Research Article |
Corresponding author: Mike Opgenorth ( mopgenorth@ntbg.org ) Corresponding author: Dustin Wolkis ( dwolkis@ntbg.org ) Academic editor: Sergey Rosbakh
© 2024 Mike Opgenorth, Emily Sailing, Nina Rønsted, Dustin Wolkis.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Opgenorth M, Sailing E, Rønsted N, Wolkis D (2024) Gardenia (Rubiaceae) seed conservation physiology with emphasis on rare Hawaiian species. Plant Ecology and Evolution 157(2): 220-235. https://doi.org/10.5091/plecevo.120167
|
Background and aims – Gardenia species are ecologically, culturally, and economically significant but the three native species of Gardenia in Hawai‘i are assessed as Critically Endangered. Seed banking is the most cost effective and efficient means of conserving plant material ex situ. To better understand the conservation physiology of Hawaiian and South Pacific Gardenia spp. and support their conservation, we asked 1) How do seeds respond to different temperatures and light and dark regimes? 2) What class of dormancy, if any, do seeds exhibit? 3) How does seed germinability respond over time in a seed bank? and 4) What is the conservation status and level of ex situ representation of Gardenia globally?
Material and methods – To answer these questions, we used 19 accessions of fresh seeds and seeds stored for varying periods of time in the National Tropical Botanical Garden’s Conservation Seed Bank and Laboratory of Hawaiian (G. brighamii, G. remyi), New Caledonian (G. aubryi, G. oudiepe), and Tahitian (G. taitensis) species. Seeds were incubated at varying temperatures and in light, and in dark.
Key results – We found that (1) seeds of all species tested germinated slowly and only at higher temperatures in the light and dark, (2) seeds have non-deep physiological dormancy, (3) seeds of the Hawaiian species are short lived at conventional seed bank conditions, and (4) only 40% of Gardenia spp. are represented in ex situ facilities, and 66% of the species have not been evaluated for the IUCN Red List.
Conclusion – Seeds of Hawaiian Gardenia spp. are short lived in storage. Since seeds germinate in darkness, they are unlikely to form a persistent soil seedbank. Although seeds of all species tested are physiologically dormant, they can be easily propagated from seed at warmer temperatures, giving some hope to the conservation and restoration of the Critically Endangered Hawaiian species. Since our dataset was limited by a lack of continuous viability monitoring, we emphasize the need for initial germination testing and ongoing viability tests to better understand seed longevity. Lastly, we discuss the ecological relevance of our results in the context of the Hawaiian archipelago.
dormancy, exceptional species, germinability, Hawaii, longevity, recollection intervals, seed banking, threatened species, viability
Plant species are becoming extinct tens to hundreds of times faster than the background extinction rate, making the need for effective plant conservation programs more acute than before (
Located over 4,000 km from the nearest continental landmass, the Hawaiian Archipelago is home to 1,367 native vascular plant taxa (
The Critically Endangered Hawaiian taxa include three native Gardenia J.Ellis: Gardenia brighamii H.Mann (Fig.
Hawaiian Gardenia. A. Flower of G. brighamii at Kānepu‘u, Lāna‘i. B. Immature fruit of G. brighamii at Kānepu‘u, characterized by hard fruits with green surface and nodular texture. C. Maturing fruit from G. remyi at Kalua‘aha, Moloka‘i, fruit texture is fleshy and compressible by hand. D. Removing seeds from fruit pulp of G. remyi to prepare for sowing and/or storage. Mesocarp is amber, with the endocarp displaying deep orange pigment with over 100 seeds inside. E. Cleaned G. remyi seed profile when pulled out of storage prior to sowing. F. Healthy seedling of G. remyi (#20150377) eight weeks after sowing collected from Hūle‘ia, Kaua‘i.
Globally, Gardenia species are native to the tropics and subtropics from Africa through the Indo-Pacific region (Fig.
