godfried 2015 germination of seeds of indigenous curacaoan tree and shrub species

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Yoeri Godfried CARMABI Curaçao GERMINATION OF SEEDS OF INDIGENOUS CURACAOAN TREE AND SHRUB SPECIES To enhance their use in reforestation

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Page 1: Godfried 2015 GERMINATION OF SEEDS OF INDIGENOUS CURACAOAN TREE AND SHRUB SPECIES

Yoeri Godfried

CARMABI Curaçao

GERMINATION OF SEEDS OF INDIGENOUS CURACAOAN TREE AND SHRUB SPECIES

To enhance their use in reforestation

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GERMINATION OF SEEDS OF INDIGENOUS CURACAOAN TREE AND SHRUB SPECIES TO ENHANCE

THEIR USE IN REFORESTATION

By Yoeri Godfried

February 6th 2015, CARMABI Foundation, Curaçao

Supervised by Martin Perescis and John de Freitas

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Preface Dear reader,

First of all, I would like to thank the CARMABI Foundation for giving me this assignment and a place

to stay during my time in Curaçao. I would like to thank Callum Reid (fellow student), Michele Pierotti

(Smithsonian Institution), Kristen Marhaver (CARMABI), Joost van den Burg (WUR) and Steven Groot

(WUR) for their valuable input trough various stages of this study and I would like to thank Martin

Perescis (HAS) and John de Freitas (CARMABI) for their guidance as I performed this study and wrote

this report.

And lastly, I would like to thank you, the reader, for reading this report. I hope you learn something

new!

Regards,

Yoeri

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Abstract This study tested for dormancy types in Zhantoxylum flavum, Capparis odoratissima, Myrcia curassavica and Bourreria succulenta, Curaçaoan endemic tree and shrub species. The species were divided into groups of 100 seeds and the groups were treated with gibberellic acid (100mg/l, 500mg/l and 1000mg/l, testing for non-deep physiological dormancy), sandpaper (testing for physical dormancy) and a combinational treatment of sandpaper and gibberellic acid (testing for combinational dormancy). Z. flavum was confirmed to be physiologically dormant, but no conclusions could be drawn from the results of other species due to very low amounts of germination (5 out of 1320 seeds). The Z. flavum seeds not responding to any form of treatment conducted in this study ruled out non-deep PD, and literary findings concerning the Rutaceae family rule out PY and PD +PY. Since intermediate PD and deep PD were not tested for, it is safe to assume that Z. flavum is either intermediately or deeply physiologically dormant. No conclusions could be drawn from this study related to the dormancy of C. odoratissima, B. succulenta or M. curassavica. The low amount of germination found in this study could have been caused by either dormancy or a large amount of embryoless seeds that were not removed using the sink or float method. A study should be conducted on these species with emphasis on seed selection, and possibly M(P)D, with repetition of the methods used in this study to determine by which cause these low germination rates occurred. The dry storage of one group of B. succulenta seeds included in this study did not positively affected germination rates when compared to Kranendonk (2014).

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Table of Contents Preface ..................................................................................................................................................... 2

Abstract ................................................................................................................................................... 3

1. Introduction ..................................................................................................................................... 5

2. Methods .......................................................................................................................................... 7

Seed selection...................................................................................................................................... 7

Experimental design ............................................................................................................................ 8

Treatments .......................................................................................................................................... 8

Gibberellic Acid (GA3) ...................................................................................................................... 8

Sanding ............................................................................................................................................ 9

Data processing ................................................................................................................................... 9

3. Results ........................................................................................................................................... 10

Z. flavum ............................................................................................................................................ 10

M. curassavica ................................................................................................................................... 11

B. succulenta ...................................................................................................................................... 11

C. odoratissima .................................................................................................................................. 11

4. Discussion ...................................................................................................................................... 12

Z. flavum ............................................................................................................................................ 12

Other species ..................................................................................................................................... 12

5. Conclusions .................................................................................................................................... 13

References: ............................................................................................................................................ 14

