypes in cultivation is small due to vegetative propagation being the only means of production. This species was already described as exceedingly rare by the IUCN in 1978 (Ojeda and Santos-Guerra 2011). Banares et al. (2003) described Lotus berthelotii and three other species of Lotus as critically rare in the wild. The paradox is that even though large quantities of plants are propagated within the nursery industry, the material has limited value for conservation practices due to low genetic diversity (Owens 1985). Ojeda and Santos-Guerra (2011) suggest using a circa situm approach, which involves cultivating the species in gardens and other areas close to natural populations. This could facilitate increased gene transfer and seed production, but should be carefully monitored and maintained. They also recommend that the hybrids not be planted near natural populations, as they could lead to genetic pollution of the species’ gene pools (Ojeda and Santos-Guerra 2011). Cosmos atrosanguineus has very low genetic diversity but has great horticultural merit. Not only are its flowers an unusual dark burgundy color, but they also have a chocolate scent. This species has not been seen alive in the wild since it was collected in 1860 (Hind and Fay 2003). Even though it has been mass propagated, all existing plants in Europe and America come from the original collection, which is self-incompatible (Hind and Fay 2003). In lieu of possible extinction, scientists are now preserving material of C. atrosanguineus via cryopreservation (Wilkinson et al. 2003). Despite much research and several attempts to relocate it in the wild, no new genetic material had been found until a self-compatible plant was “discovered” in cultivation in New Zealand (Hind and Fay 2003). It is unclear if a formal

8 conservation-breeding program has been attempted, however, even with this discovery, only two genetically unique genotypes are unlikely to produce a viable population capable of surviving in the wild. Until a solution for increasing genetic diversity is found, this species, due to its horticultural merit, serves as a fantastic example to highlight the importance of conservation to garden visitors and plant enthusiasts. Encephalartos woodii is another example of a species that has persisted in botanic gardens but is completely absent from the wild. Only one male plant was ever found and this has been clonally propagated from offsets and distributed to botanic gardens worldwide (Gorelick and Osborne 2002). Recent research has suggested that another male plant exists in a collection in South Africa, but this hasn’t been confirmed (Prakash et al. 2008). Gorelick and Osborne (2002) proposed a study to test whether sex change could be achieved in Encephalartos woodii, by using a demethylating agent on cells grown in tissue culture. This procedure had been known to cause sex change in at least one angiosperm (Vyskot et al. 1995). In order to manage the different genotypes of a species such as Cosmos atrosanguineus and Sophora toromiro, they are usually given different accession numbers, or in the case of Erica verticillata, cultivar names. A proposal by two researchers from Smith College takes this idea a step further (Nicholson and Maasch 2014). They propose a new category of naming threatened plants in the International Code of Nomenclature for Cultivated Plants, specifically focusing on uniquely naming the different genotypes of a particular species. Nicholson and Maasch argue that changing the code in this way would facilitate effective and efficient germplasm exchange, collection building, breeding, and propagation, while accurately

9 representing how many genotypes – not clones – of rare and endangered taxa are currently safeguarded in botanical collections.

Reintroduction Plant reintroduction is the establishment of a population in an area where it once occurred, or augmentation of an existing population or part of a meta-population (a system of connected, spatially distinct sub-populations) (IUCN 1998). Plant reintroduction also encompasses those methods that are deemed more controversial such as translocation of individuals/populations into new areas within their range and introduction of a species outside its natural range (Guerrant 2012). Most reintroduction projects use living plants, produced sexually or asexually, as the founders, but this depends on the biology of the species and the circumstances surrounding the site. Generally speaking, transplants into the wild seem to have greater success when compared to broadcasting seed (Albrecht and Maschinski 2012). It is generally understood that the more founders one has, the greater the chances of success. To be truly successful a reintroduced population needs to repeatedly complete its life cycle from seed to maturity without the assistance of man (Guerrant 2012). Even with thorough management of ex situ material and in situ populations, the effects of low genetic diversity cannot be avoided for species facing a genetic bottleneck. Typically, when populations are reduced in size or fragmented, there is an erosion of genetic variation (genetic drift) and negative changes in the genetic structure of a population. The results of population size decline or fragmentation include: inbreeding, reduced inter-population gene flow, as well as local extinction of populations within a larger meta-population (Young et al. 1996).

10 The same factors apply when attempting reintroduction of a species with low genetic diversity. Loss of allelic diversity can detrimentally limit species’ ability to respond to changing selection pressures (Frankel et al. 1995). Reed and Frankham (2001) questioned the relationship between molecular diversity and quantitative diversity (a measurement of the phenotypic characteristics of a particular individual in a population). Adaptation to climate change is due to phenotypic characteristics (quantitative diversity), rather than molecular genetic diversity. They found no relationship between the two measures for life history traits, and that molecular measures of diversity have a very limited ability to predict quantitative genetic variability (Reed and Frankham 2001). Later studies (Vitt and Havens 2004; Kramer and Havens 2009) agreed with Reed and Frankham. However a follow up study by Reed and Frankham (2003) found that heterozygosity does relate to evolutionary potential, and has a high correlation with current population fitness. They reported that heterozygosity, heritabilities and population size account for 15 – 20% of population fitness. They recommend that natural populations be kept at a size sufficient to retain genetic diversity and minimize the risk of extinction (Reed and Frankham 2003). Taking this all into consideration, some level of genetic diversity is important for populations that are self-perpetuating, without the influence of man. Later studies (Reusch et al. 2005) have shown that populations with higher levels of genetic diversity have also shown greater recovery from climatic extremes when compared to those with lower genetic diversity. When genetic diversity is subsequently lost, individual fitness is often reduced along with long-term viability of populations (Neale 2012).

11 Reintroducing species as small populations can lead to inbreeding and outbreeding depression, especially when the genetic diversity and source of the founder population is limited (Neale 2012; Kawelo et al. 2012). Inbreeding depression can result from the increase in homozygosity of deleterious alleles due to repeated crossing of related individuals. This can cause a reduction in fitness (Montana State University 2015). Outbreeding depression occurs when plants from two different populations adapted to different conditions are brought together in one population and cross-pollination takes place. The result can be that the progeny are not suited to growing well under either of the two conditions (Fenster and Dudash 1994; Montalvo and Ellstrand 2001). Examples include Chamaecrista fasciculata (Fenster and Galloway 2000) and Ipomopsis agregata (Waser et al. 2000). However, this is dependent on a number of factors, and mixing genetic sources in lieu of outbreeding depression may be very beneficial for reintroductions (Edmands and Timmerman 2003), especially when dealing with genetically depauperate populations that may have been separated by the action of man (Maschinski et al. 2013). There are multiple factors to consider before attempting reintroduction of a species with low genetic diversity, particularly; pollinator loss and unknown breeding systems, issues with propagule supply, uncontrolled threats, and limited sites for reintroduction (Kawelo et al. 2012). Another threat is artificial selection based on vigor, which is often incurred in propagation nurseries (Maschinski, personal communication 2014). In discussing the history of plant reintroductions, Godefroid et al (2011) reported that out of a total of 249 worldwide plant species reintroductions, survival, flowering and fruiting among reintroduced populations was relatively low, at 52%,

12 19% and 16%, respectively. Much of this could be attributed to a lack of long term monitoring, which usually ceased after four years, but other reasons include inadequate documentation, lack of understanding of underlying reasons for decline in existing populations, overly optimistic reports based on short-term results and poorly defined success criteria (Godefroid et al. 2011). Godefroid et al (2011) also found that participants in this study were also quick to judge their projects as being successful. Reintroduction of species with low genetic diversity requires attention to be paid to all these factors, but with the added burden of issues caused by low genetic diversity such as inbreeding depression, low fecundity, and maladaptation to the environment and local niche conditions (Falk et al. 2001) . In the United States, the Center for Plant Conservation (CPC) maintains the National Collection of Endangered Plants of over 750 critically threatened species native to North America. The CPC is dedicated to preventing the extinction of U.S. native plants and currently has a network of 39 botanical institutions focused on monitoring, preserving and reintroducing some of the nation’s most imperiled plant species (Center for Plant Conservation 2014a). There are 62 plant species listed within the CPC reintroduction registry (Center for Plant Conservation 2014b). The aim of these efforts is to stabilize current population numbers and introduce new populations where appropriate, all in accordance with general habitat protection and management principles (Center for Plant Conservation 2014a). The CPC best practices and guidelines for rare plant reintroduction (Maschinski et al. 2012) highlight the major factors that should be considered before attempting a reintroduction of a plant species back into the wild. The guidelines move through aspects of; justification for reintroduction, preparation, planning, regulation and funding, species biology,

13 genetics, source material, horticulture, site selection, population biology and finally implementation. These are then followed by elements of aftercare and monitoring (Maschinski et al. 2012). Part of these guidelines focus on availability of high quality and diverse source material (i.e., genetically diverse plant material). Efforts need to concentrate on banking as much germplasm as possible, focusing on genetic diversity, but not to the detriment of wild populations (Guerrant et al. 2014). Species biology needs to be carefully studied before a protocol for effective genetic sampling can be developed. Sampling protocols developed for a palm, Leucothrinax morrisii, had different results when applied to a cycad, Zamia decumbens (Griffith et al. 2015) and sampling needs to be tied closely to the species biology to be effective. The importance of effective sampling to maximize genetic diversity was demonstrated with an attempted reintroduction of Grevillea scapigera in Western Australia. This took place through the Western Australia Botanic Garden at Kings Park at a selected site, near Corrigin, in three subsequent years; 1996, 1997 and 1998 (Krauss et al. 2002). In 1999, this species was described as critically rare under the Western Australian Wildlife Conservation Act 1950 (Department of Environment and Conservation 2008). In 1995 ten plants (genotypes), thought to have adequate genetic representation were selected from the 47 known individuals for propagation purposes. Some 266 plants were vegetatively produced from these ten selected genotypes and planted at an in situ site near Corrigin (Krauss et al. 2002). Due to a lack of seedling recruitment, seed was collected from this site and germinated ex situ, and 161 seedlings were added to the existing plants at the site. After assessment of the initially reintroduced plants and their progeny, is was discovered that only eight genotypes of

14 the original ten were present in the population and 54% of the plants represented one genotype. The seedlings were on average 22% more inbred and 20% less heterozygous than their parents, because 85% of the seeds were the product of only four genotypes. Ultimately the effective population size of the founding population was approximately two plants (genotypes) (Krauss et al. 2002). It is thought that Grevillea scapigera is probably ill-adapted to remaining in small isolated populations because it is a natural out-crosser. Plants are short-lived and therefore historically, it probably has high levels of gene flow (Rossetto et al. 1995). Although it is unclear at this stage whether genetic decline threatens the translocated population, the authors thought it best to take a precautionary approach and reverse the genetic decline. In this case equalizing the influence of each founder, creating a sustainable meta-population and promoting natural seed germination in situ may have provided a more sustainable better-adapted gene pool for reintroduction efforts (Krauss et al. 2002). Genetic decline was subsequently addressed by adding more plants of the under-represented genotypes to the population. The original genotypes are also currently being held in cryopreservation as an insurance against possible population decline (Dixon and and Krauss 2008). The comprehensive review of the history and successes of plant reintroduction, Plant Reintroduction in a Changing Climate – Promises and Perils (Maschinski and Haskins 2012), highlights plant reintroduction projects and protocols, and discusses the potential for reintroduction and relocation to preserve species threatened by climate change. This work indicated that the timeframe for many projects is not long enough to determine whether or not populations are self-sustaining. Plant reintroduction is also a fairly young but growing science, and there is an increasing

15 trend towards reintroduction projects designed to test explicit hypotheses. Maschinski and Haskins (2012) suggest that future work be focused on monitoring strategies and population dynamics, especially survival, recruitment, and then spatial spread into previously unoccupied sites. However, Monitoring should be tied to the species biology and continue for many years following the first reintroduction activity. Carefully, well-designed long-term monitoring will determine how much management and intervention is necessary before reintroduced populations can be considered self- sustaining. In an independent study undertaken by Godefroid (2011), few reintroduction failures have been reported in the literature, which leads to a bias in overall reported success rates. Practitioners are encouraged to share both successes and failures. Maschinski et al. (2012) found that careful attention needs to be given to the source material in terms of representative genetic diversity. Genetic evaluation can provide information revealing evidence of divergence or convergence between geographically and ecologically separate populations. Genetic evaluation can inform best management practices of existing material in ex situ collections and identify limiting conditions such as a lack of allele diversity necessary to produce viable recruitment in the wild. Understanding the molecular and adaptive quantitative genetics, the profile of declining populations, and the diversity of potential source materials can help plan new populations with suitable levels of diversity and reduced risk of deleterious gene processes (Neale 2012). As indicated by Kennedy et al. (2012), other areas in need of research include concentrating on population dynamics and population modeling, taking an experimental approach, considering biotic and abiotic factors and including current

16 climate information in the planning process. Legal obligations, collaboration, and education all need to be made a part of the reintroduction process in the future. Gathering information on current and past reintroduction projects, and facilitating increased communication and collaboration will help clarify important principles and practices. Managed relocation is human assisted movement of plant material outside the species’ native documented range to counteract the negative effects of climate change (Haskins and Keel 2012). Considering this concept, the related concepts of chaperoned and assisted migration (Smith et al. 2013) and their acceptance in the conservation world, Kennedy et al. (2012) agree that any future studies need to be supported by planning and policy backed by significant funding. Progress can only be achieved through academic peer review and agency review of study proposals concerning managed relocation. To maintain and expand the genetic diversity of a population Falk (1996) recommends mimicking the life history characteristics of a species in the wild, including replicating its pattern of seed dispersal, planting diverse genotypes in a scattered fashion, and planting in a relatively high density to maximize cross- pollination. There are several methods for measuring the genetic diversity of individuals and populations as a whole, and this technology is becoming less expensive over time (Wisser 2014; Voytas, personal communication 2014). Creating the ideally sized founder population for a particular species is difficult and dependent on a number of factors. This is especially true of critically rare species, as in most cases scientists, often know very little about pollination biology, levels of recruitment, fecundity, etc.

17 (Falk et al 1996). Broadly speaking, founder population sizes of 50 or more genetically unique individuals (or genotypes) are optimal for establishing a persistent population and limiting genetic diversity issues (Maschinski et al. 2012). However using current methods, this may not be possible for extremely rare species (Krauss et al. 2002) . The Center for Plant Conservation’s “Best reintroduction practice guidelines” (Maschinski et al. 2012) state that a genetic assessment of wild populations is advised if the plant has any of the following criteria: fewer than 50 individuals (genotypes) flowering and setting fruit, highly fragmented and isolated populations, no pollinators are present, no viable seed is being set, high levels of herbivory, the morphology looks different in different locations, one or more populations have distinct ecology from the majority of the populations, it is difficult to distinguish a species from a congener, or there is a disagreement about the taxonomy and reintroduction may create an undesired opportunity for hybridization. High genetic diversity gives a population the ability to withstand the effects of climate change, and can defend against the genetic pitfalls such as inbreeding depression and founder effects (Maschinski et al. 2012). Many species, however, have less than 50 individuals (genotypes) alive in the wild or are preserved solely in collections (Kawelo et al. 2012). According to IUCN, a species having 50 or fewer individuals (genotypes) remaining in the wild falls under the Critically Endangered category, of which 2,205 species are currently listed. A conservation policy focusing on the concept of plant species with extremely small populations (PSESP) has been developed and is currently utilized in China (Ma et al. 2013). This policy specifically targets species that have had their populations severely reduced by human activity/development, and excludes species that are

18 naturally rare. Extremely low population numbers, high disturbance, high risk of extinction and restricted habitats are the key characteristics for a species to fall under this policy. The PSESP is consistent with some of the guidelines set up by the IUCN, but also include economic factors and public input. The protocol involves selecting a relevant plant species, conducting research on breeding and aspects on their biology and ecology, followed by reintroduction into the wild. An example of an animal species that has been effectively managed for genetic diversity is the California condor (Gymnogyps californianus), which has rebounded from just 27 birds in 1987 (Ralls and Ballou 2004) to total of 425 birds as of October 2014. Some 219 of those birds are currently found in the wild (U.S. Fish and Wildlife Services 2014). The original population comprised 14 genotypes that were entered into a breeding program in 1992 (Ralls and Ballou 2004). Careful genetic mapping and analysis and controlled breeding were used to achieve this turnaround, but the genetics of this species will need to be monitored well into the future to avoid the deleterious results from inbreeding depression (Frankham et al 2002; Keller and Waller 2002). Ideally the genetic analysis and efforts made with the California condor and this could be applied to plant species in similar situations. In extreme cases, hybridization with another species may be the only way to save a particular species from going extinct. Although it is impossible to have 100% of the imperiled species’ genetic diversity represented in a hybrid individual, certain characteristics will still be preserved (Levin 2002). Such is the case for the Florida Panther, Puma concolor (Johnson et al. 2010). By the 1990s there were only 30 panthers remaining in Florida’s swamps. Inbreeding depression was rife and threatened the survival of this sub-species of Puma concolor. Desperate measures

19 were needed in order to save this population. As a result, eight female pumas from a different sub-species, Puma concolor ssp. stanleyana, were brought into the Florida populations to boost genetic diversity. Although this increased the population to well over 100, there is still the threat of habitat loss, habitat saturation and disease.

Genetic Modification Techniques

The Long Now Foundation has developed the Revive and Restore Project, which aims to use new conservation tools to rescue the genetics of endangered and extinct bird and animal species (The Long Now Foundation 2015). The foundation mentions the possible de-extinction of the Easter Island Palm (Paschalococos disperta) on their website, but no plant projects are currently under review. The Ted X De-extinction conference, organized by the Long Now Foundation took place in 2013 at the National Geographic headquarters in Washington, DC and featured some prominent individuals involved in cutting edge and somewhat controversial practices (The Long Now Foundation 2013). Most of the controversy lies around the topic of de-extinction, and the use of genetic modification to bring species back from extinction.

Manipulation of plant genetics in order to facilitate plant species conservation has only recently been considered (Thomas et al. 2013), and practiced (Powell 2014), as with the American chestnut (Castanea dentata) whose population was decimated by a fungus (Cryphonectria parasitica) unintentionally introduced into the chestnut’s habitat.