Global native range of Gardenia. There are currently 128 recognized species of Gardenia, with diversity centres in Australia, Borneo, Cambodia, Central African Republic, Fiji, Papua New Guinea, Thailand, and Vietnam, all of which are home to eight or more native Gardenia. Map created with ArcGIS Desktop v.10.4 (
Hawaiian Gardenia species are at risk of going extinct. The most recent IUCN Red List assessments for the three Hawaiian endemic Gardenia species designate them as Critically Endangered (CR) (
In a recent study estimating ex situ seed longevity of 295 native Hawaiian species,
Seed banking and restoration efforts from seeds are often impeded by a lack of a priori knowledge of seed dormancy-breaking and germination requirements (
In the present study, with respect to Pacific Island Gardenia spp., we ask (1) How do seeds respond to different temperatures and light regimes? (2) What class of dormancy, if any, do seeds exhibit? (3) How does seed germinability respond over time in a seed bank? and (4) What is the conservation status and level of ex situ representation of Gardenia globally? By answering these questions, we aim to inform conservation and restoration practitioners, thereby improving the conservation outlook of these rare species.
In total, 19 accessions of five species collected between 1992 and 2023 were included in the 48 accession experiment combinations in the present study (Table
Summary of mean and standard deviation germinated for each accession and experiment. NTBG: National Tropical Botanical Garden, Hawai‘i.
NTBG accession number | Taxon | Experiment | Mean (s.d.) proportion germinated |
---|---|---|---|
980618 | Gardenia aubryi | 25 years old | 0(0) |
980673 | Gardenia aubryi | 25 years old | 0(0) |
010250 | Gardenia brighamii | 22 years old | 0.92(0) |
060803 | Gardenia brighamii | 20 years old | 0.16(0.15) |
120542 | Gardenia brighamii | 5 years old | 0.22(0.19) |
130066 | Gardenia brighamii | 10 years old | 0.6(0.1) |
130066 | Gardenia brighamii | 5 years old | 0.71(0.1) |
20160848 | Gardenia brighamii | 5 years old | 0.03(0.06) |
20160848 | Gardenia brighamii | immature - no soak | 0(0) |
20160848 | Gardenia brighamii | immature - soaked in 2% PPM 17 h | 0(0) |
20160848 | Gardenia brighamii | mature - soaked in 2% PPM 17 h | 0.79(0.16) |
20160848 | Gardenia brighamii | mature - no soak | 0.91(0.01) |
900569 | Gardenia brighamii | 31 years old | 0(0) |
920714 | Gardenia brighamii | 31 years old | 0(0) |
950116 | Gardenia brighamii | 28 years old | 0(0) |
980625 | Gardenia oudiepe | 25 years old | 0(0) |
080811 | Gardenia remyi | 15/5 light | 0(0) |
080811 | Gardenia remyi | 20/10 light | 0(0) |
080811 | Gardenia remyi | 25/15 dark | 0(0) |
080811 | Gardenia remyi | 25/15 light | 0(0) |
080811 | Gardenia remyi | 30/20 light | 0(0) |
080811 | Gardenia remyi | 8 years old | 0.94(0.12) |
080811 | Gardenia remyi | heat shock | 0(0) |
080811 | Gardenia remyi | sterile agar | 0(0) |
080811 | Gardenia remyi | surgery | 0(0) |
120384 | Gardenia remyi | 15/5 light | 0(0) |
120384 | Gardenia remyi | 20/10 light | 0(0) |
120384 | Gardenia remyi | 25/15 dark | 0(0) |
120384 | Gardenia remyi | 25/15 light | 0(0) |
120384 | Gardenia remyi | 30/20 light | 0(0) |
120384 | Gardenia remyi | 4 years old | 0.73(0.06) |
120384 | Gardenia remyi | 5 years old | 0.43(0.1) |
120384 | Gardenia remyi | heat shock | 0(0) |
120384 | Gardenia remyi | sterile agar | 0(0) |
120384 | Gardenia remyi | surgery | 0(0) |
20150377 | Gardenia remyi | 8 years old | 0.16(0.08) |
20150613 | Gardenia remyi | 25/15 dark | 0.8(0.07) |
20150613 | Gardenia remyi | 25/15 light | 0.73(0) |
20230237 | Gardenia remyi | initial | 1(NA) |