Appendix 1: Economically relevant endemic taxa of Curaçao .............................................................. 15

Appendix 2: P-values for Z. flavum treatment comparisons ................................................................. 16

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1. Introduction Plants are an important aspect of nature restoration, reforestation and landscaping projects. For these projects different plant species are required. Each species with their own characteristics. Examples include the amount of water present, the amount of sunshine received, the ambient temperature and an internal factor, dormancy. They all affect a plants germination, growth and health. These characteristics may differ per species and the differences are vital when it comes to plant growth and reproduction. Since plants for reforestation and landscaping are mostly grown in nurseries, it is important to define these traits for the species aimed to reproduce, in order to obtain a maximum yield from available seeds and space. Seed dormancy is sometimes defined as lack of germination, but this definition is inadequate. Unfavourable environmental conditions are one reason for lack of seed germination. Seeds could be in a paper bag on the laboratory shelf (i.e., lack of water), buried in mud at the bottom of a lake (i.e., insufficient oxygen and/or light) or exposed to temperatures that are above or below those suitable for plant growth. These obviously unfavourable conditions for germination are examples of how the environment, rather than some factor associated with the seed per se, prevents germination. A second reason why seeds may not germinate is that a property of the seed (or dispersal unit) prevents it, which we call dormancy (Baskin & Baskin, 2014). The specifics of the dormancy trait differ from species to species (Deno, 1993) but are often similar between species in the same family (Finch-Savage & Leubner-Metzger, 2006). The trait has evolved throughout time (Willis et al., 2014) and different dormancy types have been identified and categorized into a system that includes five classes of seed dormancy: physiological dormancy (PD), morphological dormancy (MD), morphophysiological dormancy (MPD), physical dormancy (PY) and combinational dormancy (PY + PD). The PD class can be further divided into three different varieties being non-deep, intermediate, and deep PD (Baskin & Baskin, 2004; Finch-Savage & Leubner-Metzger, 2006; Nikolaeva, Rasumova, & Gladkova, 1985). PD is the most common form in the field and is found in seeds of gymnosperms and all major angiosperm clades (Finch-Savage & Leubner-Metzger, 2006). PD can be divided into three levels: deep, intermediate and non-deep (Baskin & Baskin, 2004). Seeds with PD are water-permeable. Embryos from seeds with deep PD do not grow at all or produce abnormal seedlings, and require several months of cold or warm stratification before germination can take place. Embryos from seeds with non-deep PD on the other hand, produce normal seedlings and the seeds require a gibberellic acid treatment or, depending on species, dormancy can also be broken by scarification, after-ripening in dry storage, and (a much shorter period of) cold or warm stratification. Based on patterns of change in physiological responses to temperature, different types of non-deep PD can be distinguished (Finch-Savage & Leubner-Metzger, 2006). PD can be broken by hormonal treatment, or cold or warmth stratification, depending on the category (Baskin & Baskin, 2014). MD in seeds with can be identified by embryos that are underdeveloped in terms of size, but differentiated (e.g. into cotyledons or hypocotyl). These embryos are not (physiologically) dormant, but simply need time to grow and germinate (Finch-Savage & Leubner-Metzger, 2006). MPD is also evident in seeds with underdeveloped embryos, but they have an added physiological component to their dormancy (Baskin & Baskin, 2004). PY is caused by water-impermeable layers of cells in the seed or fruit coat that control water movement, and can be broken by scarification of these layers. PY + PD is seen in seeds with physiological embryo dormancy combined with PD (Finch-Savage & Leubner-Metzger, 2006). In context of the Curaçao Island Development Plan (EOP), the Curaçaoan government tries to restore damage done to nature conservation areas by implementing measures indicated by the government