20 Polyploids are organisms that have more than one set of chromosomes in their genetic makeup (Comai 2005) and can be identified by a flow cytometer (Brummer et. al 1999). Autoploidy refers to a spontaneous doubling of the basic set of chromosomes of a particular species, which are then referred to as autoploids. The process of autoploidy can happen naturally or it can be induced (Chen 2010). Chromosome doubling has varying effects depending on the species, but in several cases it has led to increased vigor in the agricultural and floriculture industries (Acquaah 2007). Natural polyploids are very common among plants and amphibians, and are usually well adapted to environmental conditions (Comai 2005). Some of the advantages of having more than one set of chromosomes include, increased vigor, deleterious gene redundancy, and loss of self-incompatibility (Mable and Otto 2001). Comai (2005) surmises that polyploidy could be important when isolated and severely bottlenecked populations are forced to inbreed, especially when the purging of deleterious alleles is made difficult by the reduced number of breeding individuals. Loss of self-incompatibility could also be important in those species where populations are small and unproductive. There are a number of negative consequences that can occur in polyploid organisms (Mable and Otto 2001), mainly genetic disruptions and chimera instability. Polyploidy can be induced using chemical doubling agents such as colchicine (Hancock 1997) and the herbicide oryzalin (Van Tuyl et al. 1992). Oryzalin, a herbicide of the dinitroaniline class, is as effective but much less toxic than colchicine. A method for inducing polyploidy successfully in Rhododendron cultivars using oryzalin was developed by Jones et al. (2008), and repeated successfully on Ruellia cultivars (Freyre et al. 2014).

21 Mutagenesis is a process where a plant is exposed to chemical or physical agents with mutagenic properties. Seed or vegetative material can be used in this process (IAEA 1977). In the evolution of agriculture, intense crop breeding and artificial selection caused genetic erosion, leading to severe bottlenecks being prevalent (Smartt and Simmonds 1995). This is a disadvantage in common with species having low genetic diversity. One of the purposes of mutagenesis is broadening the genetic base of germplasm in plant breeding projects, while minimizing decreases in viability (Mba et al. 2010; Sikora et al. 2011). Mba et al. highlight materials and several methods to provide optimal mutagenesis results, limiting detriment to the organism. No references to polyploidy or mutagenesis being used for conservation strategies were identified. The plight of Kokia cookei is relevant and significant to conservation. It is a highly endangered member of the Malvaceae, discovered in the 1860s (U.S. Fish and Wildlife Service 1997). Until recently, only one individual remained in the wild. Even though seed has been produced in the past, no seedlings achieved adulthood and it is now listed as extinct in the wild by the IUCN (IUCN 1997). In desperation, the last individual of Kokia cookei was grafted onto a related species, Kokia kauaiensis, and used to create approximately 23 clonal grafted specimens. The lack of seedling survival most likely points to inbreeding depression. Fertile seed production has now ceased, and this may be due to the fact that grafting has produced weak plants (U.S. Fish and Wildlife Service 1997). Multiple attempts of using embryo culture of immature seeds, propagation from cuttings, tissue culture and air layering have all failed (U.S. Fish and Wildlife Service 1997). However, in 2000 and 2001 six seedlings were produced through embryo rescue at the Lyon Arboretum of the University of

22 Hawaii (Guerrant et al. 2004). As of April 2015, about a dozen plants exist that have been raised through embryo rescue but it’s still too early to assess the possible effects of inbreeding depression at this stage (Sugii, personal communication 2015).

23 Chapter 3

MATERIALS AND METHODS

Due to the variability of reintroducing species with low genetic diversity to the wild, a mixed methods research approach was used to explore, characterize, compare and contrast these efforts. Surveys and case studies assessed methods of reintroduction, and posed questions to investigate solutions to the consequences of low genetic diversity. Data were collected via two surveys administered electronically through Qualtrics Online Survey Software, four case studies consisting of eight interviews and two other expert interviews. The University of Delaware Human Subjects Review Board approved all questions in advance in January, April and July 2014 (Appendix C). Survey data were analyzed on a quantitative basis, therefore many of the “other” and free text entries were standardized. The data from the surveys were then used in combination with data from the case studies to form the discussion. Data from the case studies was analyzed on a qualitative basis (Patton 2002), and backed up with published literature where possible.

24 Surveys The first online survey (Appendix A) was developed and administered to establish a baseline of botanic gardens and arboreta currently involved in plant reintroduction programs at botanic gardens in the United States. Using online discussion forums from the American Public Gardens Association (APGA), including the Plant Conservation, Plant Collections, Native Plant, and College and University Gardens Professional Sections, this survey gathered data from 367 U.S. participants, primarily botanic gardens and arboreta. This survey also revealed some of the challenges to species reintroduction efforts. This survey included 24 questions and was sent out electronically on 4 February 2014, with a reminder following on 12 February 2014. Information was collected on monitoring, culture, funding, pollination, seed production and seed banking in relation to species reintroduction efforts. Survey participants were also asked if they had an interest in participating in further research on the subject. Due to the first survey not being focused on low genetic diversity, a unanimous decision was made by the researcher’s graduate committee to omit the data from the results section of this study, but it can be found in its entirety in Appendix A.

The second survey focused on acquiring information about reintroduction of species with low genetic diversity (Appendix B). It was distributed on May 1, 2014, with a follow up reminder sent on May 15, 2014, to 1,313 gardens and arboreta engaged in plant reintroduction, and respondents were asked to answer questions based on one species. Survey recipients included gardens listed in BGCI’s GardenSearch database as well as individuals identified in the first survey. The second survey aimed to characterize and determine unique challenges and practices of those institutions reintroducing species with low genetic diversity. Of additional interest was

25 an exploration of any novel methods used by institutions that may maximize or increase genetic diversity. Due to the large number of possible answers to most of these questions and the fact that there were many “other” entries, the sample sizes were too small to perform an accurate correlation study.

Case Studies and Interviews The four case studies included eight interviews that were conducted in person and via telephone in 2014 and early 2015 to further characterize reintroduction efforts of species with low genetic diversity. Three of these case studies involved botanic gardens, and one involved a university. The preceding surveys and recommendations from experts in the field helped to identify case study candidates. The U.S. case studies included a geographically and ecologically diverse set of institutions: Fairchild Tropical Botanic Garden in Miami, Florida; Missouri Botanical Garden in St. Louis, Missouri; and The American Chestnut Research and Restoration Project based at the State University of New York in Albany, New York. International site research was conducted at Kirstenbosch Botanical Gardens in Cape Town, South Africa. The author made in-person visits to Fairchild Tropical Botanic Garden and Kirstenbosch Botanical Gardens.

The case studies were chosen according to the low genetic diversity status of a particular species currently used in a reintroduction program. Also favored were institutions having species with different pollination biology and seed dispersal mechanisms. The species assessed can be found in Table 1. Case studies aimed to compare the similarities of practices among a variety of species, life forms and ecosystems, and exploration of whether or not specific guidelines could be developed

26 for reintroduction of species with low genetic diversity. Two individuals from the U.S. Fish and Wildlife Services were interviewed due to their involvement with Consolea corallicola. Two individual interviews of experts in the field were also conducted in the fall of 2014, based on recommendations from case study participants and other experts in the field. The purpose of the interviews was to collect additional data that could be used to support and further characterize the case study interviews.

27 Table 1 Case study and individual interview participants, their institutions and the reasons for contact.

Interviewee and Species or topics discussed Reason for Institution contact Matthew Albrecht, Astragalus bibulatus Case study Ph.D. Fabaceae (telephone) Missouri Botanical Garden Quinn Long, Ph.D. Astragalus bibulatus Case study Missouri Botanical Fabaceae (telephone) Garden William Powell, Castanea dentata Case study Ph.D. Fagaceae (telephone) Co-director of The American Chestnut Research and Restoration Project Joyce Maschinski, Consolea corallicola Case study Ph.D. Cactaceae (in-person) Fairchild Tropical Pseudophoenix sargentii Botanic Garden Arecaceae Vivian Negron-Ortiz, Consolea corallicola Case study Ph.D. Cactaceae (telephone) U.S. Fish & Wildlife Service David Bender Consolea corallicola Case study U.S. Fish & Wildlife Cactaceae (email) Service Anthony Hitchcock Erica verticillata Case study Kirstenbosch National Ericaceae (in-person) Botanical Gardens Tony Rebelo, Ph.D. Erica verticillata Case study Kirstenbosch National Ericaceae (in-person) Botanical Gardens Chad Husby, Ph.D. Various species/conservation practices Interview Montgomery (in-person) Botanical Center Randall Wisser, Plant genetics Interview Ph.D. (in-person) University of Delaware

28 Chapter 4

RESULTS

Survey

The second survey focused on reintroduction of plant species that have low genetic diversity. Participants were asked to choose one particular species with a known low genetic diversity, and then complete the survey with this species in mind. This survey was started by 237 participants (18% of total recipients), but most exited by design prior to completion because they reported no involvement with reintroduction of plants with low genetic diversity. Thirty-one individuals successfully completed the survey. All survey questions and responses are in Appendix B. Question 1 asked respondents whether or not they were involved in general plant reintroduction projects and if they would be willing to share this information with BGCI’s GardenSearch database (BGCI 2015)(Table 2). Depending on their answer, respondents were directed to either question 2 or 3, which asked them to provide their institution’s name and country, so that these details could be updated in GardenSearch. Even though 171 agreed to share information with BGCI (Table 2), only 152 institutions provided the additional information in questions 2 and 3. A wide range of countries was represented from all continents, especially U.S, and European institutions. Overall, 64 institutions reported plant reintroduction activities and 88 institutions reported no reintroduction activities (Appendix B). This information was

29 sent back to BGCI and used to update reintroduction activities reported in GardenSearch. Only respondents who reported plant reintroduction activities were able to continue the survey. However, of the “yes” respondents, five did not continue the survey. Of the 77 respondents for question 4 (Table 3), 44 (56%), claimed to be working with species that have a low genetic diversity and 14 (19%) were unsure, which then directed them to continue the survey. Those that answered “no”, 19 (25%), were directed to exit the survey. Even though the “unsure” responses to question 4 were able to continue the survey, for better clarity, their answers were omitted from the remaining data analysis. Responses to question 5 include the 33 species respondents believe to have a low genetic diversity (see Appendix B). Of note, ten survey respondents exited the survey between question 4 and 5. These 33 species represent 26 plant families. Even though Cirsium pitcheri is represented twice, it is still valuable to compare reintroduction efforts reported for this species.

Table 2 Responses to survey question 1, “Is your organization currently involved in plant reintroduction projects?”

Response Number of % of Respondents Respondents Yes we are involved in reintroduction projects. I DO give consent to share this information with 73 38% BGCI. Yes we are involved in reintroduction projects. I DO NOT give consent to share this information 9 5% with BGCI. No we are not involved in reintroduction projects. I DO give consent to share this 98 52% information with BGCI. No we are not involved in reintroduction projects. I DO NOT give consent to share this 10 5% information with BGCI. Total 190 100%

Table 3 Responses to survey question 4, “Are you currently reintroducing any species that is known to have low genetic diversity in the wild?”

Response Number of Respondents % of Respondents Yes 44 57% No 19 25% Unsure 14 18% Total 77 100%

Next, respondents were asked to describe the species they identified in question 5. Question 6 asked respondents to describe the species they chose in terms of remaining populations or individuals. Responses varied widely, ranging from large populations (150 – 200 populations), disjunct populations, and only a few individuals remaining in the wild (Table 4; Appendix B). As reported in question 7, which assessed population trajectory, 42% of respondents agreed that the species’ populations are in decline (Table 5). Of 33 responses to question 8, a majority (82%) listed the action of man as the reason for the population decline (Table 6). Question 9 respondents could choose more than one answer and indicated that most species (53%) are nationally threatened. Question 10 assessed the status of the land on which the reintroductions are taking place, and from a wide range of responses no major land use type was dominant (Appendix B).

31 Table 4 Summarized responses to survey question 6, “Please briefly describe the number of populations and or individuals remaining alive in the wild?”

Remaining population size of Number of Respondents % of Respondents selected species

Reintroducing species that 14 42% have their remaining population number one or less.

Reintroducing species that have 7 21% two or three populations remaining in the wild.

Reintroducing species that have 4 12% 13 or more populations still remaining in the wild.

Reintroducing a species that has 1 3% no natural populations remaining in the wild.

No data provided 7 21%

Total 33 100%

32 Table 5 Responses to survey question 7, “Please indicate whether population numbers are currently increasing, stable or declining?” Respondents could select more than one choice.

Response Number of Respondents % of Respondents Increasing 8 24% Stable 8 24% Decline 14 42% Unsure 4 12% Other. Please specify. 4 12% Population is extremely low and in danger of extinction We have planted 100 in situ All populations are reintroductions

Response Number of Respondents % of Respondents Destruction of habitat by 27 82% man. Over-collection. 6 18% Competition with alien invasive plant, insect or 14 42% disease species. Competition from herbivores or other 7 21% animals. Climate change. 8 24% Natural disaster. 5 15% Other. Please specify in the 4 12% box below. Geographic isolation Migration bottleneck Restricted distribution in Tasmania Breeding system

33 Question 11 assessed the source of funding for the reported reintroduction projects. Most organizations (47%) receive funding from government sources, while 36% use their institution’s resources to fund reintroduction activities. Some of the “other” answers were standardized (Table 7). No trends in funding types were observed for any particular part of the world when these results were compared to the organization and country data in question 2. Question 12 assessed the distance of the institution from its reintroduction site; the data were spread fairly evenly with 64% of institutions’ reintroduction sites being between 7 – 62 miles away

Table 7 Responses to question 11, “Do you receive outside funding to support species conservation?”

Response Number of respondents % of respondents Yes, from private non- 5 15% governmental sources. Yes, from government 16 47% sources. Yes, from corporations. 0 0% No, my organization funds 12 36% all activities. Other, please specify in the 1 2% box below. Total 34 100% Multiple organizations fund activities

Information about complementary ex situ conservation practices was then gathered for the species reported by respondents. A majority of species (70%) described in question 13 are stored in a seed bank. The “Other” answers were standardized (Table 8). The “No” answers skipped to question 15, while the “Yes” respondents were directed to question 14, of which 70% of respondents stated that

34 there are plans for banked seed to be used in reintroduction work (Table 9). Question 15 asked respondents how vegetative material is preserved ex situ to facilitate conservation. Most institutions (76%) use living plant collections. It is worth noting that four institutions (12%) make use of cryopreservation to store vegetative material for longer periods of time. In question 16, most respondents (88%) grow their own plants for reintroduction purposes. Question 17 asked respondents if they grow plants in media comprising soil from the future reintroduction site. This is not common practice as only three (10%) out of 29 respondents use the soil from the site in the propagation mix. According to question 18, 81% use documented wild origin material collected by their institution for reintroduction purposes. Three (10%) institutions use material of unknown or garden origin. For the full answers for Question 15 – 18 see Appendix B.

Table 8 Responses to survey question 13, “Is there seed of this species currently stored in a seed bank?”

Response Number of Respondents % of Respondents Yes 24 70% No, it is not currently 4 12% stored in a seed bank. No, seed of this species cannot be stored using 4 12% traditional seed banking methods. Unsure 2 6% Total 34 100%

35 Table 9 Responses to survey question 14, “Are there plans for any banked seed of this species to be used in future reintroduction efforts by your institution?”

Response Number of Respondents % of Respondents Yes 14 70% No 2 10% Unsure 4 20% Total 20 100%

Survey participants were asked to provide information on monitoring and population augmentation. Responses to question 19 indicated that 19 (61%) institutions have been reintroducing their species from one to ten years (Appendix B). Question 20 asked participants to indicate how long they monitor their reintroduction sites. Responses were wide-ranging, including 26% of respondents monitoring their plants from two to five years, and 32% of organizations monitoring their reintroduction projects for 16 years and longer. Three respondents (10%) don’t monitor their reintroduction sites at all (Table 10). Responses to question 21 showed that 9 individuals (29%) introduce new material on when possible or necessary (Appendix B). From the responses to question 22 a majority of individuals (55%) do not reintroduce plants at different levels of maturity to replicate the demographics of a natural population (Appendix B).

36 Table 10 Responses to survey question 20, “How long after reintroduction (out- planting) does your institution monitor this species?”

Response Number of Respondents % of Respondents We don't monitor 3 10% reintroduction sites. 1 year 5 16% 2 - 5 years 8 26% 6 - 10 years 4 13% 11 - 15 years 1 3% 16 - 20 years 5 16% More than 20 years 5 16% Total 31 100%

Participants were asked about pollination biology and the results of hand pollination experiments. The responses to question 23 indicated that there is a wide range of reproductive mechanisms through this study, with slightly more species being self-compatible (38%) than obligate out-crossers (29%) (Table 11). Responses to question 24 indicated that 13 (42%) respondents have observed natural pollinators visiting their reintroduced populations, and for question 25, 23 (74%) respondents have observed natural seed set. Question 26 asked the 23 respondents (74%) from question 25 about the viability of this seed and the responses varied greatly (Table 12). Question 27 was only provided to “Yes” responses from question 25 and asked participants if hand pollination had resulted in successful seed set. Of the 23 respondents from question 25, only ten respondents (33%) in question 27 reported that hand pollination has resulted in successful seed set (Table 13). Twelve individuals (40%) were unsure if hand pollination had been successful and four individuals had not attempted hand pollination. Question 28 asked about the viability of the hand-

37 pollinated seed and of those ten respondents in question 27, only three (30%) knew the viability of that hand-pollinated seed (Appendix B).

Table 11 Responses to Survey question 23, “What is the main reproductive mechanism for this species?”

Response Number of Respondents % of Respondents Self-compatible. 12 38% Obligate out-crossing. 9 29% Asexual. 2 7% Unknown. 7 23% Other, please specify in the 1 3% box below. Total 31 100% mixed mating system

Table 12 Responses to survey question 26, “What percentage of this seed is estimated to be viable?”

Response Number of Respondents % of Respondents 0 - 20% 2 9% 21 - 40% 1 4% 41 - 60% 5 22% 61 - 80% 2 9% 81 - 100% 6 26% Unsure. 4 17% Other, please specify in the 3 13% box below. Total 23 100% Naturally about 25% but with treatment in smoke water, increased to about 75% Depends on the source mother stock and which strains are co-planted Varies from year to year - dry years are poor.

38 Table 13 Responses to survey question 27, “Has hand pollination of this species resulted in successful seed set?” Describe these results

Response Number of Respondents % of Respondents Yes 10 33% No 2 7% Unsure 12 40% Other, please specify in the 6 20% box below. Total 30 100% Not verify Hasn't been attempted Have not used it Not yet tried this Not needed NA fern

This survey specifically targeted species with low genetic diversity and the methods used by botanical gardens to increase the genetic diversity of species reported by respondents. Participants were asked to describe their methods and list their own reason used to define the success of the reintroduction work. Responses for question 29 indicate that 14 (47%) respondents use methods to increase genetic diversity, while 16 (49%) respondents do not (Table 14). Question 30 asked those 14 respondents from question 29 to describe the methods they use to increase genetic diversity (Table 15). Five (42%) of the responses use mixed seed sources as their method to increase the genetic diversity for reintroduction purposes, and one (8%) individual used grid-like planting to maximize cross-pollination. Question 31 asked participants if they thought their reintroduction efforts are successful or not. A total of 18 (58%) respondents thought that their efforts are successful, however 6 respondents (19%) thought it was too early to evaluate and 4 (13%) were unsure (Table 16). Half of the 6 (50%) “unsure” respondents indicated it

39 is too early to assess success or failure of these projects (Table 16). Question 32 asked the one respondent from question 31 to choose reasons for the failure of their project. They cited lack of funding, lack of monitoring and lack of institutional capacity as reasons for failure. Question 33 asked the 18 respondents who thought their reintroduction work is successful to define and list their own reasons for success. There were 17 responses that are summarized in Table 17. Most respondents (11, 65 %) thought that having a self-perpetuating population increasing in size is a good measure of success.