20230238 | Gardenia remyi | initial | 1(NA) |
20230257 | Gardenia remyi | initial | 0.84(0.1) |
20230068 | Gardenia taitensis | 15/5 dark | 0(0) |
20230068 | Gardenia taitensis | 15/5 light | 0(0) |
20230068 | Gardenia taitensis | 20/10 dark | 0(0) |
20230068 | Gardenia taitensis | 20/10 light | 0.02(0.04) |
20230068 | Gardenia taitensis | 25/15 light | 0.98(0.04) |
20230068 | Gardenia taitensis | 30/20 light | 0.98(0.04) |
Of the three native Hawaiian Gardenia spp., G. brighamii, and G. remyi seeds were used in the present study (no seeds or living plants of G. mannii existed at NTBG Seed Bank (National Tropical Botanical Garden, Hawai‘i) and Laboratory or gardens at the time this manuscript was written). G. brighamii fruits were collected from Kaua‘i and Lāna‘i Islands between May 1992 and December 2016. Seeds of G. remyi were collected from Kaua‘i and Moloka‘i Islands between September 2008 and June 2023. Seeds of Gardenia aubryi Vieill and G. oudiepe Vieill were collected in New Caledonia in June 1998. Gardenia taitensis DC. seeds were collected from a private property on Kaua‘i Island in February 2023 (Supplementary material
To determine the temperature and light/dark requirements for germination, three previously stored G. remyi accessions (#080811, #120384, #20150613) and one freshly harvested (26 days since collection) G. taitensis accession (#20230068) were used (Supplementary material
Two accessions of G. remyi (#080811, #120384) seeds were sterilized (Supplementary material
Mature and immature fruits of one accession (#20160848) of G. brighamii collected on 20 Dec. 2016 from NTBG’s McBryde Garden was used to assess the effects of maturity on germination, as well as to assess the effects of PPM on pathogen abatement and viability. Seeds were held at ambient temperature and humidity until experiments started 23–27 Dec. 2016. Three replicates of 30–33 seeds each were used in the following treatments; immature (seed arising from immature fruit), immature + PPM (17-hour soak in a solution of 2% PPM in distilled water), and two replicates of 25 and 30 seeds each of mature (seeds arising from mature fruit), mature + PPM (Supplementary material
For some species, desiccation tolerant seeds lose viability more rapidly than expected at conventional storage temperatures. The incomplete melting of fatty acids upon retrieval from cold storage could be a source of damage during seed imbibition, and brief exposure to high temperatures (“heat shock”) could melt lipids that would otherwise be frozen at incubation temperatures (
In some species with physiological dormancy, low growth potential, or push power, of the embryo plays an important role in delaying germination (
Seeds removed from storage as well as fresh collections were placed in 60 mm Petri dishes on seed germination paper (Anchor Paper Company, St. Paul, MN, USA) and placed above a saturated potassium sulphate solution in a sealed chamber (achieving an RH of ~98% at 25°C) overnight to avoid imbibition damage (for previously stored seeds unless indicated above). Following this step, seeds and germination paper were wetted with 0.1% PPM. Dishes were then sealed with plastic paraffin film to prevent excessive water loss. Seeds were exposed to daily alternating regimes of 12 h of light (~41 mmol/m2/s cool white [4100 K] fluorescent light) and 12 h of dark at corresponding temperatures of 25/15°C in a germination chamber (GR36L, Percival Scientific, Perry, IA, USA). Germination was defined as radicle emergence, and the proportion germination was calculated by dividing the number of seeds germinated by the number of seeds sown.