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and to be implemented by project developers undertaking projects under the required permit (“aanlegvergunning”) (Jonker, 1996). The number of projects in conservation areas that require reforestation through the EOP have been very limited. Most of the plants used in those reforestation projects are provided by the small nursery of CARMABI (Figure 1). Ideally, species that grew in the area before the damage was inflicted would be used to restore that area back to its previous form. Sadly, a lot of the information required to propagate indigenous Curaçaoan plant species is still missing or derived from species in the same family (Burg, Freitas, & Debrot, 2014), making it problematic to fulfil this goal on a large(r) scale (including possible initiatives to do reforestation projects). The derived methods have not been tested on the local Curaçaoan species with the exception of Bourreria succulenta, this species was tested for by Kranendonk (2014) but no dormancy type could be determined from that study, since differences between treatments were minimal. For a large number of species it is currently unknown how to best germinate them, and how to grow them from seedlings and introduce them into a new environment (the latter will not be covered by this study). This study aims to fill a knowledge gap in regards to seed dormancy and provide methods to propagate indigenous tree and shrubs species more effectively. This study will test for breaking of the most common dormancy types using common techniques. The main goal is to determine the dormancy types for endemic Curaçaoan tree and shrub species to allow for easier reproduction of these species. Because the most commonly found dormancy type in plants is non-deep physiological dormancy (Finch-Savage & Leubner-Metzger, 2006), the results of this study are expected to be mostly species with non-deep physiological dormancy.

Figure 1. CARMABI’s nursery.

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2. Methods Our study aimed to identify differences in germination rates between treatments directed at

breaking dormancy types and to fill in the dormancy types for seeds available in the period of

October 2015. This study focussed on a number of focal species among economically relevant

indigenous taxa, derived from Broeders (1967) (all economically relevant taxa can be found in

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Appendix 1). All used seeds are depicted in Table 1 along with their origins and the amount used. After the seeds were collected in the field, they were removed from fruit and dehusked if necessary. Unhealthy seeds were removed by sink-or-float method. The healthy seeds received treatment directed at breaking non-deep physiological dormancy (PD), physical dormancy (PY) or combinational dormancy (PY + PD). Each treatment group within species consisted of 100 seeds. Seeds selection and treatments are discussed separately in their respective sections below. No tests were conducted for morpho-(physio-)logical dormancy due to limited seed availability, and seeds that are morpho(physio)logically dormant are likely not suitable for commercial growing purposes or reforestation projects because they need more time to reach an embryonic stage suitable for germination (Finch-Savage & Leubner-Metzger, 2006).

Table 1. The focal species used, the amount of seeds that were used, the area of collections and their collector/collecting organisation.

Species: Amount of seeds: Source:

Zhantoxylum flavum 940 Collected by STINAPA Bonaire at Klein Bonaire

Myrcia curassavica 940 Christoffelpark

Capparis odoratissima 300 Daaibooi, CARMABI, Christoffelpark

Bourreria succulenta 100 Collected by Kranendonk in February and March of 2014

Seed selection Testing was done on a number of focal species, employing a substantial number of seeds (n>200). As an addition to previously conducted experiments by Kranendonk (2014), a smaller sample of Bourreria succulenta seeds was included to test if an extended period of dry storage would affect germination rates. M. curassavica was largely available in the Christoffelpark, Z. flavum was offered to CARMABI by STINAPA Bonaire and C. odoratissima is a particularly hard species to obtain seeds from, since they only shed their seeds for a short period after it has rained. After the seeds were collected the sink-or-float method was used to select healthy seeds for the germination experiments based on their density (a live embryo in the seed would have a high density, causing the seed to sink). The seeds were submerged in water, healthy seeds sink, and unhealthy seeds float. The floating seeds were removed. The treatments listed in Table 2 are known to break seed dormancy in their respective dormancy classes and are separately discussed below.

Table 2. The groups used in this study with species name, group code, treatment received, group size, plant date, and the dormancy type the treatment was meant to break. PD is physiological dormancy, PY is physical dormancy and PY + PD is combinational dormancy.