Table 14 Responses to survey question 29, “Does your institution use any methods to increase or maximize the genetic diversity of this species?”

Response Number of Respondents % of Respondents Yes 14 47% No 16 49% Unsure 1 4% Total 31 100%

40 Table 15 Summarized responses to survey question 30, “Please briefly explain the methods used by your institution to increase the genetic diversity of this species. For example conservation breeding, laboratory techniques, curatorial practices etc.”

Response Number of % of Respondents Respondents Banking of seeds collected throughout the species 5 42% range. Mixing of seed from several of the most diverse populations for reintroduction purposes. Tracking the provenance of individual plants at all 1 8% stages of reintroduction, with the aim to create genetically diverse populations. Only using F1 seedlings for out-planting to reduce 1 8% genetic drift. Planting different individuals in a grid-like pattern to 1 8% maximize mixing. Using only wild collected seed to produce the 1 8% propagules. No useful data entered 3 25% Total 12 100%

Table 16 Responses to survey question 31, “Do you think that your institution’s reintroduction project is successful or soon to be successful?”

Response Number of Respondents % of Respondents Yes 18 58% No 1 3% Too early to evaluate 6 19% success Unsure, please specify in 4 13% the box below. Total 31 100% Only after a long-term monitoring Low survival, but several seeds dispersed Plantation and establishment can be successful but is affected by harsh climatic condition just like typhoon and forest fire. They are holding their population number, but only through vegetative propagation from the pads. Not increasing spatially.

Table 17 Summarized responses to survey question 33, “How do you define the success of your institution’s reintroduction efforts for this species? Please list your own reasons for success.”

Reasons for successful reintroduction Number of % of Respondents Respondents Establishment and naturally regenerating populations 11 65% with evidence of seedling recruitment The population is growing 1 6% The plants are growing well and reaching maturity 3 17% < 50% survival and production of seed. 1 6% Experimental questions answered 1 6%

Expert Interviews

Dr. Randall Wisser

Dr. Randall Wisser, a corn geneticist at the University of Delaware, clarified and explained the details and feasibility of genetic diversification concepts discussed in the case study interviews. In an attempt to start a conservation breeding program or to select clones best suited for reintroduction work, Wisser was asked if one could tell on a genetic level what combination of parents would be best in order to produce the most fertile seed. Wisser said,

You can’t take two individuals, sequence their genomes and determine the level of their compatibility. You need to know what the genes are that control that process, and the variance in those genes that would cause them to mismatch. It’s a very complicated process to figure that out (Wisser 2014).

He went on to say that manual reciprocal cross pollination is the best way to determine whether a specific cross will work and produce the ideal number of progeny. In this way the viability of each clone as a seed producing or pollen- producing parent can be determined. Any clones that are sterile or partly sterile can be easily identified. Genome mapping can be useful when assessing the relatedness of individuals, but this practice is expensive and currently confined to crops with high economic value and will not show which individuals are the most fecund when crossed.

43 In lieu of completely mapping the genome, Wisser recommended crossing in a pair-wise fashion (depending on pollen biology), which will show the fertility of each individual and could determine relatedness (depending on seed production). Crosses that repeatedly produce no seed may be clones of the same individual, or be closely related. Wisser explained,

If all the remaining individuals of a species can be crossed, you want to maximize the number of crosses that are possible. There are two main sources at the genome level of creating diversity – one is mutation and the other is recombination. Mutation takes a long time to accumulate naturally but recombination can happen as fast as you can cross your plants. If you continue to cross in a pair wise fashion for one or two generations, then you are continuing to increase the genetic diversity that once existed in the population. You are reshuffling the genome. You are not creating new variance per say but you are stacking the information in new combinations (Wisser 2014).

Wisser stated that there is no all-encompassing definition for low genetic diversity and its effects, as some plants with a low genetic diversity do not suffer with any of the problems usually attributed to such a situation. Husby (2014) and Voytas (2015) both support this statement. Concerning polyploidy, as stated by Wisser, and supported by Comai (2014) and Folta (personal communication 2014), if there are two copies of a genome then you tend to have more vigorous phenotypes or characteristics. It may be an advantage in the wild for a plant to be more vigorous, as it may be better adapted to competition. Viability will not be affected, as long as the chromosome numbers of each individual match. After inducing polypoidy, the subsequent uniformity and fecundity would need to be thoroughly tested before releasing a plant into a natural setting.

44 In preserving or maintaining genetic diversity by crossing a species with a congener, Wisser stated that backcross breeding can have negative results. If the desirable species has few remaining individuals (genotypes), backcrossing the hybrid progeny to those few individuals (genotypes) will continue to decrease the genetic diversity. Another problem, highlighted by Wisser, is when certain characters need to be present in the progeny, apart from diversity. When compared to a single gene controlled character, it is more difficult to produce a plant containing only that character through breeding if multiple genes control the specific character required. Those genes might also control several other functions or characteristics which may be completely undesirable. Wisser additionally indicated that chemical mutagenesis could also improve genetic diversity and is used to produce variation in crop species; those altered outlying genes would then be selected and studied. The process involves creating mutations at the genome level through exposure to chemicals or gamma radiation (Mba et al. 2010) . Even though the mutations in this process are random, some of them could be useful in creating genetic diversity where none had existed before. Wisser stated that these plants should be genetically different enough to produce fertile seed when crossed. Citing examples of Encephalartos woodii and Consolea corallicola, Wisser was asked what could be done with plants that are no longer producing viable seed. His response was DNA preservation, as well as employing new and improved technology. He stated, “At some point people need to feel pain to be proactive and reactive. People are responsive to the system that they function in every day, the societal norms. There is a lot of complication in the scenery.” In agriculture, Wisser

45 talked about geneticists attempting to synthesize new chromosomes. For example in corn all the genes for yield will be stacked in one particular chromosome and that chromosome will be introduced into one particular corn line. Instead of thinking of a one-gene modification project, scientists are expanding this to a cassette of 30 – 40 genes that essentially form a new chromosome. What dangers might this type of engineering present? Wisser said,

Well I don’t really know, I mean it gets so complicated to me with commercial motives versus scientific motives. We are able to do a lot of things in this plant because it has such an importance as an agricultural crop and it therefore brings in a lot of funding. There’s a lot of stuff that we don’t understand. However, when you breed plants traditionally you are creating new variation all the time with the genome that you have, in some cases even more variation than we have created by using genetic modification. Think about the side effects that we’ve tolerated as a consequence of new varieties (Wisser 2014).

Another interview was conducted with Chad Husby, formerly of The Montgomery Botanical Center (MCB) and now with Fairchild Tropical Botanic Garden in south Florida. The mission of the MBC is “to advance science, education, conservation, and horticultural knowledge of tropical plants, emphasizing palms and cycads, and to exemplify excellent botanical garden design”. These collections are made available for scientific research, which is disseminated through scientific and popular publications. Information from this interview was used to support data and ideas in the Discussion section of this research.

46 Case Studies

Kirstenboch Botanical Gardens

Kirstenbosch Botanical Gardens is located in Cape Town, South Africa and focuses on the collection and conservation of plant species native to South Africa. Its mission is, “To promote sustainable use, conservation, appreciation, and enjoyment of the exceptionally rich plant and animal life of South Africa, for the benefit of all people” (SANBI 2015). The South African Biodiversity Institute (SANBI) manages Kirstenbosch, and also is responsible for eight other botanic gardens in South Africa. This case study focuses on the reintroduction of Erica verticillata (Figure 1), a species once thought to be completely extinct by the first half of the 20th Century. Interviews were conducted with two individuals managing the reintroduction efforts, Anthony Hitchcock, the Nursery, Living Collections and Threatened Plant Species Manager of Kirstenbosch, and Dr. Tony Rebelo, a Senior Scientist from SANBI.

Figure 1 Erica verticillata flowering in midsummer at Tokai in the Table Mountain National Park, South Africa, 2015.

Erica verticillata

Erica verticillata is a woody, fire-dependent, out-crossing species that used to occur in seasonally marshy areas near Cape Town in South Africa. Fire kills adult plants but stimulates germination of seeds that can lie dormant in the soil for many years. The goal of the reintroduction work is to have self- perpetuating populations of Erica verticillata in the wild. This species was rediscovered in an old botanical collection in South Africa in 1984, and later in other gardens in Europe and the United States, resulting in the identification of eight genotypes that are now in cultivation at Kirstenbosch.

48 Even though this extremely rare germplasm is carefully monitored and maintained at Kirstenbosch, there are several additional obstacles to the species’ conservation. Hitchcock and Rebelo initially indicated that little was known about the original habitat of this species, as the descriptions on herbarium sheets are vague, only mentioning the general locality. Some of the herbarium sheets from the early 1900s mention seasonal wetlands in Cape Flats Sand Fynbos (an Afrikaans word for fine leaved bush), but little was known about the specific habitat in which they once thrived. Another problem was that most of the original habitat of E. verticillata is now buried beneath urban development on the Cape Peninsula, with only a small portion of Cape Flats Sand Fynbos being conserved near the coastline. According to Hitchcock and Rebelo, this species is also completely dependent on fire for seedling recruitment, and the extinct in the wild status given by IUCN can only be challenged once a population completes three life cycles from seed to flowering without any human interference. This equates to a minimum of about 30 years if the fire cycles are manipulated to occur every ten years.

Current Management

To improve the public relations aspect of the Erica verticillata project and facilitate dissemination of plants to nurseries, the eight Erica genotypes received cultivar names. In open pollination experiments with these cultivars, Hitchcock noted that all but two of them produce scant seed. One of the genotypes, which is assumed to be the youngest, is the best seed producer. All reintroductions consist of plants asexually produced from a selected number of the genotypes.

49 Hitchcock also noted that limited nursery space availability at Kirstenbosch impedes the project’s future progress. Every genotype must be propagated vegetatively and conserved in a living collection. Hitchcock said that the following processes need to be evaluated: seed production and resulting seed in terms of its use for reintroduction; seed banking; method of accessioning seedlings; and availability of space for germination. The first reintroduction attempt took place at Rondevlei Nature Reserve in 1994 and later 2001, where the species was once thought to occur naturally. Only the two most fertile genotypes were asexually propagated and used in the reintroduction. Through an experimental approach it was established that this species survives best on the margins of wetlands, and is able to handle extremely wet soils in the winter. This is the first site that was burned in March 2013, and seedling recruitment was noted in late 2013 and early 2014. However, recruitment numbers were not as high as Hitchcock had hoped. The next site, located at Kenilworth Racecourse, was selected in 2005 due to suitable conditions and remnant Cape Flats Sand Fynbos matching the original habit description. Two areas were chosen for planting; one that hadn’t burned, and another area that had been burned fairly recently. Plants performed fairly well at the vegetated site, while the burned site reintroduction had no observed out-planting survival. Hitchcock said vegetative cover might be a factor that needs to be taken into consideration in the future, believing that the surrounding vegetation may act as some sort of protection against the elements. The vegetated site hasn’t been burned yet and so recruitment hasn’t been achieved.

50 The final site was chosen in 2005 on land reclaimed from a commercial pine plantation in Tokai. This area held a Cape Flats Sand Fynbos natural seed bank deposited before the pines were planted, which was rediscovered following a small accidental fire in 1998. This seed bank did not contain material of Erica verticillata, as it never occurred here, but the area that burned was deemed suitable as an introduction site. As a result of further management, a number of other areas were chosen for planting at Tokai. Hitchcock and Rebelo both consider this to be a case of managed relocation. Several groups of plants were planted out at two Tokai sites, one comprising two genotypes and the other comprising three. There was much trial and error involved as the land had only recently been regenerated and had not yet reached a stable ecological state. Examples of such issues include competition from disturbance- response invasive and native plant species, herbivory from a spike in the Otomys (African Vlei Rat) population, variation in the size and location of the wetlands and changing niche conditions due to the continued removal of pines and other non-native trees. Other issues include differing opinions between the experts involved and public outcry in response to the transition from a “forest” to natural vegetation. Withstanding these threats, according to Hitchcock, one of the greatest successes was achieved at a site where three genotypes were planted in multiple transects (Figure 2). These transects run from the wettest to the driest part of the edge of a seasonally marshy area. A remnant pine population also surrounds this area. This method shows where the ideal niche habitat is for E. verticillata (Figure 3), as those in the very wettest and driest sections failed to thrive, with those in between having greatest

51 survival. This method was repeated under similar conditions in other parts of the reserve with some success.

Figure 2 The successful method of planting Erica verticillata in transects on the edge of the wetland at Tokai, South Africa, 2009. Photo credit Anthony Hitchcock.

Figure 3 Erica verticillata thriving six years after being planted at Tokai using the transect method. South Africa, 2015.

Future Genetic Concerns

Hitchcock and Rebelo were questioned as to why only three genotypes are represented at all sites and the possible impacts of low genetic diversity. Rebelo and Hitchcock said that at the time of planting, there wasn’t enough material of any of the other genotypes to enable planting. Hitchcock and Rebelo list other obstacles to reintroduction, including the fact that planting cycles need to fit into the burn cycles, differing opinions between ecological and horticultural practices, different levels of influence at each site regarding the people involved, and differences in vigor and seed production between genotypes. When the question of genetic diversity was posed to Hitchcock, he surmised that as much genetic diversity should be present as soon as possible to limit the

53 detrimental effects of possible inbreeding depression. Hitchcock agrees in that there is more potential for inbreeding to occur after a couple of generations if the genetics of only two genotypes are present, rather than the genetics of all eight genotypes. Even if this conflicts with the IUCN status of the species and the natural ecological processes of the site, Hitchcock agrees that it’s a serious issue that needs to be considered. Rebelo had the following to say regarding adding additional plants to the reintroduced populations:

We don’t want to replant in the area, because if we replant then we reset the clock in saying it has established, so it has got to be three generations unassisted. You’ve got to choose now whether to just leave it and let it become part of the system or whether you want to augment it, which might weaken the system. You don’t really understand what’s happening. I personally would say it is hands off unless we discover something is going wrong (Rebelo 2015).

The concept of managed relocation and several methods relating to issues of low genetic diversity were also discussed with Hitchcock and Rebelo. Even though the topic of managed relocation has been broached with E. verticillata at the reclaimed site, both Hitchcock and Rebelo had varying opinions on its applicability to South African plant species. Rebelo stated that in flat areas, managed relocation has a lot more viability than in mountainous areas. The reason being that in mountainous areas there are often sister species in close proximity, possibly leading to hybrid swarms being formed. Rebelo posed the questions, “Do we give up? Do we let the species hybridize to save the genetics, even though we no longer have the pure species?” Rebelo explained that in situ conservation is the highest priority and that managed relocation should only be applied to those species in exceptional circumstances, because it may lead to displacement or disruption of other species.

54 Managed relocation needs to be examined on a case-by-case basis, as several factors come into play. Rebelo and Hitchcock were asked about the value of a backcross breeding program. Rebelo sees justification in experimenting to save a species but doesn’t know where hybrids, such as those produced with Encephalartos woodii, belong in the landscape. Some view hybridization as leading to extinction for a species. Hitchcock feels similarly, citing loss of a pure species. When asked about the potential of genetic modification for conservation, Rebelo said that it depends on what needs to be conserved. If it is an ecosystem or natural population then genetic modification is out, but if the focus is on a single species, then it could be feasible. Hitchcock agreed with Rebelo, but was more supportive, highlighting the importance of education. Using the example of the American chestnut, Hitchcock agrees that you haven’t essentially changed the species apart from a gene that is being used to protect it. There are degrees to how a plant is changed and in a broad sense Hitchcock claims not to be a purist. Hitchcock would favor genetic modification over back cross breeding, as long as the reasons for experimentation are clearly stated and supported. The process of altering a particular species’ ploidy level in an attempt to increase vigor and fitness was discussed. Hitchcock was initially adamant that this would so drastically change the plant that it could no longer be considered the same organism. However, once the process was explained and examples of natural polyploid species were given, Hitchcock retracted slightly, and stated that adequate reasons need to be given for attempting the process.

55 Hitchcock talked about the Sophoro toromiro (Ricci and Eaton 1997; Maunder et al. 1999), where the ploidy could be raised to increase vigor, however he stated that the whole process needs to be acknowledged. For something like the Erica, Hitchcock wouldn’t initiate polyploidy because there are a number of original (until proven otherwise) genotypes that are vigorous. Hitchcock wouldn’t have an issue with the process if there is a need for this type of manipulation and that it is carefully documented and explained in the literature.

Measuring Success and Future Plans

Rebelo views reintroduction in an ecological context, taking all species impacts on the ecosystem into consideration. The important aspect is weighing up the advantage of bringing in one species versus what’s already there. Rebelo stated, “We don’t want to restore a species only to drive another species towards extinction, so it’s all very difficult. The results of what we are doing will only be seen in two or three generations.” Hitchcock is pleased with the results of the E. verticillata reintroductions so far; plants have been established at all sites and are producing seed, and next generation recruitment has been observed at Rondevlei. A milestone will be the successful germination and recruitment of plants at Tokai once the prescribed burns have taken place. The percentage of seedling establishment at Rondevlei was relatively low so it will be valuable to assess post-burn recruitment at Tokai. In the future Hitchcock wants a genetic analysis performed on additional plants recently acquired from other gardens in Europe. These plants are phenotypically identical to the form originally brought from Vienna. However, without a thorough

56 genetic analysis, he can’t be sure if they are in fact exactly the same genotype. In the meantime these plants are taking up valuable space in the Kirstenbosch nursery. After the prescribed burn (Figure 4) in 2013, a strange phenomenon was observed at Rondevlei. Instead of being completely destroyed by the fire, as were most species, the adult plants remained intact, with completely preserved structure (Figure 5). It is believed that the plants might contain a type of flame retardant and that the skeletonized plants might actually protect the newly recruited seedlings as they develop. Hitchcock said this is theoretical, but may point to the hypothesis that this species used to occur in dense colonies rather than as isolated specimens. Hitchcock said it’s a factor that will be taken into consideration in further reintroduction efforts. Hitchcock described his reasons for continuing to pursue conservation and reintroduction of rare species. In summary, Hitchcock stated that people should move away from the notion that plants need to have a medicinal or sustenance value to have value. Plants and animals are not here solely for human purposes. Hitchcock feels that if monetary potential is seen as the prime reason for a species’ existence, then we have lost our value system. Hitchcock went on to say, “If it doesn’t matter that certain things go extinct, then it doesn’t matter that anything goes extinct. It’s all about having a value system. If we don’t value anything, we are not going to have any value for each other as humans.” Another advantage shared by several other rare species in horticulture is Erica verticillata’s visual appeal. Its size and bold color increases its status as a flagship species for other less horticulturally appealing species that are equally as rare. “It’s the panda bear of the Fynbos plant community,” says Hitchcock.