In order to place Hawaiian and Pacific Gardenia germination data into context of the larger genus, we identify three primary sources that constitute the majority of published Gardenia studies to date. First, the Seed Information Database (
Some of the global Gardenia taxa listed in the Seed Information Database were synonyms of other accepted species names according to the Plants of the World Online (
Mean monthly temperature and mean annual temperature data for each Hawaiian accession that included latitude and longitude were harvested from the Online Climate Atlas of Hawai‘i (
The software RStudio (
For the two New Caledonian Gardenia species stored for 25 years, seeds neither germinated from G. aubryi (#980618, #980673) nor from G. oudiepe (#980625) (Table
For the eight tested accessions of G. brighamii stored for 0–31 years, germination ranged from 0–92%. In one accession stored for five years with no other longevity tests (#120542), mean germination was 22% (± 19). Only two G. brighamii accessions had more than one test. For #130066, germination declined from 71% (± 10) at year 5 to 60% (± 10) at year 10, but the difference between them was not significant (p = 0.2488). In #20160848, the highest initial viability test was 91% (± 10), and declined to 3% (± 6) at year 5. For the remaining 5 accessions stored for 20–31 years germination occurred in only a single accession (#010250; 22 years old), and was, remarkably, 92% (± 0) (Table
In one accession of G. remyi (#080811), mean germination was quite high after 8 years at 92% (± 12). However, at year 14, no seeds germinated (including those used in other experiments, see below) (Table
Seeds neither germinated from the surgical treatment experiment, nor from the sterile condition experiment, nor from the heat shock experiment. Unbeknown to us, germinability of the seeds used in these experiments had already decreased to zero by the time the experiments were begun.
For one accession of G. remyi (#20150613) stored for 8 years then incubated at 25/15°C, germination in light (12 h light/12 h dark) was observed to have begun between weeks 3 and 5, and maximum germination was observed at week 27 (the seeds exposed to constant darkness was left unopened until after week eleven). Mean maximum germination was 73% (± 0) and 80% (± 7) in light and dark, respectively, with no significant difference between them (p = 0.2254; Fig.
Germination time courses with mean proportion germinated, and ± standard deviation (error bars). A. Gardenia remyi (#20150613) used in the light requirement experiment. The dark treatment was incubated at 25/15°C in constant darkness (wrapped in aluminium foil) and unopened until 74 days had elapsed since sowing. The light treatment was incubated at 25/15°C and exposed to a daily alternating photoperiod of 12 h light/ 12 h dark. There was no significant difference in mean maximum germination between the two treatments (p = 0.2254). B. Gardenia taitensis (#20230068) used in the temperature requirements experiment. Mean proportion germinated ± standard deviation (error bars) is presented. The four treatments shown were incubated in a daily alternating photoperiod of 12 h light/ 12 h dark. No seeds germinated at 15/5°C. Mean maximum germination was equivalent at 25/15 and 30/20°C (p = 1.0000). C. Effects of maturity and plant preplant preservative mixture (PPM) on germination of Gardenia brighamii (#20160848). Mean proportion germinated ± standard deviation (error bars) is presented. No seeds germinated from immature fruits. There was no significant difference in mean maximum germination between the two mature treatments (p = 0.3169).
For Gardenia taitensis (#20230068) collected in February 2023, no seeds germinated at 15/5°C regardless of light regime. Similarly, only one seed germinated (in light) for seeds sown in light and dark at 20/10°C. For seeds incubated at 25/15°C and 30/20°C, 50% germination was reached at between weeks 7–9 and 3–5, respectively, and had concluded by weeks 30 and 15, respectively. Mean germination in light at 25/15°C and 30/20°C at week 5 was 24% (± 14) and 89% (± 8), respectively with a significant difference between them (p = 0.0052), and mean maximum germination were both 98% (± 4) (Fig.
No seeds germinated from G. brighamii immature fruits. Germination from G. brighamii seeds arising from mature fruit ranged from 79% (± 16; mature + PPM) to 91% (± 1; mature), and was not significantly different (p = 0.3169) according to a Welch Two Sample t-test (Fig.