Species Group code Treatment Group size (n)

Planted on Directed at

Z. flavum Z1 Control H20 100 28/10/2014

Z. flavum Z2 100 mg/l GA3 100 28/10/2014 PD

Z. flavum Z3 500 mg/l GA3 100 28/10/2014 PD

Z. flavum Z4 Sanding + 100 mg/l GA3 100 31/10/2014 PY + PD

Z. flavum Z5 Sanding + 500 mg/l GA3 100 31/10/2014 PY + PD

Z. flavum Z6 Sanding + 1000 mg/l GA3 100 31/10/2014 PY + PD

Z. flavum Z7 1000 mg/l GA3 100 31/10/2014 PD

Z. flavum Z8 Control 100 29/10/2014

Z. flavum Z9 Sanding 100 29/10/2014 PY

M. curassavica M1 Control H20 100 29/10/2014

M. curassavica M2 100 mg/l GA3 100 29/10/2014 PD

M. curassavica M3 500 mg/l GA3 100 29/10/2014 PD

M. curassavica M4 Sanding + 100 mg/l GA3 100 29/10/2014 PY + PD

M. curassavica M5 Sanding + 500 mg/l GA3 100 30/10/2014 PY + PD

M. curassavica M6 Sanding + 1000 mg/l GA3 100 04/11/2014 PY + PD

M. curassavica M7 1000 mg/l GA3 100 04/11/2014 PD

M. curassavica M8 Control 100 04/11/2014

M. curassavica M9 Sanding 100 04/11/2014 PY

B. succulenta B1 5 months dry storage 120 06/11/2014 MD

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C. odoratissima C8 Control 100 07/11/2014

C. odoratissima C2 Control H20 100 07/11/2014

C. odoratissima C3 500 mg/l GA3 100 07/11/2014 PD

Experimental design The seeds were sown in sowing trays and received fungicidal treatment. The fungicide used was CAPTAN 50 wettable powder. The active component was N-Trichloromethylthio-4-cyclohexene-1,2-dicarboximide, and the concentration used was 4 g/l (based on usage instructions for trees). The used soil consisted of potting soil mixed with river sand in a 70/30 ratio. The trays with 40 seeds each were set up at random throughout CARMABI’s nursery (Figure 2). Some trays shared a treatment in this setup, in order to use the available space in the greenhouse as efficient as possible and not to waste materials. The trays were watered by an automated dripping system keeping the soil moist throughout the day. This required the cells to be watered at 7 AM, 1PM and 7PM for a total of 100 ml water per day per cell (the dripping system ran for one minute at each of the previously mentioned times, at a rate of 2 l/hour). Two control groups were used, one received a 24 hour soak in water to simulate GA3 treatment without the active hormonal component and one with no interference from the experimenter, e.g., planted dry. The first control group (soaked in water) was used to remove soaking in liquid as a parameter from the gibberellic acid treatment, and the dry control was used as a control group to compare all other treatments to.

Treatments

Gibberellic Acid (GA3) This treatment is directed at breaking the most common of three forms of physiological dormancy

(PD), i.e., non-deep PD. In high concentrations it is also known to break intermediate PD (Baskin &

Baskin, 2004). Gibberellins in seeds with non-deep PD induce germination by removing either a block

in seed embryo growth or a block conveyed by the seed embryo-covering layers (Finch-Savage &

Leubner-Metzger, 2006). An alternative would be GA4+7. The GA used was GA3 since this is the most

commonly used to break dormancy (Koyuncu, 2005; Takata, da Silva, Corsato, & Ferreira, 2014; Zhao

& Jiang, 2014).

GA3 will be used in three different concentrations, 100 mg/l, 500 mg/l and 1000 mg/l, prepared by appropriately diluting with distilled water, 1 g of GA3 dissolved in 70% ethanol until no solid particles were visible to the naked eye (10ml were used in this case). The seeds were soaked in GA3 for 24 hours and planted directly afterward.

Figure 2. CARMABI's nursery. Left: Picture of the nursery. Right: Schematic representation of the experiment. The path is displayed in the middle and two countertops filled with trays above and below. The blue lines represent the dripping system, each rectangle represents a sowing tray and the red and green colours are examples of treatments. Note that all of the trays held seeds that received a treatment, but only a few have been coloured in the image.