Figure 4 Specimens of Erica verticillata during an intense fire at Rondevlei Nature Reserve, South Africa, 2013. Photo credit Dalton Gibbs.

Figure 5 Erica verticillata plants retaining leaves and flower color after a prescribed burn, Rondevlei Nature Reserve, South Africa, 2013. Photo credit Dalton Gibbs.

58 Fairchild Tropical Botanic Garden

The Fairchild Tropical Botanic Garden is located in south Florida and has a subtropical climate, with frost occurring rarely. Its mission is “To save tropical plant diversity by exploring, explaining and conserving the world of tropical plants; fundamental to this task is inspiring a greater knowledge and love for plants and gardening so that all can enjoy the beauty and bounty of the tropical world” (Fairchild Tropical Botanic Garden 2015). The Fairchild conservation program focuses on plant species native to southern Florida as well as the Florida Keys, and has resulted in the reintroduction of 19 species of plants in the last 22 years. Dr. Joyce Maschinski, coordinator of the South Florida Endangered and Threatened Flora Program, was interviewed in person to discuss reintroduction efforts of Consolea corallicola and Pseudophoenix sargentii.

Consolea corallicola

Apart from Dr. Maschinski, Dr. Vivian Negron-Ortiz and David Bender from the U.S. Fish and Wildlife Service (USFWS) were also interviewed as both have experience managing populations and studying the pollen biology of Consolea corallicola. Pertinent parts from their interviews are included in the following interview summary with Maschinski.

Figure 6 A close-up of the flower of Consolea corallicola.

As discussed with Maschinski, Consolea corallicola (Figure 6) is currently one of the most endangered plants in the U.S., and it is restricted to coastal areas in southern Florida and the Florida Keys, USA. Its threats include rising sea levels due to climate change, hurricanes, destruction from the invasive South American cactus moth, Cactoblastis cactorum, attack from an unknown fungal pathogen (Possley et al. 2004) and a lack of genetic diversity. Additionally, all plants are said to be female sterile, with only the male parts producing fertile pollen (Cariaga et al. 2005). Plants do produce fertile seed, but it is thought to be apomictic and doesn’t germinate in situ (Negron-Ortiz 2014). Instead plants propagate vegetatively from fallen pads. Only a

60 few populations remain on the Florida Keys, with an introduced population on higher ground in the upper Florida Keys. The goal for reintroduction is to increase the number of populations within the species’ historical range.

Current Management Maschinksi and Negron-Ortiz were asked about the contemporary work with Consolea corallicola to date. Random Amplified Polymorphic DNA (RAPD) analysis has been used to test for genetic variance in this species, resulting in the identification of 14 genotypes. These have been used in reintroduction work and tests for resistance to Cactoblastis cactorum (Stiling, personal communication 2014; Jezorek et al. 2012). However, an Inter Simple Sequence Repeat (ISSR) analysis showed that there is very little genetic diversity in the two remaining populations, with one extirpated genotype (with six different alleles) being represented in the plants at Fairchild. Even though ISSR is generally a better method of determining remaining genetic diversity within a population (Cariaga et al. 2005), the lineages identified by RAPD are currently honored and kept separate at Fairchild.

62 Future Management

Even though there is little genetic diversity in Consolea corallicola, Maschinski considers the mixing of genotypes identified with RAPD a means to maintain the current level of limited genetic diversity. Until the unknown fungal pathogen is identified, Maschinski isn’t confident that the conservation efforts for this species have been effective. Bender suggests the best thing that can be done now is to increase both the number of populations and the extent of representation within the species’ historical range. When the concept of assisted migration was posed, Bender had the following to say:

I am spearheading the ongoing assisted migration efforts that, based on current sea level rise projections, will preserve the species from sea level rise (at least for our lifetimes) by establishing populations at higher elevation sites within the species’ historical range (Bender 2014).

According to Negron-Ortiz, climate change is still very unpredictable, and assisted migration may lead to the displacement of other species, however, the concept is slowly being accepted even though it might not make sense for every species. Considering the current pollination biology of this species, a breeding project with another species was posed. Bender thought this would be interesting, but he indicated that every plant in the Swan Key C. corallicola population should be surveyed to find a functional female, before this would be considered by USFWS. After the embryology work on this species, Negron-Ortiz also considered a breeding program as a possibility. Maschinksi would consider a breeding program, as there is no other course of action at this stage, other than perpetual vegetative propagation.

63 Only the pollen of C. corallicola is viable (Negron-Ortiz 1998) so if this project were attempted it could only be the pollen donor. According to Bender, an ESA10(a)(1)(A) permit would need to be issued by the USFWS, and would stipulate that the breeding program take place at a suitable distance away from wild populations in order prevent contamination of wild populations with pollen from a congener. Genetic modification to increase the diversity of C. corallicola was proposed to the three interviewees. Bender states that he is not against the concept in principal, but viability is largely a question of economics. Both Maschinski and Negron-Ortiz agree that current methods are more applicable at this stage, with Maschinski adding that she personally wouldn’t genetically alter the plants. Additionally, Bender was asked about the feasibility of chemical mutagenesis. He responded by saying, “I suppose it is worth a shot. I am familiar with the process.”

64 Pseudophoenix sargentii

As discussed with Maschinski, this is another species (Figure 8) restricted to one population in the Florida Keys several reintroduced individuals; and other populations in parts of the Caribbean islands. The population in Florida is threatened by natural disasters (e.g., hurricanes), climate change and sea level rise, predation from herbivores such as the Mexican Red-bellied Squirrel, low genetic diversity, lack of mature individuals (Fotinos et al. 2015) and an unidentified pathogen. Previous threats include illegal collection of plants for the nursery industry, and habitat destruction due to residential development. The populations are now protected in nature preserves and illegal collection has subsided. The goals of reintroduction are to improve age structure in the population and to maintain and increase the genetic diversity of those populations.

Figure 8 Pseudophoenix sargentii growing in the nursery at Fairchild Tropical Botanic Garden, Coral Gables, Florida, USA.

Current Management

The seeds of P. sargentii are short-lived and if the mature trees disappear, there will be no subsequent recruitment – not until the young plants become mature (Dickie et al. 1992). Work on improved seed storage is ongoing at the USDA Plant Germplasm Preservation Research Unit in Fort Collins, Colorado but little information is available about the management of the Caribbean populations. It is speculated that the Mexican Red-bellied Squirrel feeds on young plants but has since been controlled (Pernas and Clark 2011). Planting slightly older plants and protecting smaller plants with cages also addressed this problem. Maschinski also

66 mentioned that plants grown under nursery conditions might actually be more palatable to herbivores when compared to naturally recruited individuals. Maschinski states that having plants in ex situ collections has preserved the genes that were extirpated from the wild by the Mexican Red-bellied Squirrel in the 1980s. Due to this unexpected occurrence, Fairchild has been able to reintroduce lost genetic diversity back into the natural populations (Fotinos et al. 2015) An unknown pathogen is present in plants at Fairchild. It invades the meristem, eventually causes death, and is a primary reason for halting current reintroductions of this species. However, according to Maschinski, this pathogen has now been documented in the wild population. She is unsure whether or not this pathogen has been observed in the Caribbean populations or if some resistance might exist in those populations. Due to the length of time it takes for plants to reach maturity this pathogen could have a serious impact if some method of control is not established quickly.

Future Management

With greater resources, Maschinski would like to include the Caribbean populations in a complete species analysis. While it might be entirely feasible to bring germplasm from the Caribbean populations, Maschinski agrees that experimentation is first needed to prevent issues like outbreeding depression. Even so, due to the species’ long life cycle from seedling to fruiting, this will require decades of research efforts. According to Maschinski, it was discovered that plants growing in nurseries where cultivation practices are optimal can produce seed in greater numbers with higher

67 viability than plants growing under impoverished conditions in the wild. This could be a useful way to produce good seed for reintroduction or long-term storage. Maschinski stated that because artificial selection for garden conditions could occur in post generations, seed should be collected from the individual grown from wild collected seed and stored or used for reintroduction. According to Maschinksi, due the time it takes for plants to reach maturity and economic reasons, genetic modification of any kind would be out of the question at this stage.

68 Missouri Botanical Garden

The mission of the Missouri Botanical Garden (MOBOT) is “To discover and share knowledge about plants and their environment in order to preserve and enrich life” (MOBOT 2015). This institution is well known for its plant conservation programs, which form a vital part of its mission. MOBOT works within the framework of the GSPC (as adopted in 2002) adopted by the United Nation’s Convention on Biological Diversity (CBD) to halt the loss of genetic diversity worldwide. Information on Astragalus bibulatus (Figure 9) is assembled from an interview with Dr. Matthew Albrecht, the assistant curator of conservation biology, and Dr. Quinn Long, an ecologist/botanist, both of MOBOT.

Figure 9 Mature flowering plant of Astragalus bibulatus reintroduced to the Cedar Glade Habitat in Tennessee, USA, 2014. Photo credit Ashley Morris.

Astragalus bibulatus

This perennial, leguminous species is sparsely populated in specific rural habitats of Tennessee, USA. It is threatened by habitat destruction and low genetic diversity, and reproduces solely from seed. The long-term goal of the A. bibulatus reintroduction work is to increase the number of viable self-sustaining populations in the wild. The ultimate measure of success for this species’ recovery will be the removal of its name from the U.S. Endangered Species List.

Current Management

Albrecht and Long stated that taking an experimental approach to the reintroduction of A.bibulatus informed an adaptive management plan for the species, which helped to gain additional knowledge for the next round of successful reintroductions in following years. This included adapting to threats of herbivory of reintroduced propagules (Figure 10). This led to the following statement by Long,

We now have a much better idea of what target habitat should be, not just for this species, but also for restoration management of the broader limestone glade natural community within which the species occurs (Long 2014).

According to Albrecht and Long, fresh seed is collected from wild populations nearly every year, added to MOBOT’s seed bank, and sown at Missouri Botanical Garden MOBOT for reintroduction purposes (Figure 11). These reintroductions

70 contain the combined genetics from all populations, as mixing has not been deemed detrimental to genetic diversity of the species. Albrecht and Long stated that recently produced seed has less genetic diversity compared to previous years, indicating possible inbreeding depression. Research continues to evaluate the genetic status of both existing and reintroduced populations to highlight any developing trends, and the reintroduction protocols will be adjusted accordingly.

Figure 10 Astragalus bibulatus being reintroduced to the Cedar Glade habitat by Dr. Matthew Albrecht (right) and Dr. Quinn Long (Left) in Tennessee, USA, 2012. Photo credit David Kennedy.

Figure 11 Astragalus bibulatus plants propagated from seed in a nursery setting at Missouri Botanical Garden, St. Louis, Missouri, USA, 2012. Photo credit Dr. Quinn Long.

Future Management

Research continues to evaluate elements of herbivory, seedling recruitment and genetic diversity of reintroduced populations in order to guide future reintroduction efforts. Albrecht and Long hope that natural populations remaining dormant in the soil seed bank in other parts of the Cedar Glade Habitat may be uncovered by the use of fire. This is could bring Albrecht and Long closer to achieving their goals of increasing the population numbers and genetic diversity. Long had the following to say,

I suspect with most rare species, our estimate of rarity is enough to clearly state that a species is in fact very rare, however I would imagine there are few instances in which we truly know every single population.

72 All populations on private land that no botanist has or ever will step foot on are most likely going to be undetected (Long 2014).

The question of whether or not Albrecht and Long approve of managed relocation was asked with the following responses,

I think that we have to exercise a great deal of caution in doing so, it’s certainly not a strategy we can use for every endangered species, I think it’s something we are going to have to use in some cases for climate vulnerable species (Albrecht 2014).

The whole discussion really underscores the importance of maintaining genetically diverse seed bank collections, so we have the capacity to make these decisions in the future, because for many of these species we don’t really know their adaptive capacity in situ. We don’t know what their response will be to the altered climatic environment that they will experience in their present geographic distribution (Long 2014).

When asked for their views on genetic modification and genome editing for conservation purposes, Albrecht stated that if another species’ genes are being used in the alteration then that is probably contrary to the principal of plant conservation of rare plants. Both researchers agreed the A. bibulatus would not be a candidate for such work. However, Long closed the topic with the following statement,

I suppose [it may be beneficial] if there were genetic recombinations done in a way that were utilizing alleles present in the populations but making new combinations of those, rather than introducing genes from totally different species. Using genetic engineering to somehow create allelic combinations that ought to be present if inbreeding depression were not at play, is something to think about (Long 2014).

73 American Chestnut Research and Restoration Projet

The goal of this project is to produce genetically modified blight-resistant American chestnuts (Castanea dentata) and reintroduce populations of these resistant trees back into forest ecosystems of New York and later the rest of the eastern United States. Dr. William Powell, the Co-director of the American Chestnut Research & Restoration Project, was interviewed about his work on a project using genetic modification to enhance fungal resistance in C. dentata.

Current Management

Powell stated that many efforts are being made to conserve C. dentata, including a hybridization project through the American Chestnut Foundation (ACF) using the Chinese chestnut (Castanea mollissima) (The American Chestnut Foundation 2015). Powell indicated that even though some progress has been made hybridizing the C. dentata with C. mollissima, the resulting progeny are not guaranteed to be blight resistant. The progeny will also never be pure C. dentata as thousands of genes from C. mollissima are required to achieve blight resistance. Powell’s recent breakthrough was made using genetic modification (GM) as a means to introduce resistance into a C. dentata individual. The process basically involves the insertion of a gene from a wheat plant (Triticum sp.) into C. dentata embryos using a bacteria Agrobacterium sp. as the transferring agent. This gene then produces an enzyme that breaks down the harmful oxalic acid that is produced by the fungus.

74 The public, says Powell, has generally been receptive to this project as long as the research is thoroughly backed up with facts and reasoning. Powell stated that a majority of the population agrees that we are ethically responsible for bringing C. dentata back by any means possible. Powell thinks that with more education any doubts and concerns about the approach can be addressed, but he understands that there will always be some form of opposition. Powell says that as long as all the necessary tests are performed in order for the product to be accepted and passed through the regulatory agencies, public fears tend to be addressed. All products genetically modified in this way essentially need to pass through the Food and Drug Administration (FDA), the Environmental Protection Agency (EPA), and the U.S. Department of Agriculture (USDA), which could take up to five years or longer.

Future Management

There was concern from the American Chestnut Foundation (ACF) that their 25 years of work would be eclipsed by the work of Powell. Powell is positive that the two methods could complement each other, with the Chinese genes eventually being weeded out by natural selection. Unfortunately, it could require much more time to ultimately develop pure C. dentata populations. On the other hand, Powell is hopeful that some of the lost C. dentata diversity could be present in the makeup of these C. dentata x mollissima hybrids. In summarizing his approach, Powell explained that genetically modified individuals (genotypes) can be crossed to clones of wild individuals (genotypes) that are flowered prematurely in the laboratory. Through a genetic assay the seedlings can be tested for the presence of the blight resistance gene. Due to the dominance of the

75 gene, all trees that contain it will express resistance. After three to four rounds of outcrossing, Powell hopes to produce trees that are homozygous for the resistance gene that can be used to reintroduce populations of self-perpetuating blight resistant trees back in the wild. When comparing examples from other GM projects (Mellon and Gurian- Sherman 2011), Powell stated that although their C. dentata work has taken 25 years, it has been accomplished on a small budget. As he and Chuck Maynard, co-director of the American Chestnut Research & Restoration Project, experimented with various methods, goals became a lot easier to achieve and the relative costs lessened over time. Powell thinks that their experimentation should lead to a compounded reduction in costs for other species in similar situations, as well as a reduction in the amount of time needed for success. In conclusion, Powell stated that the C. dentata project will serve as a model system. Once it is passed through the regulatory agencies and the research demonstrates that this will benefit our forests, any fears should be alleviated. Powell stated that it is going to be very important for other tree species that are affected by exotic pathogens. Powell was also asked to discuss the practicality of other types of manipulation of plants to alleviate low genetic diversity. When the topic of chemical mutagenesis was posed as a way to initiate diversity, Powell thought this method might likely result in random mutations, usually with a low occurrence of valuable results. Referencing the Long Now Foundation, Powell thought it might be possible to recover lost genetic diversity from DNA of specimens preserved in herbaria or similar institutions. He stated that if this were feasible, it would be one of the best ways to

76 increase diversity for genetically depauperate plant species. This view was also supported by Voytas (2015). Powell was asked about using polyploidy as a means to increase fitness and vigor of species to benefit reintroduction efforts. Powell said this might be applicable for species with a lack of vigor, but that experimentation and thorough testing is needed before anything is released to the wild.

77 Chapter 5

DISCUSSION

Low genetic diversity has been defined as a loss of the allelic variability or genetic characteristics in the genetic makeup of species (Young et al. 1996). This is due to a loss of genes across populations caused by one or several possible factors. These include impacts by man, invasive species and climate change. However, Wisser (2014) and Husby (2014) both agreed that the impact of low genetic diversity on a particular species might vary widely. If any of these symptoms: inbreeding depression, low fecundity, low recruitment, genetic drift, decreased fitness, or loss of vigor is present, then low genetic diversity is usually the cause (Young et al. 1996; Neale 2012; Frankel et al. 1995; Reusch et al. 2005). Some species have low genetic diversity but do not seem to suffer from any of the consequences usually attributed to low diversity. Husby (2014) provided the example of the Wollemi pine (Wollemii nobilis), which has very little remaining genetic diversity, but is still capable of producing fertile seed. A well-known example in the animal kingdom is the Albatross. Two species, Diomedea exulans and Diomedea amsterdamensis, show high breeding rates and high fitness despite their extreme low genetic diversity (Milot et al. 2007). Furthermore invasive species usually begin with a very low genetic base and yet thrive and become invasive, lacking issues usually attributed to low genetic diversity (Husby 2014). However this success can usually relate to asexual reproduction (Zimmerman et al. 2010; Ren et al. 2005), or adaption for self-pollination (Price et al. 1981; Rodger et al. 2013). Therefore, to assume that all species with low genetic diversity would

78 benefit form the same management practices would be inaccurate. This thesis research found that out of 77 respondents involved in reintroduction work, 44 (56%) are reintroducing plants species that are thought to have low genetic diversity. Symptoms of low genetic diversity are not yet evident in the Erica verticillata, Pseudophoenix sargentii and Castanea dentata highlighted in the case studies, but the genetic diversity is known to be low (Hitchcock 2015; Maschinski 2014; and Powell 2014). In anticipation of issues associated with low genetic diversity, action needs to be taken now to manage for genetic diversity in these species. Limiting the occurrence of deleterious symptoms requires that populations be kept at a certain size while preserving a suitable level of genetic diversity (Reed and Frankham 2003). However, the conservation community’s current understanding is that there is no “magic number” for all species. The Center for Plant Conservation recommends a minimum of 50 individuals (genotypes) to preserve and recreate a viable founder population (Albrecht and Maschinski 2012). Real world situations do not always accommodate this recommendation, but it remains an important aspect for successful genetic management. For E. verticillata, a viable founding population number was not considered and reintroduction took place as material periodically became available (Rebelo 2015).

Collection Management

Collection management was a common theme among research participants. If a species is still present in the wild, every effort should be made to represent a good proportion of the genetic diversity in an ex situ collection (seed or living plants). Since quantitative diversity can be a factor (Reed and Frankham 2001; Vitt and Havens

79 2004; Kramer and Havens 2009), collections should be made from a range of individuals (genotypes) with different phenotypic and genotypic characteristics. This applies to seed as well as vegetative material. Depending on space constraints and number of individuals remaining, one could have all known genotypes of a species in one collection. The case studies support this approach, specifically with Erica verticillata (Hitchcock 2015) and Consolea corralicola (Maschinski 2014). However, Husby (2014) added that if only a few individuals (genotypes) represent a species, they should also be represented in multiple locations as a backup measure to prevent loss. According to Hitchcock (2015) and Husby (2014) this provides insurance against loss due to disease outbreak, mismanagement, natural disasters, or extinction, which has been proven with Erica verticillata and Sophora toromiro. Material should be documented and curated with known provenance information, as practiced by 81% of survey respondents. If multiple genotypes of a rare species are represented in an ex situ collection, accessions could be confused, resulting from unintentional error, such as excessive propagation of more vigorous genotypes as mentioned by Maschinski (2014), mislabeling, or miscommunication during plant transfer activities between ex situ sites. Whatever the reason, inaccuracy and loss of provenance information can greatly diminish the reintroduction potential of an ex situ collection. As with Erica verticillata, each genotype was given a specific cultivar name (Hitchcock 2015). Collection management may be improved by assigning a unique identity to each genotype, which could dually serve to raise the prominence of the species to the public if they are sold in the nursery trade (Hitchcock 2015). Each of the eight genotypes of Erica verticillata has defining characteristics, which makes this procedure more applicable, but this may not be the case for all

80 species. It was concluded by the researcher that attaching cultivar names should be left to the discretion of the botanic garden. The assignment of unique genotype names to threatened species proposed by Nicholson and Maasch (2014) is in line with this idea of adding epithet information to certain individuals. In lieu of making changes to the International Code of Botanical Nomenclature, perhaps an ex situ collection could stipulate that an epithet remain attached to the specific genotype when it is shared and displayed in other botanic gardens.

Seed Banking

Having germplasm stored in a seed banks is currently one of the best ways of storing genetic diversity of orthodox species for an extended length of time. Fortunately, 60% of survey respondents reported the presence of seed banked material of their particular species in question. While this provides some insurance against further loss of genetic diversity or complete species extinction, it is not a permanent solution, as this stored material is unable to evolve in response to a changing environment. As indicated by Albrecht (2014) and Long (2014), banked seed of Astragalus bibulatus can be an important resource for reintroduction activities. Seed collection over multiple generations under cultivated conditions should be avoided when possible, as artificial selection for those conditions can occur fairly rapidly (Maschinski 2014). Albrecht (2014) and Long (2014) took this into consideration by only using seed from wild populations in their reintroduction program for A. bibulatus. When survey respondents were asked to list their own methods for improving genetic diversity, most responded with “mixing of seed sources”. This is good practice, as long as inbreeding risks and outbreeding depression have been assessed.

81 Outbreeding depression may not be problematic for species that have been fragmented via man’s impact (Maschinski 2014), which was cited by 82% of survey participants as reason for species decline. Seed source mixing may improve the genetic diversity of species that still have a more than one population left in the wild, but will not have much application to species with few individuals (genotypes) remaining. For species with recalcitrant seed, the National Center for Genetic Resources Preservation in Ft. Collins, Colorado, among others, is investigating improved methods for long-term storage (Maschinski 2014). In the meantime, the Montgomery Botanical Center and many other botanical gardens conserve species with recalcitrant seed in genetically diverse living collections (Husby 2014). For Erica verticillata, Toromiro sophora, Cosmos atrosanguineus and others, fertile seed production would be a big step in the right direction in terms of conservation. But in order for pollination experiments to be attempted, every effort should be made to locate all existing genotypes to maximize the level of genetic diversity (Wisser 2014). If pollination experiments are successful, a portion of this seed should be banked before reintroduction is attempted. Erica verticillata was rediscovered in collections in Europe nearly 50 years after it was considered extinct in the wild (Hitchcock 2015). These collections held some diversity, which has led to ex situ and in situ seed production after reintroduction to areas close to its native range. It is important to consider whether or not plants have been perpetually asexually propagated since they were collected from the wild, as exemplified by Consolea corallicola (Maschinski 2014), Cosmos atrosanguineus (Hind and Fay 2003), Lotus berthelotii (Owens 1985), Erica verticillata (Hitchcock 2014) and Encephalartos woodii (Gorelick and Osborne 2002), or produced from seed at some

82 stage during their existence under ex situ conditions, such as Franklinia alatamtaha (Owens and Rix 2007), Kokia cookei (U.S. Fish and Wildflife Service 1997) and Pseudophoenix sargentii (Maschinski 2014). Plants perpetually propagated from vegetative material (e.g. Erica veriticillata; Hitchcock 2015) or highly inbred (e.g. Kokia cookei; Sugii 2015) may be or become less fertile or even infertile over time. It is worthy of consideration, especially for species like E. verticillata, where the reintroduction of certain genotypes may not be viable due to sterility (Rebelo, Hitchcock 2015). C. atrosanguineus is an extreme case, as all cultivated material in Europe and the U.S. was produced asexually from the original genotype and is self- sterile (Hind and Fay 2003). Even though minimal seed has been produced artificially, all cultivated material of L. berthelotii originates vegetatively from the same genotype and is self-incompatible (Owens 1985). Other methods of preservation (i.e., cryopreservation and tissue culture) can store genetic material for extended periods of time, and are valuable if seed is not available or compatible to bank (Pence 2010). These methods require less space than a living plant collection and serve as a means of preservation while other possibilities for conservation are explored. Such methods, however, are still being developed, as nearly every species requires slightly different conditions in order to survive. Cryopreservation is also still relatively expensive (Pence 2010), yet four survey respondents indicated its use in their conservation efforts, which is promising. Tissue culture is also used to germinate difficult seed or in embryo rescue techniques, as reported for Kokia cookei (Sukii, 2015).

83 Pollination Experiments

As reported for Erica verticillata, Cosmos atrosanguineus, Sophora toromiro, and many others with a handful of genotypes remaining, pollination experiments in a pair-wise fashion should be attempted (Wisser 2014). Using Erica verticillata as an example of a bisexual out-crosser, the following could be attempted with other species. According to Hitchcock (2015), there are thought to be eight genotypes ex situ with varying levels of fertility suspected. One would need to make 56 crosses, including reciprocal crosses, in order to include every possible combination of genotypes. Through this practice, any genotypes that are weakly fertile, completely sterile or female or male sterile could be identified. Completely sterile genotypes could be removed from future reintroduction attempts, but remain as display plants in an ex situ collection. The reason for sterility needs to be determined, but it could lead to that genotype being a non-viable part of a reintroduction attempt. Certain genotypes may be incompatible, and could be kept apart at the time of reintroduction. If the original number of genotypes is greater than 15, the number of crosses required may go beyond the capacity of the garden or staff involved, but for a small number of genotypes, this method could be a feasible approach to managing for genetic diversity. This method of crossing in a pair-wise fashion is the best way to increase diversity for a species (Wisser 2014). New variability is not being created per se, but the available information is being stacked in new combinations. In an ideal situation, crossing the eight Erica verticillata genotypes in this fashion would lead to the production of 56 genetically unique genotypes if you only raised one seedling from each cross. This all depends on the relationships of the original eight, but is the best way to produce a diverse a group of plants in a less than ideal situation. The seed of

84 these 56 crosses could then be used to produce plants for reintroduction or it could be banked. If feasible, and considering the pressures of artificial selection, one individual could be raised from each cross and the process could be repeated, however this would then involve 3080 crosses, a formidable and unrealistic effort for many institutions. With the Cosmos atrosanguineus and Sophora toromiro, one could feasibly progress through at least two generations of crosses ex situ due to their lower initial number; however, one must be careful not to select for ex situ conditions unintentionallly (Maschinski 2014). Additionally the fewer the number of founding individuals (genotypes), the more likely siblings will cross in each generation and the less likely there will be any significant recombination. The seed could also be banked at each filial generation, as it represents the maximum level of diversity possible at that point in time. Even if an outcrossing species is capable of self-pollination, this should be discouraged in the experiment, as self-pollination tends to fix certain alleles, which may be deleterious in a small population (Acquaah 2007). Plants that are naturally self-pollinating may not suffer from the deleterious effects of inbreeding depression and loss of vigor as much as naturally outcrossing species (Price et al. 1981; Rodger et al. 2013). Therefore, when a self-pollinating species is down to a low number, it will be more easily managed with respect to vigor and compatibility issues (Acquaah 2007). For example, Tecomanthe speciosa, a native of the Three Kings Islands off the coast of New Zealand, existed as one individual (genotype). The ability of this individual (genotype) to self- pollinate has enabled a future in horticulture and in its native habitat, and seedlings obtained from fruits show more vigor and are quicker to flower than plants produced from cuttings (Hunter 1967). One disadvantage is that if there is more than one population remaining, self-

85 pollinating species may be more susceptible to the effects of outbreeding depression if mixing of seed sources is attempted. Due to fixation of genes populations become quickly adapted to niche conditions, and if mixing of seed sources is absolutely necessary care must be taken if there is potential for cross pollination (Ellstrand and Elam 1993). Reintroduction practitioners need to know the pollination biology of their species because each type requires a different management approach. Obligate out- crossers and self pollinating species were well represented in the survey results, but a number of respondents (23%) don’t know the pollination biology of their species. These results are not surprising, however, nearly 70% of all respondents haven’t attempted hand pollination or were unsure of its results. Of those that had produced seed from hand pollination, only three individuals (30%) knew the viability of that seed. Knowledge about the pollination biology, pollinators and the seed viability of a species is essential for any form of reintroduction success. Pollination experiments both in situ and ex situ could help to determine these parameters for a species. Species that no longer produce fertile seed due to a loss of pollinators are challenging because they require artificial pollination to cause viable seed set (Kawelo et al. 2012). There is no long-term solution for this problem, unless plants become self-fertile or produce seed through apomixis. Wisser (2014), Husby (2014) and Voytas (2015) agree that genome sequencing is becoming less expensive and could be effective in the conservation management of small populations in particular. According to Wisser (2014), once the genome has been sequenced, it is relatively easy to develop assays using SSRS (short sequence repeats) to show relatedness of different individuals in a population. This could be

86 very applicable when dealing with small populations, especially when trying to develop a conservation-breeding program. It could identify closely related individuals, so that a breeding plan could be created for plants as is done for animals in zoos.

Monitoring

According to survey results of this study, a majority (61%) of respondents have been reintroducing their particular species for one to ten years, and that many of these projects are still in their infancy and more time is needed to establish any level of long-term success. The data illustrates that 58% of respondents reported that their projects are successful or soon to be successful and were asked to list their reasons for success. The 39% that were unsure felt it was too early to tell. The reasons for success included the following: the plants have survived, the plants are growing well, the population/s is/are growing, the declining population has stabilized and seems to be growing, plants have survived and set seed, and there has been recruitment over many generations without human interference. Most respondents (65%) cite success as a stable population with active recruitment taking place without human assistance. Even though recruitment may not be achieved within our lifetime for certain species, it is premature to state overall success or failure merely by assessing whether or not the plants are growing well. To develop viability models and predict population trajectories, populations should be monitored for decades according to new CPC reintroduction guidelines (Maschinski et al. 2012). To establish long-term trends, the effects of possible inbreeding depression, and assessment of population sustainability, 10 to 100 years of monitoring time is needed (Maschinski et al. 2012). According to survey results, the length of monitoring varies widely among respondents, varying

87 from zero (10%) to more than 20 years (16%). On a slightly more positive note, 29% of respondents reported plans to add new plants to the reintroduction site when necessary or possible. However this depends of the ecology of the species and the environment, as exemplified by Erica verticillata (Hitchcock and Rebelo 2015).

Site Management

An important discussion point with Rebelo and Hitchcock concerned the correct balance of management practices at Tokai. Specifically, the Tokai reintroduction site in South Africa was reclaimed from a large plantation of Pinus radiata, and besides the Erica verticillata, contains numerous species with different levels of conservation concern. Understandably, the site cannot be managed for the benefit of only one species, but there is still a slight contention between the parties involved. On the one hand the Tokai site is managed with minimum intervention from man, apart from prescribed burning every 10 to 15 years, to return natural ecosystem function as quickly as possible. After initially reintroducing populations, they will not augmented with any new material, unless a major problem arises. Examples include lack of recruitment after fire or death of all reintroduced plants. The purpose of this is to integrate the reintroduced plants into the ecosystem as quickly as possible.

According to Rebelo, augmentation after a couple of years could upset the delicate balance achieved since the initial reintroduction. However, the genetic diversity of these reintroduced populations are represented by only two genotypes (founders), with only one or two populations at Tokai represented by three genotypes (founders). The goal for these populations will eventually be prescribed burning with the hope of initiating seedling recruitment. It’s

88 to be decided whether or not new material will be added to those populations. The argument is that the available genetic diversity (eight genotypes) is not currently represented at any of these sites, and the fact that the soil seed bank that develops at the reintroduction sites over the next 10 – 15 years will represent the genetics of just two or three genotypes. This could lead to genetic problems in the future, which could be minimized if material of all available genotypes were introduced at the available sites. Considering the act of reintroduction is unnatural in the first place, and the fact that Erica verticillata was never historically recorded or thought to have ever occurred at Tokai, it is probable that the benefits of adding all the available genetic diversity to all sites would far outweigh the risks of upsetting whatever ecological balance may have developed thus far. In terms of the IUCN extinct-in-the-wild status being changed, perhaps the population at Rondelvlei could be left without augmentation for three successive generations, while additional plants (other genotypes) could be added to the other reintroduced populations. Erica verticillata also serves as a flagship for other species at Tokai that may go unnoticed by the general public, so having large populations of plants will help to further boost the conservation message. Albrecht and Long (2014) recommend employing reintroduction in an experimental capacity, as confirmed by Kennedy et al. (2012). Hitchcock stated that this approach has led to the discovery that the Erica may benefit from nurse plants soon after reintroduction, and that plantings in recently burned areas failed to thrive. However this discovery has led to further contention, because in an ecological sense, the reintroduction should take place as soon as possible after the fire as to limit disruption to the establishment of other species on site.

89 This research indicates that a balance needs to exist between management for the sake of a species and management for the benefit of the ecosystem as a whole. Each reintroduction project needs to be considered on a case-by-case basis. In some cases, certain practices may suit both the species and the ecosystem in general, and in others the detriment of the ecosystem may prevent certain action being taken for the benefit of the species.

Genetic Diversity

As already noted from the survey most survey respondents mix seed sources in order to increase the genetic diversity of their particular species. This approach has also been used for A. bibulatus (Albrecht and Long 2014), C. dentata (Powell 2014), and might be considered in the distant future for P. sargentii (Maschinki 2014). However if there are only a few genotypes remaining alive in the wild or in cultivation, every effort should be made to include all these genotypes in a conservation breeding program or reintroduction effort, as exemplified with the E. verticillata. This should not be limited to botanic gardens, as private collections have also been known to hold important germplasm, as in the case of C. atrosanguineus (Gorelick and Osborne 2002). As stated by Husby (2014) and illustrated by Hitchcock and Rebelo (2015), the importance of maintaining a strong network of botanic gardens and plant collectors cannot be over emphasized.

90 Advanced Methods of Improving Genetic Diversity

The ultimate goal of all this management and experimentation is to reintroduce a species back into the wild, and have that species perpetually reproduce without human intervention. Yet, the final question still remains, what could be done if a species, despite all management efforts, still has one or more of the following symptoms?

• Poor vigor

• Inbreeding depression • One remaining self-incompatible genotype • Susceptibility to a pathogen • No viable pollinators • Sterility of male or female parts or both

Hybridization

Although not the best strategy to preserve a pure species, hybridization has been used to try to save both plant and animals species (The American Chestnut Foundation 2015; Rebelo 2015; Johnson et al. 2010). The process involves crossing the critically endangered species with another related species with the goal of providing a benefit to the imperiled species, which may include, providing disease resistance, increasing the level of genetic diversity where none existed before, increasing vigor and overcoming incompatibility issues. Hybrids do occur naturally, especially where two species overlap. This can be advantageous, as the hybrid plants may be more fit to occupy that niche habitat (Schweitzer et al. 2002). However, they

91 may not be suited well to either of the paternal environments (Tauleigne-Gomes et al. 2008). It depends on how closely related the species are, and their genotypic and phenotypic characteristics. One of the more notable examples is the hybridization of the American chestnut (Castanea dentata) and Chinese chestnut (Castanea mollissima) trees (The American Chestnut Foundation 2015). The goal of this project is to produce “American” chestnut trees with a small component of C. mollissima genes to incur resistance to Chestnut Blight (Cryphonectria parasitica). According to Powell (2014), this project has been active for the past 25 years. Another example originates from South Africa where Encephalartos woodii is being hybridized with Encephalartos natalensis in order to add some much needed diversity to the genetically depauperate E. woodii, of which only male plants remain (Gorelick and Osborne 2002). Bender (2014) describes an introgressive breeding program that could be attempted with Consola corallicola, because all existing plants are thought to be female sterile. Consolea would have to be the pollen parent. This approach has also been proposed for Kokia cookei (U.S. Fish and Wildlife Service 1997). However hybridization is not without its challenges (Wisser 2014), which can outweigh the benefits. For example, for the sake of clarity, let’s call the imperiled plant, species A, and the plant with which we wish to hybridize it, species B. The main concern is that the hybrid will never be pure, even with multiple backcrosses to the species A; there will always be some level of genetic material present from species B. Another concern is trying to find a suitable congener as a parent. Species B should match species A as closely as possible in terms of phenotypic and genotypic characteristics, and habitat

92 requirements. Despite these considerations, as in the case of outbreeding depression, the hybrid/s may still not be adapted to the habitat of either species A or B, due to a mixing of genes adapted to different niche environments. As stated by Maschinski (2014), if you attempt an introgressive breeding program, you need accept that what you are ending up with is a hybrid. As stated by Wisser (2014), although repeated backcrossing to species A results in hybrids which are more similar to species A, this can result in a loss of the genetic diversity initially provided by species B in every round of back crossing. Backcrossing may also become more difficult as the hybrids become genetically closer to the genotype used from species A. This is especially true for species like the Encephalartos woodii and Kokia cookei, which only have one individual (genotype) remaining. The only other option is a series of intercrosses between the hybrids, but the percentages of species A versus species B in the genetic makeup of the progeny is difficult to predict. The American Chestnut Foundation promises the release of hybrid trees, nearly identical to the original American chestnut and resistant to Chestnut Blight. Even though some success has been achieved, this project has been running for over 25 years, and they also started with the luxury of more than one American chestnut tree. A project like this cannot be repeated with many other species due to their low remaining numbers and the time commitment required. Taking all this into account, hybridization is not recommended in the pursuit of saving a species. There are too many undesirable consequences, and the product will never be a pure species. However, if all other methods have been exhausted, the research indicates that saving the genetics of a species through hybridization is still better than having the species go completely extinct.