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Sanding Sanding is a scarification method used to break physical dormancy (PY). It weakens or breaks the

endocarp by chafing and allows water to permeate. Due to the large amount of seeds and a limited

supply of corrosive chemicals, sanding has been chosen for this study. Sanding paper (grain size: 200)

was used, multiple seeds were placed between 2 sheets of sanding paper which were then moved

against each other applying a small amount of pressure with the hands (enough to keep them

together). The seeds were sanded until chafing was visible on the exterior of the seeds. This took

approximately 10 back and forth strokes of the sanding paper per batch. The aim was not to damage

the inside of the seed, this would negatively affect germination.

Data processing Over the 12-week testing period, all germinated individuals were grouped by the treatment received

and counted. The last count was done on Friday, January 23rd, 2015. Germinated individuals were

counted manually and their cells were marked using toothpicks to avoid counting the same

germinated individual twice. The amounts of germinated seeds per group were tested for significant

differences (p<0.05) using a Chi-Square test for equality of proportions. All descriptive and inferential

statistics were done using RStudio (the packages used were reshape2, ggplot2, ggthemes and scales).

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3. Results

Z. flavum After 12 weeks a total of 82 (9.1% of 900 planted Z. flavum seeds) germinated. The most successful

treatment, the Control H20, yielded 16 germinations out of 100 sown seeds. The control being the

most effective indicates that the treatments were not effective in increasing germination.

Germination per treatment per week is displayed in Figure 3.

Figure 3. The cumulative amount of germinated individuals per treatment group over the testing period.

A 9-sample Chi-Square test for equality of proportions between the number of germinated

individuals per group after 12 weeks resulted in significant differences between the groups (p =

0.0491), indicating that some treatments were more effective than others. Each possible

combination of groups was tested for significant differences using a standard Chi-Square test, the

groups found to differ from each other have been displayed in Table 3 (all comparisons between

groups are listed in Appendix 2).

Table 3. Significant differences between treatment groups for Z. flavum germination (p <0.05).

Comparison p

Control H20 vs. Sanding + 100 mg/l GA3 0.0461

Control H20 vs. Sanding + 1000 mg/l GA3 0.0169

100 mg/l GA3 vs. Sanding + 1000 mg/l GA3 0.0169

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M. curassavica Out of 900 sown M. curassavica seeds only 2 germinated over the 12 week testing period, one in the

100 mg/l GA3 treatment group and one in the Sanding treatment group (Table 4

Treatment: Total germinated (n):

Control 0

Control H20 0

100 mg/l GA3 1

500 mg/l GA3 0

1000 mg/l GA3 0

Sanding 1

Sanding + 100 mg/l GA3 0

Sanding + 500 mg/l GA3 0

Sanding + 1000 mg/l GA3 0

). No statistical analysis for was performed because of the small amount of results that were

obtained.

Table 4. Germinated individuals per treatment group for M. curassavica.

Treatment: Total germinated (n):

Control 0

Control H20 0

100 mg/l GA3 1

500 mg/l GA3 0

1000 mg/l GA3 0

Sanding 1

Sanding + 100 mg/l GA3 0

Sanding + 500 mg/l GA3 0

Sanding + 1000 mg/l GA3 0

B. succulenta During the 12 week testing period 1 out of 120 B. succulenta seeds germinated.

C. odoratissima During the 12 week testing period only two out of 300 sown C. odoratissima seeds germinated, both

of which in the 500 mg/l GA3 group (Table 5). No statistical analysis was performed because of the

small amount of results that were obtained.

Table 5. Germinated individuals per treatment group for C. odoratissima.