93 Managed Relocation

Managed relocation is a concept relatively new to plant conservation (Sax et al. 2009). Its main purpose is to pre-empt the effects of climate change; which will affect species incapable of adapting or moving into new areas (Haskins and Keel 2012). The advantages and disadvantages of this practice will not be discussed here, but certain elements of this practice relate to this thesis topic. For a full discussion on the concept of managed relocation see Maschinski and Haskins (2012). One of the disadvantages of managed relocation is the potential for introduced species to become invasive (Ricciardi and Simberloff 2009). However, other studies point to a lack of evidence that endangered species pose an invasive threat or cause extinctions (Sax et al. 2009; Reichard et al. 2012). In following the guidelines of the Weed Risk Assessment, as detailed by Reichard et al (2012), it is fairly unlikely that a relocated species will become invasive. Under the Endangered Species Act, species planted outside their natural range are designated as “non-essential experimental populations”, and this makes it lawful that a species be removed if it becomes ecologically harmful (Reichard et al. 2012). Some species are listed as endangered in their native habitat and yet have naturalized in other countries. Ixia viridiflora is one such example, which is threatened in its native South African habitat (South African National Biodiversity Institute 2012), but has naturalized in South Australia (WWF 2006). These naturalized plants could represent genetics of I. viridiflora that have long since disappeared from the South African populations and could be important in reintroduction work. The same can be said for Moraea aristata, which is critically endangered (South African

94 National Biodiversity Institute 2012), but has become naturalized in Australia (WWF 2006; Walsh, personal communication 2015). If an endangered species thrives in another country away from its natural environment, but does not pose a threat to the native flora of that country, could this be viewed as a positive conservation outcome? There is a high probability that Moraea aristata will become extinct in the wild in South Africa (South African National Biodiversity Institute 2012), and the material in Australia could be a resource for conservation and reintroduction purposes. This wouldn’t be viable for species with any invasive potential, but in the cases of K. cookei, L. berthelotii, C. corallicola, and C. atrosanguineus this could be feasible. The appearance of a fertile plant of C. atrosanguineus in New Zealand is a prime example of an unexpected occurrence far from a species native habitat (Hind and Fay 2003). Perhaps there could be some benefit in growing a species like K. cookei away from its native Hawaii. The best platform for an experiment like this would be a botanic garden or managed location where the species can be carefully monitored for any positive changes such as seed production and recruitment from seed.

95 Polyploidization

Due to polyploids being common in nature as a part of the evolutionary process, and the fact that there are numerous advantages to having a higher ploidy level (Comai 2005), there is merit for considering this process as a tool in plant conservation. A species with few individuals (genotypes) remaining could benefit from having a higher ploidy level. Through methods already explained (Jones et al. 2008), it could be possible to develop specimens from each of those individuals with a higher ploidy number. Most plants are naturally diploid and the level of ploidy of each individual needs to match in order for fertile seed to be produced. The plants that are produced from polyploidization are usually tetraploid (four sets of chromosomes) and confirmation can be achieved by using a flow cytometer (Brummer et al. 1999). Altering the ploidy level might have the benefit of increasing vigor or causing self- sterile organisms to become self-fertile (Comai 2005; Hörandl 2010), the latter example is important for species that have lost their pollinators. If raising the ploidy can’t be warranted in a natural situation, having an artificially produced polyploid example in a botanic garden might be beneficial. The specimen may be more vigorous and more easily cultivated, and could be used as an example to highlight the plight of the naturally diploid species. Artificial polyploidization of a species is considered by many to be unnatural, and that the resulting organism would be completely different from that of the original species (Maschinski 2014; Sugii 2015; Voytas 2015; Hitchcock 2015). Hitchcock (2015) retracted slightly, stating that as long the reason for creating polyploid individuals is given and backed up by plenty of research, it might be warranted.

96 Powell (2014), Husby (2014), Wisser (2014) and Foltas (2015) supported experimentation, as long as the resulting plants are thoroughly tested before being released into the wild. All three listed the benefits of polyploid organisms. In consideration of these views this research does not outwardly advocate the use of polyploidization to save species in peril, however considering all the advantages of polyploidy organisms, there is still potential for this process to have application. A polyploidy organism would have multiple copies of the original chromosomes. Polyploids have been documented to occur naturally during severe bottlenecks, and have also been known to revert back to diploids over time (Comai 2005). Polyploidization could be considered for those species having poor vigor and low seed production that won’t respond to the management already described.

Mutagenesis

Mutagenesis is another process that has been used in the agricultural industry but never in conservation practices. According to Wisser (2014) and Voytas (2015) even though the process is random and apt to have a lot of undesirable results, there is the potential for worthwhile changes to be made. An optimum result would be a widening of the genetic base of species with no genetic diversity, such as K. cookei, without compromising its health or viability. Due to the random nature of the mutations this process could potentially produce a functional female in both the E. woodii and C. corallicola. As the costs of genome sequencing continue to drop (Wisser 2014; Powell 2014; Voytas 2015), and the high-resolution analysis of useful mutations continues to improve, the process of mutagenesis could be applicable to the conservation of imperiled species.

97 Genome Editing

Recent technology has been developed where the genes within a plant can be deleted to express a desired effect (Voytas 2015). This was recently accomplished with Potato where the gene for the production of sweet sugars during storage was deleted. It is hoped that this will result in a product with a longer storage capacity. This type of work bypasses the stringent regulations imposed on genetically modified crops containing the genes of other organisms, as it is considered a form of mutagenesis, which is currently unregulated. This leads to less time between development and release to market for agricultural products. Voytas (2015) stated that in the future, scientists should be able to make multiple simultaneous changes to an organism. That being said, these newer developments might have more applicability to plant conservation than any of the methods discussed. Genetic editing is more directed and precise, with less random mutations being produced. Due to the process currently being unregulated, the release of an edited species could be achieved in significantly less time and with less cost than a genetically modified species such as Castanea dentata. Whether this technology is adopted into conservation or not depends on expense, institutional capacity, future regulation and public acceptance.

98 Genetic Modification

Genetic modification is a process familiar to many individuals, with a history of praise and controversy (Benbrook 2012; Ando and Khanna 2000). Unfortunately we don’t know enough about how humans assimilate genetically modified food to establish if the products are detrimental to our health. There are so many aspects to consider and so much false information from both sides is being presented in the media (Katiraee 2015; Greenpeace International 2013). Pamela Ronald has a convincing argument on how genetic modification can move forward together with organic farming methods, resulting is less pesticide use, and less monoculture. She also envisions a future not dominated by Mosanto-like corporations, but by small partnerships between farmers and scientists (Ronald and Adamchak 2008). To give an opinion on genetic modification is outside the scope of this study, however genetic modification is still happening, whether we like it or not. Castanea dentata is the first species that has been genetically modified for conservation purposes. Through the American Chestnut Research and Restoration Project, the aim is to produce blight resistant chestnuts and to eventually reintroduce a population back into the forest ecosystems of New York and the rest of the eastern United States. The process of genetic modification with this species has taken a lot of dedication, but as shown promising results. As Powell mentions, this work will serve as a model for other conservation projects once the EPA, FDA and the USDA have approved the trees. Powell and colleagues (2014) have also covered their bases in determining the effects of this genetically modified organism on the micro flora in the soil, the animals dependent on the chestnuts as a food source, and the impacts of the chestnut on humans.

99 Opposition to this project by organizations like the Global Justice Equality Project present inaccurate information and jump to irrational conclusions before the research has been completed. Their campaign against the genetically modified American chestnut states that, “…this engineering damages the Chestnut genome and leads to numerous mutations” and that “The move to GM trees will destroy forests and expand unsustainable land use” (Global Justice Ecology Project 2015). Even though this information is unsubstantiated, it could still lead to unnecessary public fears. In agreement with Wisser (2014), Powell (2014) states that as we progress with projects like this, genome sequencing and genetic modification will become cheaper and more accessible to a wider range of institutions. Perhaps Consolea corallicola could benefit from resistance to the fungus affecting it in Florida or attack from Cactoblastis cactorum? While Hubsy (2014) doesn’t think that genetic modification is a viable option right now, due to the expense, perhaps it will be more widely used in the future. He mentions genes that could be used in a multitude of species, providing increased vigor, or tolerance to altered soil conditions. However, if these genetically modified trees are released, they will be superior over those from the traditional breeding program with the Chinese chestnut. Powell (2014) hopes the results from each project will be complimentary, however the researcher feels that the negatives outweigh the benefits. It is difficult to discredit the work of a foundation that has been working towards a goal for over 25 years, but if the genetically modified Chestnuts are approved the Chinese American hybrids will effectively be obsolete. Even though these Chinese American hybrids may contain some lost American genetic diversity, the unwanted Chinese characteristics will continue to pollute the gene pool for many years to come. The concern is that the

100 height, width and nut size of the Chinese chestnut do not fill the niche left behind by the American chestnut. Once hybrid trees and genetically modified trees are released it is unclear which genes will dominate and which genes will become recessive and disappear in successive generations. Having Chinese genomes present in the wild could also infiltrate and affect the end goal of having genetically modified chestnuts that are homozygous for the resistance gene. It is recommended that Chinese American Hybrids not be planted in wild areas, unless the genetically modified trees are not approved, or fail to live up to expectation. The current research shows that having a self-perpetuating population of American chestnut trees with one gene from a wheat plant is more favorable than having a population of Chinese American hybrids with varying levels of diversity, fitness and phenotypic characteristics. At this stage most of the individuals interviewed for the case studies don’t think that genetic modification has much application to species that have less economic value and status than that of the American chestnut. Perhaps other poorly known species will be more suited to new technology known as gene editing, as it may be considered less invasive and potentially less expensive.

101 Conclusions and Recommendations

As indicated by this research, endangered plant species are very specific in their individual requirements for survival and there are numerous factors to consider for successful genetic management and reintroduction to take place. Through careful study of the biology, phenology and characteristics of each species, there are means of assessing and managing genetic status to maximize the available genetic diversity and reintroduction success. The continued development and decreasing expense of genetic engineering technologies will expand their use to conservation work, but increased education is essential in achieving broader acceptance of these tools for conservation purposes. Given what is already known and the information derived from this research, the author proposes the following reintroduction guidelines unique to species with issues related to low genetic diversity. These guidelines could also be applied to other species as well.

1. Determine the available live germplasm (plants in the wild, plants in ex situ collections, banked seed) and determine the level of genetic diversity remaining.

If the genome of the particular species has been mapped, it is possible to determine the level of genetic diversity in the remaining individuals. In lieu of genetic studies, pollination experiments can determine fecundity in outcrossing species. It is important to know the genetic baseline of the founder population in order for the genetic diversity to be monitored in subsequent generations.

102 2. Using guidelines from the CPC, aim for a founder population of at least 50 individuals (genotypes). This serves as a rough guideline, since the appropriate population number is very species specific. For species with fewer than 50 individuals (genotypes) remaining, this number serves as a suitable goal.

3. In the case of a species with one population or fewer, one should attempt to locate ALL available germplasm, including vegetative material in collections in other parts of the world, and any banked seed. A maximum amount of diversity ensures a greater chance of successful reintroduction. 3.1. Where two or more populations remain, outbreeding depression must be considered before mixing germplasm. This is especially true of species that self-pollinate. Evaluation is very much case-by-case, but populations that were historically separated by man generally shouldn’t suffer the effects of outbreeding depression when mixed. In cases where only a few individuals remain in each population, the risks of outbreeding should be negated and cross-pollination between populations should be attempted.

4. The pollination biology of the particular species needs to be determined. Species with obligate out-crossing, self-pollination, and asexual reproduction need to be managed differently under ex situ pollination experiments. The extinction

103 of a pollinator can have severe impacts on the success of a reintroduction. Hand pollination may need to be employed as a management tool.

5. Where possible pollination experiments should be performed to determine fecundity and relatedness of each genotype. This is limited by space constraints but is especially important when a handful of individuals (genotypes) remain.

6. The value of sterile genotypes or genotypes incapable of reproducing by any means needs to be determined before including in reintroduction efforts. These genotypes can remain as display plants in an ex situ collections, but may not have value for reintroduction unless they regain fertility or reproduce asexually.

7. Pair-wise crossing experiments should be attempted as appropriate. In order to ensure that every possible combination is made and the seed produced has the maximum amount of possible genetic diversity, these experiments should take place in a controlled ex situ environment, free from external pollinators. This is the most natural way of producing enough plants to form a viable founder population of 50 individuals (genotypes).

8. Pair-wise crossing should continue as long as space, time and ex situ selection pressures are taken into account. Institutional capacity and selection pressures from ex situ conditions can limit the number of generations that can be produced.

104

9. Seed/vegetative material from each generation should also be banked/preserved as an insurance against unintended disaster. Examples include disease outbreak, poor management of collections, unexpected climatic events, and herbivory. All of these examples can occur in an ex situ collection or after plants are reintroduced into the wild.

10. Where possible species should be reintroduced within their historical range. This may not always be possible due to human and climatic impacts. 10.1. Threats must be eliminated, managed or avoided before reintroduction to an historical range. 10.2. Managed relocation or chaperoned migration should be considered where suitable native habitat has disappeared or climate change has had an impact. These sites should be as close as possible to the historical distribution, or in an area that has conditions similar to that of original distribution.

11. Reintroduction should always be undertaken with an experimental approach. This informs future reintroduction work and makes room for error initially.

12. Monitoring should continue until a species is naturally self-perpetuating, and any instances of inbreeding or outbreeding depression are remediated. This may be difficult to achieve easily with species that are long lived and take time to produce seed.

105

The following circumstances might prevent pollination experiments from having any success. Recommendations, starting with the most practical to the least practical follow under each condition.

Remaining individuals lack vigor in the wild

• If more than one population remains, mixing may lead to beneficial heterosis (vigor).

• Plants could be grown at institutions in other countries under controlled conditions to determine whether or not vigor improves.

• If this is impractical or if only a few individuals remain, the process of polyploidization could be attempted. Individuals would need to be carefully monitored for stability.

• Hybridization with a congener could be attempted in order to restore some vigor to the imperiled species.

Remaining individuals are already highly inbred

• Chemical mutagenesis could be used to induce much needed diversity. As with polyploidy, the plants will need to be monitored for stability or deleterious mutations.

• In the future genome editing might provide some means of developing diversity so that closely related individuals could suddenly become compatible without the deleterious effects of inbreeding.

106 • Hybridization with a congener will increase diversity, but at the expense of the phenotype and genotype of the imperiled species.

Only two genotypes remain

• Pairwise crossing in this instance won’t result in much diversity and all the progeny will be full sibs. Results do depend on the genetic makeup of the plant though. If pairwise crossing does result in some diversity, progeny can be further intercrossed or back-crossed.

• Polyploidy may induce some vigor and or make each genotype self-fertile, which could provide some more diversity to a pair-wise cross experiment.

• Chemical mutagenesis or the emerging idea of genetic editing could be attempted to provide more diversity.

Remaining genotypes are completely sterile, or male or female sterile

• Propagation of remaining genotypes and dispersal to multiple back-up locations.

• Plight of the species should be publicized as an example of a species on the brink of extinction.

• Polyploidy could restore fertility to sterile genotypes. • Chemical mutagenesis could potentially create fertile mutations.

Remaining plants are affected by a pathogen in the wild

• All available genotypes should be tested for resistance.

107 • Plants could be reintroduced into areas where the pathogen is not known to occur.

• Living collections representative of the existing genetic diversity could be grown in other parts of the world where the pathogen is not present.

• Individuals could be genetically modified to incur resistance against the pathogen.

• Individuals could be hybridized with a congener to incur disease resistance.

The only pollinator is extinct

• Propagation of remaining genotypes and dispersal to multiple back-up locations.

• Conduct hand pollination projects every year in the wild. • Observe plants in botanic garden collections in other parts of the world to determine if they are being pollinated by exotic species.

• Polyploidization may result in self-fertile plants, so that hand pollination may not be necessary.

108

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121 Appendix A

SURVEY I

122 1. Is your institution currently involved in some way with plant reintroduction projects? # Answer Response %

1 Yes 44 46%

2 No 47 49%

3 Unsure 5 5% Total 96 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.59 Variance 0.35 Standard Deviation 0.59 Total Responses 96

2. What title best describes your position? # Answer Response %

1 Director 12 27% Director of

2 10 22% Horticulture

3 Botanist 4 9%

4 Conservationist 6 13%

5 Land steward 0 0%

6 Ecologist 1 2%

7 Other 12 27% Total 45 100%

123 Other Board Chair Horticulturist Curator Curator Director of Collections Director, Germplasm Repository Curator of Living Collections Horticulturist/Arborist Arboretum Manager Plant Records Manager Forester Director of Information Technology

Statistic Value Min Value 1 Max Value 7 Mean 3.51 Variance 5.76 Standard Deviation 2.40 Total Responses 45

3. Except from your parent organization, do you receive other funding for these reintroduction projects? # Answer Response %

1 Yes 27 61%

2 No 17 39%

3 Unsure 0 0% Total 44 100%

Statistic Value Min Value 1 Max Value 2 Mean 1.39 Variance 0.24 Standard Deviation 0.49 Total Responses 44

124 4. Where does the funding come from for these reintroduction projects? Please choose all that apply. # Answer Response %

International

1 0 0% sources National

2 3 12% sources

3 State sources 5 20% Regional

4 4 16% sources

5 Endowment 1 4% Private (donations,

6 4 16% last will and testament)

7 Other 8 32% Total 25 100%

Other Grants All the above national, state, private many sources including state and regional... though it is a complicated answer really state, regional and private Endowment & Private - won't let me choose more than 1 the last 4 sources apply state sources

Statistic Value Min Value 2 Max Value 7 Mean 4.88 Variance 3.61 Standard Deviation 1.90 Total Responses 25

125 5. What best characterizes your motivation to be involved in plant reintroduction projects? Please choose all that apply. # Answer Response %

Preservation

1 of fragile 32 80% ecosystems To alleviate the current

2 red list status 12 30% of selected species It's part of my

3 job 8 20% description As a steward of the planet,

4 26 65% its the right thing to do. To enhance

5 my plant 14 35% collection To advance the

6 17 43% reputation of my institution

7 Other 5 13%

Other All of the above For pollinators, wildlife and stormwater management It's a great partnership with other organizations and we have propagation staff & facilities AND the right thing to do to assist like minded organizations with their efforts Assist native fauna

126 Statistic Value Min Value 1 Max Value 7 Total Responses 40

6. Do you revisit these sites to monitor the progress after plant reintroduction? # Answer Response %

1 Yes 31 79%

2 No 5 13%

3 Unsure 3 8% Total 39 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.28 Variance 0.37 Standard Deviation 0.60 Total Responses 39

7. How long after reintroduction do you monitor these plant species? # Answer Response %

1 1 year 5 17%

2 2 - 5 years 12 41% 5 - 10

3 4 14% years 10 - 15

4 0 0% years 15 - 20

5 1 3% years

6 20 + years 7 24% Total 29 100%

127 Statistic Value Min Value 1 Max Value 6 Mean 3.03 Variance 3.53 Standard Deviation 1.88 Total Responses 29

8. Do you provide supplemental watering after reintroduction for these plant species? # Answer Response %

1 Yes 4 11% Yes, initially, but then we

2 23 64% rely on seasonal rainfall

3 No 4 11% No, we rely

4 on seasonal 3 8% rainfall

5 Unsure 2 6% Total 36 100%

Statistic Value Min Value 1 Max Value 5 Mean 2.33 Variance 0.97 Standard Deviation 0.99 Total Responses 36

128 9. Do you rely on natural rainfall as a means to irrigate these reintroduced species after establishment? # Answer Response %

1 Yes 30 83% No, we irrigate

2 during 4 11% drought periods.

3 No 1 3%

4 Unsure 1 3% Total 36 100%

Statistic Value Min Value 1 Max Value 4 Mean 1.25 Variance 0.42 Standard Deviation 0.65 Total Responses 36

10. Have you observed natural seed set on reintroduced species without human intervention? # Answer Response %

1 Yes 18 51%

2 No 6 17%

3 Unsure 11 31% Total 35 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.80 Variance 0.81 Standard Deviation 0.90 Total Responses 35

129 11. Has there been any hand pollination? # Answer Response %

1 Yes 1 17%

2 No 5 83%

3 Unsure 0 0% Total 6 100%

Statistic Value Min Value 1 Max Value 2 Mean 1.83 Variance 0.17 Standard Deviation 0.41 Total Responses 6

12. Has hand pollination been successful in producing viable seed? # Answer Response %

1 Yes 1 100% Yes, but it

2 depends on 0 0% the species.

3 No 0 0%

4 Unsure 0 0% Total 1 100%

Statistic Value Min Value 1 Max Value 1 Mean 1.00 Variance 0.00 Standard Deviation 0.00 Total Responses 1

130 13. Generally speaking, what percentage of seed set by hand pollination is viable? # Answer Response %

1 0 - 20% 0 0%

2 20 - 40% 0 0%

3 40 - 60% 0 0%

4 60 - 80% 0 0%

5 80 - 100% 0 0%

6 Unsure 0 0% Total 0 0%

Statistic Value Min Value - Max Value - Mean 0.00 Variance 0.00 Standard Deviation 0.00 Total Responses 0

14. Prior to reintroduction, has there been a need to remove competitive or invasive plant species at the reintroduction site? # Answer Response %

1 Yes 23 66%

2 No 9 26%

3 Unsure 3 9% Total 35 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.43 Variance 0.43 Standard Deviation 0.65 Total Responses 35

131 15. Following reintroduction, has there been a need to remove competitive or invasive plant species? # Answer Response %

1 Yes 21 58%

2 No 11 31%

3 Unsure 4 11% Total 36 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.53 Variance 0.48 Standard Deviation 0.70 Total Responses 36

16. Are reintroduced plants replaced following unexpected premature death? # Answer Response %

1 Yes 10 28%

2 No 14 39%

3 Unsure 12 33% Total 36 100%

Statistic Value Min Value 1 Max Value 3 Mean 2.06 Variance 0.63 Standard Deviation 0.79 Total Responses 36

132 17. In your opinion, what has been the public's reaction to plant reintroduction projects? # Answer Response %

1 positive 29 81%

2 neutral 6 17%

3 negative 1 3% Total 36 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.22 Variance 0.23 Standard Deviation 0.48 Total Responses 36

18. Does your organization teach the value of reintroduction projects to the general public? # Answer Response %

1 Yes 29 78%

2 No 5 14%

3 Unsure 3 8% Total 37 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.30 Variance 0.38 Standard Deviation 0.62 Total Responses 37

133 19. To the best of your knowledge, does your institution have ex situ material that is used in some way in reintroduction projects? # Answer Response %

1 Yes 22 59%

2 No 9 24%

3 Unsure 6 16% Total 37 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.57 Variance 0.59 Standard Deviation 0.77 Total Responses 37

20. Please react to the following statement: ex situ material has aided in our plant reintroduction projects. Strongly Strongly Total # Question Disagree Neutral Agree Mean disagree agree Responses choose 1 the best 0 0 3 8 9 20 4.30 answer

Statistic choose the best answer Min Value 3 Max Value 5 Mean 4.30 Variance 0.54 Standard Deviation 0.73 Total Responses 20

134 21. Are you affiliated with any seed banks? # Answer Response %

1 Yes 20 51%

2 No 18 46%

3 Unsure 1 3% Total 39 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.51 Variance 0.31 Standard Deviation 0.56 Total Responses 39

22. Have you made use of seed banks in the process of plant reintroduction projects? # Answer Response %

1 Yes 13 65%

2 No 6 30%

3 Unsure 1 5% Total 20 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.40 Variance 0.36 Standard Deviation 0.60 Total Responses 20

135 23. Do you propagate your own plant stock for reintroduction projects? # Answer Response %

1 Yes 25 66%

2 No 11 29%

3 Unsure 2 5% Total 38 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.39 Variance 0.35 Standard Deviation 0.59 Total Responses 38

24. Would you participate in future surveys about plant reintroduction projects? # Answer Response %

1 Yes 38 90%

2 No 4 10% Total 42 100%

Statistic Value Min Value 1 Max Value 2 Mean 1.10 Variance 0.09 Standard Deviation 0.30 Total Responses 42

25. Please provide a contact name and email address for future contact.

Statistic Value Total Responses 31

136 Appendix B

SURVEY II

137 1. Is your organization involved in plant reintroduction projects? Note: this research is being conducted in collaboration with Botanic Gardens Conservation International (BGCI) and, with your consent, your answer to this question will be contributed to BGCI’s GardenSearch database (www.bgci.org/garden_search.php) # Answer Response %

Yes we are involved in reintroduction projects. I DO

1 73 38% give consent to share this information with BGCI. Yes we are involved in reintroduction projects. I DO

2 NOT give 9 5% consent to share this information with BGCI. No we are not involved in reintroduction projects. I DO

3 98 52% give consent to share this information with BGCI. No we are not involved in

4 reintroduction 10 5% projects. I DO NOT give

138 consent to share this information with BGCI. Total 190 100%

Statistic Value Min Value 1 Max Value 4 Mean 2.24 Variance 1.06 Standard Deviation 1.03 Total Responses 190

4. Are you currently reintroducing any plant species that is known to have LOW GENETIC DIVERSITY in the wild? # Answer Response %

1 Yes 44 57%

2 No 19 25%

3 Unsure 14 18% Total 77 100%

Statistic Value Min Value 1 Max Value 3 Mean 1.61 Variance 0.61 Standard Deviation 0.78 Total Responses 77

139 5. Please choose ONE plant species with LOW GENETIC DIVERSITY which your institution is reintroducing to the wild, and use it to answer the remaining questions of this survey. Please list the species name below: Text Response Cirsium pitcheri corydalus caucasica Arnica montana Arenaria grandiflora Castanea dentata American Chestnut Cyanea superba Vanda denisoniana Grevillea scapigera Cercocarpus traskiae Carex lupuliformis Caladenia pumila Apium (Helosciadium) repens Helichrysum arenarium Sebaea ovata Anisoptera thurifera Silene tomentosa Phytolacca tetramera Nannorrhops ritchieana H. Wendl. Arnica montana Pitcher's thistle (Cirsium pitcheri) Penstemon debilis Embelia keniensis Corchorus cunninghamii Mansonia altissima Viburnum molle Hardenbergia violacea Stylophorum diphyllum Arctostaphylos glandulosa ssp crassifolia Limonium multiflorum Erben Astragalus bibullatus Bletia urbana Muscari neglectum

140 Edithcolea grandis Sarracenia rubra alabamensis erica verticillata Britton's Beargrass Woodwardia radicans Woodsia ilvensis Consolea corallicola Pseudophoenix sargentii

Statistic Value Total Responses 41

141 6. Please briefly describe the number of populations and or individuals remaining in the wild. Text Response About 150 populations, most quite small Caucasian mineral wsters 2 This is a relict species in Lowlands. Near Paris, in Fontainebleau forest, one population remains with one individual. 300 approx Not hardly any on the East Coast of America anymore This species did technically go extinct in the wild, however, the Oahu Army Natural Resources Program propagated individuals from the last known founders and have successfully out-planted and created naturally reproducing individuals. There are about 170 individuals in three populations in the wild as of last year's reporting. Unknown, very rare species. 13 popuilations mostly on degraded road verges. Most of these populations do not have any living plants and very little soil seedbank. Typically over the last few years in total there were about 5 living plants in total ie counting plants from all populations. However, the lagest known population was distrubed (this plant is a disturbance oppourtunist) for roadworks in 2010 and about 180 seedlings appeared from the soil seedbank. Most of these died due to a record low rainfall year (about 25 survived the summer). The following year more seedlings germinated I think there are probably about 30 plants surviving, seedling and putting seed back into the soil seedbank. Note I do not monitor the natural populations that is done by our conservation agency DPaW One population, approx 6 adult individuals (pure), over 100 seedlings (undetermined genetics) 3 populations, 31 individuals in the wild (2, 5 and 22 individuals) The species (a terrestrial orchid) was believed to be extinct (not observed for >50 years) until discovered at a site near to, but not the same as the original, 4 years ago. There are only 2 plants in the wild. About 350 plants one population left in the wild; exact number of individuals unknown (genetical analyses ongoing) but extremely low: one patch (probably a clone) of less than 50 flower heads Restricted to two known populations in NZ (also present in Australia). http://www.nzpcn.org.nz/flora_details.aspx?ID=39 We are working with land managers and the national department of conservation by growing and providing seed for use in enhancing the wild population.

142 about 200 mother trees The species is recorded in the wild infrequently. It was not recorded in the wild between 1985 and 1994. Seeds were collected from 3 plants in 1994 and these have been used for propagation. A single wild plant was seen in 2008, with none recorded since. unsure This is the only wild palm found in Pakistan. Its very difficult to comment on the number of populations and or individuals remaining in the wild, but due to anthropogenic activities, populations have been drastically affected. The number of wild poplations in Federal state of Brandenburg is about 5. The 2012 reintroduced individuals orginate from an isolated population without genetic information.

4 populations Few thousand individuals, 5-7 known adult specimens known in the wild Three main population areas in northern NSW and south-east Queensland. The centre of these three populations is located in Mount Cotton, south of Brisbane and within the Redland City Council boundary. In Benin, this species exists only in one small rainforest patch in a few specimens. During the last visit we could not find an adult tree (though we might have missed one). This is the easternmost location for the species, which is still rather common in Côte d'Ivoire and other countries, but the next population probably exists only in Ghana. We currently have two colonies on the property with no more than five individuals in each colony. There is a single wild population in Tasmania with between 40 and 50 individuals. Only a few hundred plants remain in two small colonies that are highly disjunct from main range of the species. The decline is due to agricultural expansion and impact of exotic species. small pockets of this plant occur along a narrow strip of coastal habitat-not sure of total numbers This species is endemic to the west Portugal coast. About 15 small populations are known (with about 10 - 25 individuals) . Nevertheless, 1 population with about 1000 individuals is recognized 5 populations, ~5,000 individuals two populations it is a geophyte. it lives naturally in Gaziantep-Turkey. it flowers in spring. Not more than 100 Historically, the species was documented from 28 sites (Alabama Natural Heritage Program 2011, Byrd 2011). Population size estimates from 2010 (most recent census

143 data available for the species) range from two clumps at one site to nearly 170 clumps at another. Four sites contain 10 clumps or less; two are estimated to have between 10 and 50 clumps; and three populations have between 50 and 170 clumps (Alabama Natural Heritage Program 2011, Byrd 2011). zero The range of Nolina brittoniana is from the south end of Lake Wales Ridge in Highlands County north to Marion County and northern Lake County. An isolated locality has been reported from Hernando County, north of Tampa. On the Lake Wales Ridge, it occurs in both Highlands and Polk Counties in most of the tracts that are targeted for acquisition by the state or by the U.S. Fish and Wildlife Service. It may still occur in western Orange County and in the northwest corner of Osceola County where specimens were collected in 1958 but remaining habitat is being rapidly destroyed. In Lake County, it occurs in the remnants of high pine on hills west of Lake Apopka near Clermont. It was recently discovered at a site in Ocala National Forest. 98 individuals in GB There are two populations remaining; one had 12 individuals and the other has 500 individuals. Most of them have a single genotype across these two populations. The plant collections in cultivation have a unique allele when compared to the wild populations. Genetic diversity also depend on the technique being used. Rapid analysis, which deals with enzyme diversity, was completed 20 years ago. 14 different individuals were noted versus one different allele in an AFLP analysis (using neutral genetic markers). The current population is only producing one kind of flower (male) There are reproduction problems within this genus. There is a single population in south florida with other populations present in the Caribbean.

Statistic Value Total Responses 40

144 7. Please indicate whether population numbers are currently increasing, stable or declining? If there are multiple populations, please choose all that apply. # Answer Response %

Population

1 numbers are 6 15% increasing. Population

2 numbers are 11 28% stable. Population

3 numbers are 16 40% in decline.

4 Unsure. 5 13% Other. Please

5 7 18% specify in the box below.

Other. Please specify in the box below. Slowly increasing but with great management effort against alien pest species. Numbers appear to be increasing, hybridization may be an issue. Population is extremely low and in danger of extinction. We have planted 100 in situ in protected areas populations are stable however continued development is a constant threat to the habitat as well as the species. Even in protected areas there has not been much seedling recruitment all populations are reintroductions 67 in Lake District increasing, the rest stable

Statistic Value Min Value 1 Max Value 5 Total Responses 40

145 8. Keeping in mind the species you selected, what threat(s) have led to its current low genetic diversity? Please choose all that apply. # Answer Response %

Destruction

1 of habitat by 29 73% man. Over-

2 7 18% collection. Competition with alien invasive

3 13 33% plant, insect or disease species. Competition from

4 herbivores or 7 18% other animals. Climate

5 8 20% change. Natural

6 5 13% disaster. Other. Please

7 specify in the 13 33% box below.

146 Other. Please specify in the box below. Geographic isolation Chestnut blight and deer herbivory Herbicide and fertiliser drift weed encroachment due to a number of factors including water level change and land management. Please see the threats section of this webpage: http://www.nzpcn.org.nz/flora_details.aspx?ID=39 Unknown. Perhaps climate change. landuse change, decline of traditional sheep grazing migration bottleneck Rare specific habitat Changed fire regimes Restricetd distribution in Tasmania Breeding system Woody encroachment rescued from old collections in european botanical gardens

Statistic Value Min Value 1 Max Value 7 Total Responses 40

9. Does this species have an official threat status? Please choose all that apply. # Answer Response %

Yes, globally

1 9 22% threatened. Yes,

2 nationally 22 54% threatened. Yes, locally

3 12 29% threatened.

4 No 4 10%

5 Unsure 1 2% Other, please

6 specify in the 8 20% box below.

147 Other, please specify in the box below. Federally listed endangered CITES Appendix II and Thai threatened species list, restricted geographical distribution, unknown pollinator/biology Yes, Threatened - Nationally Critical (2012) Should be on Red list but isn't Endangered in Canada and Species at Risk in Ontario Listed as federally Endagered by the USFWS Extinct in the wild Why no IUCN cats here?

Statistic Value Min Value 1 Max Value 6 Total Responses 41

10. What is the status of the land where this species' reintroduction site(s) is located? Please choose all that apply. # Answer Response %

4 Public land. 16 39% On my

1 institutions 12 29% property.

2 Private land. 12 29% Other, please

6 specify in the 10 24% box below.

5 Federal land. 8 20% On a

3 partner's 3 7% land.

148 Other, please specify in the box below. State College This is the State of Hawaii, Natural Area Reserve Land as well as United States Army Land The current stage is on the property of the institution, aiming at federal land (national park) Local Government reserves Central media of the Motor Way in between Pesadhawar and Islamabad. There is a conservation covenant on the land SDBG is involved with our own property-not sure if other agencies are actively reintroducing this species as well Urban Reserve provincial nature reserves and national parks NGO's land

Statistic Value Min Value 1 Max Value 6 Total Responses 41

149 11. Do you receive outside funding to support this species' reintroduction? # Answer Response %

Yes, from private non-

1 4 10% governmental sources. Yes, from

2 government 17 41% sources. Yes, from

3 0 0% corporations. No, my organization

4 15 37% funds all activities. Other, please

5 specify in the 5 12% box below. Total 41 100%

Other, please specify in the box below. from the European Union (LIFE programme) No, my organisation funds its activities in this work, but our part of this work is relatively minor and does not require additional funding to our general propagation budget. This activity is self support scheme launched by Biodiversity Conservation Society. all on my costs up to now multiple organizations fund activities

Statistic Value Min Value 1 Max Value 5 Mean 3.00 Variance 1.70 Standard Deviation 1.30 Total Responses 41

150 12. How far is this species' reintroduction site(s) from your institution? If there is more than one site, please choose all that apply. # Answer Response %

less than 1

1 10 24% km (1 mile) 1 - 10 km

2 (1 - 6 5 12% miles) 11 - 40 km

3 (7 - 25 11 27% miles) 41 - 100

4 km (26 - 62 12 29% miles) More than

5 100 km (62 8 20% miles)

Statistic Value Min Value 1 Max Value 5 Total Responses 41

151 13. Is there seed of this species currently stored in a seed bank? # Answer Response %

1 Yes 25 61% No, it is not currently

2 6 15% stored in a seed bank. No, seed of this species cannot be stored using

3 2 5% traditional seed banking methods.