Treatment: Total germinated (n):

Control 0

Control H20 0

500 mg/l GA3 2

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4. Discussion

Z. flavum Although significant differences have been found between some treatments of Z. flavum, the differences were not consistent between treatments directed at different dormancy types, thus no conclusions can be drawn as to the dormancy type of Z. flavum solely based on these results. It is interesting that the water control was the most effective treatment with 16 germinated seeds. Although not by far, and no significant different were found between the second (15 germinations) and third (10 germinations) best treatments. This suggests that the seeds are not dormant at all, because an obviously unfavourable condition (in this case, lack of water) for germination is an example of how the environment rather than some factor associated with the seed per se prevents germination (Baskin & Baskin, 2014). To prove this, a study should be conducted to rule out the other dormancy types (medium physiological dormancy, deep physiological dormancy and morphological dormancy). If no treatment directed at breaking any other dormancy types proves to be effective, then it would be safe to conclude that this species does not produce dormant seeds. Intermediate PD and deep PD do not appear to be likely in any Curaçaoan species because there is very little temperate variation throughout the year (MDC, 2015) but Baskin and Baskin (2014) do list Z. flavum as being PD, based on research by Marrero, Castilleja, and Francis (as cited in Baskin & Baskin, 2014). If this was a case of non-deep PD, PY or PY + PD, their respective treatments should have yielded much higher results. M(P)D, intermediate PD, and deep PD are still contenders because they were not tested for in this study. Because Baskin and Baskin (2014) only found PD in Zhantoxylum species (Z. flavum, Z. kellermanii, Z. dipetalum, Z. gilletii and Z. kavaense), intermediate or deep PD is by far the most likely. For a conclusive statement to be made, a study should be conducted for those dormancy types.

Other species C. odoratissima, B. succulenta and M. curassavica appear to be dormant because no form of treatment increased the amount of germinated seeds. The sink or float method used to determine seed health should also be questioned in these cases, as only 5 out of 1320 planted seeds germinated. A better method to use in the future would be to look for embryos in seeds. This can be done using x-ray analysis (Sivakumar et al., 2007) or by manually cutting seeds open under a microscope (Baskin & Baskin, 2014). The reasons for embryoless seeds are many, including degeneration of zygote (Baskin & Baskin, 2014), death of embryo (Vijayalakshmi, 2000), presence of a mutation that causes failure of embryo and endosperm development (Hong, Aoki, Kitano, Satoh, & Nagato, 1995), degeneration of ovule (Daskalova, 1977) and infestation by insects (Baskin & Baskin, 2014). Given the low overall yield of these species, it would not be safe to rule out non-deep PD, PY, and PD + PY. This leaves all of the dormancy classes open as possibilities. No prior research towards C. odoratissima has been found. B. succulenta belongs to the PD category according to Castilleja and Francis & Rodriguez (as cited in Baskin & Baskin, 2014), but since data from this study rules out non-deep PD, only intermediate and deep PD have been left open as options. Interestingly, M. cuprea, a species in the same family as M. curassavica had 51% germination in black potting soil (Neire Maria Mendes Ferreira & Gurgel, 2013) without any treatment. M. curassavica did not display a similar result in this study. Finch-Savage and Leubner-Metzger (2006) suggest that these species should both not be dormant (derived from their family, the Myrtaceae) but Baskin and Baskin (2014) have since then found PD in M. crassifolia, M. deflexa, M. selloi and M. tomentosa. This difference in findings could have occurred because of the sink and float method not functioning properly with this species (in this case the unhealthy seeds would sink and be planted instead of

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being removed from the study), or because M. curassavica has a different dormancy class than other species in the family of Myrtaceae. M(P)D could be a possibility for M. curassavica, as well as PD, PY, and PD + PY when assuming that the sink or float method did not lead to the desired healthy seeds being used. When assuming that the sink or float method worked as intended, it is necessary to note that Baskin and Baskin (2014) expected that in tropical and/or subtropical regions of the world there may be some species whose seeds have intermediate PD that require only a long period of exposure to high temperatures for dormancy break to occur. This could indicate that each year only seeds that had been dispersed the previous year would germinate, but this is only speculation.

5. Conclusions The Z. flavum seeds not responding to any form of treatment conducted in this study rules out non-deep PD, and literary findings concerning Zhantoxylum species in the Rutaceae family make PY and PD +PY highly unlikely. Since intermediate PD and deep PD were not tested for, it is safe to assume that Z. flavum is either intermediately or deeply physiologically dormant. However, no conclusions can be drawn from this study related to the dormancy of C. odoratissima, B. succulenta or M. curassavica. The low amount of germination found in this study could be caused by either dormancy or a large amount of embryoless seeds that were not removed using the sink or float method. A study should be conducted on these species with emphasis on seed selection, and possibly M(P)D, with repetition of the methods used in this study to determine by which cause these low germination rates occurred. The dry storage of one group of B. succulenta seeds included in this study did not positively affected germination rates when compared to Kranendonk (2014).

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New Phytol, 171(3), 501-523. doi: 10.1111/j.1469-8137.2006.01787.x Hong, S. K., Aoki, T., Kitano, H., Satoh, H., & Nagato, Y. (1995). Temperature-sensitive mutation,

embryoless 1, affects both embryo and endosperm development in rice. Plant Science, 108(2), 165-172. doi: http://dx.doi.org/10.1016/0168-9452(95)04128-H

Jonker, M. (1996). Toetsinsgcriteria voor het gebruik van conserveringsgebieden. De uitwerking van de bestemming conserveringsgebied van het EOP (art. 9). Willemstad.

Koyuncu, F. (2005). Breaking seed dormancy in black mulberry (Morus nigra L.) by cold stratification and exogenous application of gibberellic acid. Acta Biologica Cracoviensia Series Botanica, 47(2), 23-26.

Kranendonk, L. (2014). Seed germination methods for native Caribbean trees and shrubs with emphasis on the species Bourreria succulenta.

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Appendix 1: Economically relevant endemic taxa of Curaçao Appendix 1 Names of Curaçaoan endemic trees and shrubs that grow on limestone/sand or on any type of ground. Species from 'De inheemse bomen van de ABC eilanden', J.A. de Freitas, 1996. and dormancy classes from 'Seed dormancy and the control of germination', Finch-Savage & Leubner-Metzger, 2006. *Species was not in original list by J.A. de Freitas but has since become interesting to perform germination experiments on.