4 Unsure 2 5% Other, please

5 specify in 6 15% the box below. Total 41 100%

Other, please specify in the box below. We keep seeds We have given 200 seeds for MSB and KGB this year It is stored but not in a formal seed bank (it is kept in a kitchen fridge!) Spore bank & conservationj collection of living plants onsite seed not currently produced by this species Seeds are recalcitrant.The ncgrp is currently researching ways to better store palm species.

152 Statistic Value Min Value 1 Max Value 5 Mean 1.98 Variance 2.22 Standard Deviation 1.49 Total Responses 41

14. Are there plans for any banked seed of this species to be used in future reintroduction efforts by your institution? # Answer Response %

1 Yes 14 56%

2 No 3 12%

3 Unsure 4 16% Other, please

4 specify in 4 16% the box below. Total 25 100%

Other, please specify in the box below. Not at present but may in future and plant material is in long term storage Question is ambiguous! They will be used if needed We are collecting and propagting seed from our in situ plants-no need to use banked seeds

Statistic Value Min Value 1 Max Value 4 Mean 1.92 Variance 1.41 Standard Deviation 1.19 Total Responses 25

153 15. How is vegetative material of this species preserved for future reintroduction efforts by your institution? Please choose all that apply. # Answer Response %

1 Living plants. 28 74% Tissue culture

2 6 16% of explants. Cryogenic

3 4 11% samples. Vegetative material not

4 preserved for 6 16% reintroduction efforts. Other. Please

5 specify in the 7 18% box below.

Other. Please specify in the box below. seed collection and growing Seeds We only work with first generation seedlings to increase genetic diversity and avoid genetic drift Thousands of seed at Kings Park material not preserved for reintroduction, rather seed is grown for return to the land manager seed and mycorrhiza collection Spore

Statistic Value Min Value 1 Max Value 5 Total Responses 38

154 16. How is this plant being reintroduced into the wild? Please choose all that apply. # Answer Response %

Plants are grown by my institution and then

1 37 90% planted into a suitable habitat in the wild. Rooted cuttings are planted out

2 into a 5 12% suitable habitat in the wild. Seeds are directly sown into a suitable

3 8 20% habitat at the optimum time of year. Other, please

4 specify in 3 7% the box below.

Other, please specify in the box below. unsure, but seed may be grown into plants by land manager as well as direct sowing. my site is a rehabilitated forest Symbiotic germination

155 Statistic Value Min Value 1 Max Value 4 Total Responses 41

17. Does your institution incorporate soil from the future reintroduction site into the growing media during propagation of this species? # Answer Response %

Yes, please

1 describe why 5 14% here:

2 No 26 72%

3 Unsure 2 6% We do not grow this

4 0 0% species prior to reintroduction. Other, please

5 specify in the 3 8% box below. Total 36 100%

Yes, please describe why here: Other, please specify in the box below. mychorrhizae innoculation to growing No, but we use sand for growing, it is a media dune species trying some with and some without Native soil, not specific to reintroduction because it grows on a very specialized site, is used in media. geologic formation for two reasons: to mimic conditions for seed germination and to lessen No: no idea what natural soil would be transplant shock Its a Leptosol on basaltic rock it adapts earlier.

156 Statistic Value Min Value 1 Max Value 5 Mean 2.17 Variance 0.94 Standard Deviation 0.97 Total Responses 36

18. What is the provenance of the material used for this species' reintroduction by your institution? Please choose all that apply. # Answer Response %

Documented wild-origin material

1 30 79% collected by my institution. Documented wild-origin

2 material 7 18% collected by a third party. Material of garden or

3 3 8% unknown origin.

4 Unsure. 1 3% Other. Please

5 specify in the 2 5% box below.

Other. Please specify in the box below. volunteers Non-Documented wild-origin material collected by my institution

157 Statistic Value Min Value 1 Max Value 5 Total Responses 38

19. How long has your institution been reintroducing this species into the wild? # Answer Response %

1 0 - 5 years 16 42% 6 - 10

2 8 21% years 11 - 15

3 6 16% years 16 - 20

4 3 8% years More than

5 4 11% 20 years Other, please

6 specify in 1 3% the box below. Total 38 100%

Other, please specify in the box below. until recently SDBG has done some sporadic reintroductions into our own natural areas. We have started a much more focused program within the last two years. We will be

Statistic Value Min Value 1 Max Value 6 Mean 2.32 Variance 2.22 Standard Deviation 1.49 Total Responses 38

158 20. How long after reintroduction (out-planting) does your institution monitor this species? # Answer Response %

We don't monitor

1 4 11% reintroduction sites.

2 1 year 5 14%

3 2 - 5 years 11 30%

4 6 - 10 years 5 14%

5 11 - 15 years 2 5%

6 16 - 20 years 5 14% more than 20

7 5 14% years Total 37 100%

Statistic Value Min Value 1 Max Value 7 Mean 3.84 Variance 3.70 Standard Deviation 1.92 Total Responses 37

159 21. After the initial reintroduction of this species, how often does your institution add additional plants or seeds to the reintroduced population(s)? Please choose all that apply. # Answer Response %

More than 10

5 1 3% years

4 6 - 10 years 1 3%

3 2 - 5 years 5 13%

2 Annually 6 16%

1 Never 7 18% Other. Please

7 specify in the box 8 21% below. Only when

6 12 32% possible/necessary

Other. Please specify in the box below. Comment to q21: we aim at monitoring until safe, but this is a new project This was done annually for over 10 years also adding to genetic divesity only the three first years the species is yet to be reintroduced unsure, we are not the ones doing the active reintroduction, this is done by the land manager and national department of conservation of NZ two introductions within 5 years initially SDBG will be planting annually-then tapering of with new plants being added every 6-10 years to maintain population demographics Pathogen has been detected on cultivated plants.

Statistic Value Min Value 1 Max Value 7 Total Responses 38

160 22. Do you reintroduce plants at different levels of maturity to replicate the demographics of a natural population? # Answer Response %

1 Yes 8 21%

2 No 23 61%

3 Unsure 3 8% Other, please

4 specify in 4 11% the box below. Total 38 100%

Other, please specify in the box below. seeds and 1-yr juveniles small plantlets were introduced no: in fynbos natural demography is a single aged cohort germinating after a fire see above

Statistic Value Min Value 1 Max Value 4 Mean 2.08 Variance 0.72 Standard Deviation 0.85 Total Responses 38

161 23. What is the main reproductive mechanism for this species? # Answer Response %

Self-

1 11 30% compatible. Obligate out-

2 9 24% crossing.

3 Asexual. 3 8%

4 Unknown. 9 24% Other, please

5 specify in the 5 14% box below. Total 37 100%

Other, please specify in the box below. They can self but out crossing is thought to yield higher fruit and seed set. Native bird pollinator is thought to be extinct. Self compatible, but to date, it has been manually pollinated. self compatible, but it flowers from 2 to 3 years or more, like any dipterocarpaceae species. mixed mating system

Statistic Value Min Value 1 Max Value 5 Mean 2.68 Variance 2.17 Standard Deviation 1.47 Total Responses 37

162 24. Have natural pollinators been observed visiting your institution's reintroduced populations of this species? # Answer Response %

1 Yes 17 45%

2 No 7 18% No, the natural

3 0 0% pollinator is extinct.

4 Unsure 11 29% Other, please

5 specify in 3 8% the box below. Total 38 100%

Other, please specify in the box below. THe natural pollinator is likely extinct but we do not know for certain what the natural pollinator was. It was likely one of our native honey-creeper birds. Some other native birds have been seen visiting but they are likely not properly equipped to successfully pollinate these flowers. autopollination Fern so no

Statistic Value Min Value 1 Max Value 5 Mean 2.37 Variance 2.24 Standard Deviation 1.50 Total Responses 38

163 25. Have you observed natural seed set by this species at your institution's reintroduction site(s)? # Answer Response %

1 Yes 25 66%

2 No 7 18%

3 Unsure 1 3% Other, please

4 specify in 5 13% the box below. Total 38 100%

Other, please specify in the box below. Seed has been set following hand-pollination not yet (plants have been transplanted a few weeks ago) the reintroduced plants are juveniles, not flowering in the first year too soon trees still too young!

Statistic Value Min Value 1 Max Value 4 Mean 1.63 Variance 1.10 Standard Deviation 1.05 Total Responses 38

164 26. What percentage of the seed is estimated to be viable? # Answer Response %

1 0 - 20% 2 8%

2 21 - 40% 2 8%

3 41 - 60% 5 20%

4 61 - 80% 2 8%

5 81 - 100% 6 24%

6 Unsure. 4 16% Other, please

7 specify in 4 16% the box below. Total 25 100%

Other, please specify in the box below. Naturally about 25% but with treatment in smoke water, increased to about 75% difficult to say-this species appears to require fire for germination-trials have shown good but sporadic germination over multiple year periods depends on the source mother stock and which strains are co-planted Varies from year to year - dry years are poor.

Statistic Value Min Value 1 Max Value 7 Mean 4.44 Variance 3.51 Standard Deviation 1.87 Total Responses 25

165 27. Has hand pollination of this species resulted in successful seed set? # Answer Response %

1 Yes 11 31%

2 No 2 6%

3 Unsure 16 44% Other, please

4 specify in 7 19% the box below. Total 36 100%

Other, please specify in the box below. Not verify Hasn't been attempted Have not used it not yet tried this have not attempted this not needed NA fern

Statistic Value Min Value 1 Max Value 4 Mean 2.53 Variance 1.28 Standard Deviation 1.13 Total Responses 36

166 28. What percentage of hand -pollinated seed is estimated to be viable? # Answer Response %

1 0 - 20% 0 0%

2 21 - 40% 0 0%

3 41 - 60% 1 9%

4 61 - 80% 1 9%

5 81 - 100% 1 9%

6 Unsure. 7 64% Other, please

7 specify in 1 9% the box below. Total 11 100%

Other, please specify in the box below. Always use genetic diversity to obtain good seed. Very few seed are produced using pollen from the same plant

Statistic Value Min Value 3 Max Value 7 Mean 5.55 Variance 1.27 Standard Deviation 1.13 Total Responses 11

167 29. Does your institution use any methods to increase or maximize the genetic diversity of this species? # Answer Response %

1 Yes 14 37%

2 No 21 55%

3 Unsure 1 3% Other., please

4 specify in 2 5% the box below. Total 38 100%

Other., please specify in the box below. co plant strains from different mother stocks Not currently. Could honor the 14 different genotypes and plant as many as possible, including the unique allele from cultivation.

Statistic Value Min Value 1 Max Value 4 Mean 1.76 Variance 0.56 Standard Deviation 0.75 Total Responses 38

168 30. Please briefly explain the method/s used by your institution to increase or maximize the genetic diversity of this species. For example: conservation breeding, laboratory techniques, curatorial practices etc. Text Response We bank seeds from throughout the range, we assessed genetic diversity of many populations and mix seeds from several of the more diverse, nearby populations for the reintroduction Methods - geoinform.,morphometric, anatom., chemical The reintroduced population is composed of individuals coming from the threatened population (after a tissue culture multiplication) and of individuals coming from another population known to be genetically diverse. Tracking providence of individual plants in all stages of the effort. Aiming for creating genetically diverse populations when possible though this species is known to have very low genetic diversity. Not very special; only using seeds from crossings of different individuals, and only use F1 seedlings for outplanting to minimize genetic drift. The P plants are donated to us and where they originally come from are usually unclear. We never accept forms deviating from the wild type. The start of the project involved using 10 clones represting 87% of the known genetic diversity of the species (using 47? clones. Note this project started as a PhD project using the best scientific equipment available DNA testing, using plants raised in tissue culture. These plants were planted out in a grid patten to maximise gentic diversity ie mixing the clones. Genetic testing of plants in the field indicated genetic decline. More original clones were reintroduced to maximise genetic diversity and clonal material from new wild plants was introduced...Later plantings of seedlings were from seed collected from the translocation sites due to the difficulty and cost of raising tissue culture plants (some clones were difficult to raise in tissue culture and often more difficult to establish on the 3 translocation sites. Noe millions of seed have been produced by plants on the translocation sites (one site produced over 1,000,000 seed in one year) - In our reintroduction design, we use seeds from 3 large populations (>1000 individuals) - we reintroduce 4 different populations close to each other, so that pollinators can fly from one to the other (maximizing gene flow) - 500 individuals are reintroduced in each population (a mixture of the 3 different origins, at equal proportions) Use of multiple populations as seed sources for restoration Reintroduced populations are comprises of individuals from different populations

169 sources collected over a 20-year period we use production techniques. Conservation and multi-propagation We don't use vegegative propagation, only wild collected seed. Mixing different sources from GB.

Statistic Value Total Responses 13

31. Do you think that your institution's reintroduction project is successful or soon to be successful? # Answer Response %

1 Yes 21 55%

2 No 1 3% Unsure, please

3 specify in 16 42% the box below. Total 38 100%

170 Unsure, please specify in the box below. Only after a long-term monitoring Low survival, but several seeds dispersed Still early days, but similar reintroductions using similar protocols have been successful too early to conclude It is relatively early days for this project. We are successful in producing seed for the land manager, but have had no feedback on the whole project Plantation and establishment can be successful but is affected by harsh climatic condition just like typhoon and forest fire. It will take long time to know to early to assess stochastic demography, and threats of climate change and invasive seed herbivores make predictions difficult too soon, focus right now is on successful propagation should be successful, but three generations is 3 fire cycles is 30-60 years before we can be certain Good survival % nut no regrenaration yet. They are holding their population number, but only through vegetative propagation from the pads. Not increasing spatially. project was successful, but it is such a long lived species that it is unfair to judge this species against a fast growing perennial species.

Statistic Value Min Value 1 Max Value 3 Mean 1.87 Variance 0.98 Standard Deviation 0.99 Total Responses 38

171 32. Apart from lack of genetic diversity, please choose other reasons for the lack of success of your institution's reintroduction efforts for this species. Please choose all that apply. # Answer Response %

Lack of funding to

1 1 100% continue project. High death rate of

2 reintroduced 0 0% plants soon after planting. Natural

3 0 0% disaster. Failure to monitor

4 reintroduced 1 100% populations over time. Competition from

5 herbivores or 0 0% invasive plant species. Lack of

6 natural 0 0% pollinators. Other. Please

7 specify in the 1 100% box below.

Other. Please specify in the box below. lack of institutional capacity

172 Statistic Value Min Value 1 Max Value 7 Total Responses 1

173 33. How do you define the success of your institution's reintroduction efforts for this species? Please list your own measures of success. Text Response Natural recruitment and a population growth rate (lamda) >1 for the reintroduction for 10 years From 1943 our institure did this work on the territory of our botanical garden ,there were reintroduction more than 700 different mwdicinal plants in our conditions An existring but declining population was stabilized and in situ regeneration has been observed. On 2 sites populations have been reintroduced and seem to be growing. Establishment of the specimens at the sites. With the Chestnut Blight - it is not a matter of IF they will get it, it is a matter of WHEN. We will try to keep the trees as healthy as possible for as long as possible, and if they reach adulthood, we will be successful. Naturally regenerating populations in the wild that eventually can persist with little human assistance (predator control, fence repair, etc). Success is the establishment of individuals on living trees, and their ability to reproduce naturally. We have observed wild populations without fruit formation, a most disturbing fact hinting the pollinator is likely missing due to habitat destruction. Its a very successful project as all three sites have produced recruits, varies from year to year depending on rainfall events. Although a disturbance oppourtunist seedlings will recruit between disturbance events. Its all due to the good scientific and horticultural input, including new methods on ground trials, adequate funding (dont get any funding now but still check on the sites and moritor mature plants and seedling recruitment). The project was relient on a good working relationship withe the state conservation agency, local shire (government) assistance, some local volunteers over the years and a huge involvment of volunteers through Kings Park Volunteer Master Gardeners eg planting, monitoring and data anaylsis, seed collecting etc. This species will never become extinct due to plant material being in cryostorage (plants have been taken out successfully put into tissue culture, planted out and seed produced which was sown and resulting seedlings planted out. A large amount of seed is in storage in Kings Park and seed sent to MSB and the state conservationa agency DPaW. Add hoc scientific research continues and 50 year seed burial trials are harvested every 2 years(over 6 years) indicating seed viability is high both on the soil surface and buried at 5cm. The population is growing All small plants that we brought back to my forest grew well, i.e., no mortality was observed. The earliest introduction resulted in trees that are now about 12 m high We believe that our reintroduction of Viburnum molle has been successful.

174 Observations of the introduced individuals have shown that they are growing well and are reaching maturity. When last monitored in 2012 there was over 50% survival of plants and some individuals had set seed. We have had success with a number of individuals that were planted out 10 or so years ago. We have just resumed an active program of propagating this species for re-introduction within our facility. Future plans are to work with other institutions to re-introduce this species. - increase in the number of individuals in wild population (increase in population density) - increase in population range - reproductive sucess (number of mature plants and ability to produce seeds) - measure genetic diversity of plants from experimental populations Success is defined as: 1) Experimental questions answered 2) Enhanced ecological knowledge of the species and habitat 3) Demographic metrics: transition to reproduction, fruit set, next generation seedling recruitment The wild survival has been stabilized in 50% of mycorrhizal plants after more than 10 years, 0% of asymbiotic plants survive, flowering after 5 years of almost the half of the reintroduced population, and plant recruitmen of these reintrodution population after near 10 years even when one individual (orchids are very special for these) perpetuating and producing are our own measures of success. A bit challenging but we will keep on doing it with or without funds Survival rates over 75%, evidence of seedling recruitment.

Statistic Value Total Responses 19

34. Would you participate in future surveys on plant reintroduction projects or like to be considered for a case study? # Answer Response %

1 Yes. 34 89%

2 No 1 3%

3 Unsure 3 8% Total 38 100%

175 Statistic Value Min Value 1 Max Value 3 Mean 1.18 Variance 0.32 Standard Deviation 0.56 Total Responses 38

176 Appendix C

HUMAN SUBJECTS REVIEW BOARD

177

RESEARCH OFFICE 210 Hullihen Hall University of Delaware Newark, Delaware 19716-1551 Ph: 302/831-2136 Fax: 302/831-2828

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- 1 - Generated on IRBNet

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RESEARCH OFFICE 210 Hullihen Hall University of Delaware Newark, Delaware 19716-1551 Ph: 302/831-2136 Fax: 302/831-2828

- 1 - Generated on IRBNet

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RESEARCH OFFICE 210 Hullihen Hall University of Delaware Newark, Delaware 19716-1551 Ph: 302/831-2136 Fax: 302/831-2828

- 1 - Generated on IRBNet

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RESEARCH OFFICE 210 Hullihen Hall University of Delaware Newark, Delaware 19716-1551 Ph: 302/831-2136 Fax: 302/831-2828

- 1 - Generated on IRBNet

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