No. Name Papiamentu name Family Dormancy class**

1 Amyris simplicifolia Rutaceae PD, ND

2 Avicennia germinans Mangel blanku Accanthaceae -

3 Bontia daphnoides Oleifi Myoporaceae -

4 Bourreria succulenta Watakeli Boraginaceae -

5 Bursera karsteniana Pal'sia blanku Burseraceae -

6 Bursera simaruba Pal'sia kora Burseraceae -

7 Bursera tomentosa Takamahak Burseraceae -

8 Caesalpinia coriaria Watapana Scrophulariaceae PD, ND

9 Capparis hastata Palu di lora Capparaceae -

10 Capparis indica Oliba machu/palu pretu Capparaceae -

11 Capparis odoratissima Oliba Capparaceae -

12 Casearia tremula Palu di Boneiru Flaculteaceae -

13 Coccoloba swartzii Kamari Polygonaceae PD, ND

14 Coccoloba uvifera Dreifi di laman Polygonaceae PD, ND

15 Condalia henriquezii Beshi Rhamnaceae PD, ND, PY, PY + PD

16 Conocarpus erectus Mangel blanku Combretaceae -

17 Cordia dentata Kohara Borachinaceae -

18 Crescentia cujete Kalbas Digmoniaceae -

19 Crossopetalum rhacoma Palu di pushi Celastraceae -

20 Croton niveus Lumbra blanku//bara blanku Euphorbiaceae -

21 Ficus brittonii Mahawa/Mahok di mondi Moraceae -

22 Guaicum officinale Wayaka Zygophyllaceae -

23 Guaicum sanctum Wayaka shimaron Zygophyllaceae -

24 Guapira pacurero Mashibari Nyctagimaceae -

25 Haematoxylon brasiletto Brasil Fabaceae PD, ND, PY, PY + PD

26 Hippomane mancinella Manzanilla Euphorbiacaae PD, ND

27 Jacquinia armillaris Palu huku Theophractaceae -

28 Krugiodendrom ferreum Koubati Rhamnaceae PD, ND, PY, PY + PD

29 Laguncularia racemosa Mangel blanku Combretaceae -

30 Machaonia ottonis Palu heru Rubiaceae PD, ND

31 Malpighia emarginata Shimaruku Malpighiaceae -

32 Manihot cartaginensis Kasabi marga/Marihuri Euphorbiaceae PD, ND

33 Maytenus versluysii Celastraceae -

34 Metopium brownei Manzanilla bobo Anacardiaceae -

35 Pithecellobium unguis-cati Beshi di yuana/unja gato Fabaceae PD, ND, PY, PY + PD

36 Prosopis juliflora Kuida Fabaceae PD, ND, PY, PY + PD

37 Sabal sp Sabal Arecaceae -

38 Sideroxylon obovatum Rambeshi Sapotaceae -

39 Tabebuia billbergi Kibrahacha Bigononiaceae PD, ND

40 Thespesia populnea Palu santu Malvaceae ND, PY, PY + PD

41 Ximenia americana Kashu di mondi Ximeniaceae -

42 Zanthoxylum flavum Kalabari Rutaceae PD, ND

43 Zanthoxylum monophyllum Bosua Rutaceae PD, ND

44 Myrcia curassavica* Myrtaceae -

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Appendix 2: P-values for Z. flavum treatment comparisons Appendix 2 Comparisons between each treatment group with respective p-values (p-values below 0.05 have been displayed in bold text).

Comparison p

Control vs. Control H20 0.0824

Control vs. 100 mg/l GA3 0.1199

Control vs. 500 mg/l GA3 1.0000

Control vs. 1000 mg/l GA3 1.0000

Control vs. Sanding 0.7961

Control vs. Sanding + 100 mg/l GA3 1.0000

Control vs. Sanding + 500 mg/l GA3 0.6152

Control vs. Sanding + 1000 mg/l GA3 0.5383

Control H20 vs. 100 mg/l GA3 1.0000

Control H20 vs. 500 mg/l GA3 0.0824

Control H20 vs. 1000 mg/l GA3 0.1357

Control H20 vs. Sanding 0.2085

Control H20 vs. Sanding + 100 mg/l GA3 0.0461

Control H20 vs. Sanding + 500 mg/l GA3 0.3022

Control H20 vs. Sanding + 1000 mg/l GA3 0.0169

100 mg/l GA3 vs. 500 mg/l GA3 0.1199

100 mg/l GA3 vs. 1000 mg/l GA3 0.1911

100 mg/l GA3 vs. Sanding 0.2846

100 mg/l GA3 vs. Sanding + 100 mg/l GA3 0.0694

100 mg/l GA3 vs. Sanding + 500 mg/l GA3 0.3999

100 mg/l GA3 vs. Sanding + 1000 mg/l GA3 0.0169

500 mg/l GA3 vs. 1000 mg/l GA3 1.0000

500 mg/l GA3 vs. Sanding 0.7961

500 mg/l GA3 vs. Sanding + 100 mg/l GA3 1.0000

500 mg/l GA3 vs. Sanding + 500 mg/l GA3 0.6152

500 mg/l GA3 vs. Sanding + 1000 mg/l GA3 0.5383

1000 mg/l GA3 vs. Sanding 1.0000

1000 mg/l GA3 vs. Sanding + 100 mg/l GA3 0.7835

1000 mg/l GA3 vs. Sanding + 500 mg/l GA3 0.8065

1000 mg/l GA3 vs. Sanding + 1000 mg/l GA3 0.3747

Sanding vs. Sanding + 100 mg/l GA3 0.5944

Sanding vs. Sanding + 500 mg/l GA3 1.0000

Sanding vs. Sanding + 1000 mg/l GA3 0.2535

Sanding + 100 mg/l GA3 vs. Sanding + 500 mg/l GA3 0.4381

Sanding + 100 mg/l GA3 vs. Sanding + 1000 mg/l GA3 0.7480

Sanding + 500 mg/l GA3 vs. Sanding + 1000 mg/l GA3 0.1675