The World Flora Online (
IUCN Conservation status for 128 global Gardenia spp. Eight (6.2%) are Critically Endangered, four (3.1%) are Endangered, four (3.1%) are Vulnerable, two (1.6%) are Near Threatened, 25 are of Least Concern, and one is Data Deficient. The remaining 84 taxa are Not Evaluated (NE) (
The Botanical Garden Conservation International (BGCI) PlantSearch tool for accessing and sharing information about living collections at ex situ facilities around the globe have records for 51 species (40%) of Gardenia, not including hybrids, subspecies, and synonyms. Of these 51 species, 27 (21%) are represented at a single ex situ site (
Of the 15 species (12%) of Gardenia with reported seed storage behaviour in the Seed Information Database, 100% of them are reported as some form of orthodox (orthodox, orthodox?, or orthodox p), i.e. no recalcitrance or intermediate storage behaviour is reported (
Longevity of the study species and accessions varied widely. This is not surprising, since the rate of decline as well as the theoretical initial viability are collection level traits (
With the exception of three accessions having one previous test each, previous germination tests were never performed. Thus, it is impossible to determine when germinability declined to p70 (the recollection interval suggested by
The trait of seed longevity can vary with seed maturity (
The pattern of seeds stored cool having higher longevity compared to seeds stored in subfreezing temperatures as observed in Hawaiian Gardenia (see
One measure of longevity that is biologically meaningful and intuitive is p50, defined as the time for viability to decline to 50% (
Gardenia taitensis seeds experienced little to no germination (≤ 0.02%) in light at the cooler temperatures of 15/5 and 20/10°C, but they germinated to 98% in light at both 25/15 and 30/20°C. Thus, G. taitensis seeds appear to require these warmer temperature regimes for germination to occur. In a similar experiment that included G. brighamii, the optimal temperature regime for germination to occur was 25/15°C but 30/20°C was not tested (
There was no significant difference in one 8-year-old accession of G. remyi seeds incubated at 25/15°C in the light vs dark. Thus, there is no light requirement for the germination of this species. The germination of seeds in continuous darkness suggests that G. remyi is unlikely to form a long‐lasting soil seed bank; however, more studies are needed to determine whether this is the case in their natural habitat. If the seeds germinate while buried, this would lead to the loss of a viable soil seed bank. Therefore, the re‐establishment of extirpated populations will require reintroduction. After the germination experiment for G. taitensis had ended, the Petri-dishes were placed at ambient temperature and light conditions. When we were cleaning up and ready to dispose of the seeds, we noticed that germination had occurred in three Petri-dishes, all of which had been in the cool and dark treatments (two dishes/4 total seeds at 15/5°C and one dish/5 total seeds at 20/10°C). Because there was no difference in light vs dark in G. remyi and because few to no seeds of G. taitensis germinated at these temperatures, we hypothesize that this germination was due to an increase to ambient temperature (~25°C) rather than the addition of light. However, more research is needed to understand the effects of moving seeds from colder to warmer temperatures (
Seeds of Gardenia remyi stored for 8 years and then incubated at 25/15°C light started to germinate between weeks 3 and 5 and had concluded by week 27. Because seed coats are water permeable (inferred from congener G. brighamii;
Freshly harvested seeds of G. taitensis did not germinate at 15/5°C regardless of light regime, and only one seed germinated for seeds sown in light and dark at 20/10°C. For seeds incubated at 25/15 and 30/20°C, 50% germination was reached between weeks 7–9 and 3–5, respectively, and had concluded by weeks 30 and 15, respectively. Given this information and because seed coats are water permeable and embryos are fully developed (see discussion above), seeds of G. taitensis have non-deep PD. Of the Gardenia spp. studied by
A global analysis on the effect of macroclimate on seed dormancy found that species with seeds exhibiting PD had a high proportion of species in relatively drier climates, and that increasing annual range of temperature had a significant positive effect, precipitation seasonality had a small negative, yet significant effect, and mean annual temperature had no significant effect (
In the G. brighamii maturity experiment, no seeds from immature fruit germinated compared to 91% in seeds arising from mature (control) fruit. This highlights the need for collections to be made when fruits are at the stage of natural dispersal, especially for collections bound for ex situ seed storage. Seeds collected immature will be undeveloped and will lose viability when dried, or even fail to germinate altogether, and seeds collected too late may have reduced viability (
Hawaiian Rubiaceae are reported to be desiccation tolerant yet short lived at conventional storage temperatures (intermediate “freeze-sensitive”;
In seeds of species exhibiting physiological dormancy such as the Gardenia spp. discussed in the present study, a surgical treatment can reduce the push power needed for the radicle to emerge and therefore to overcome dormancy (
The Global Strategy for Plant Conservation (GSPC) is a program of the United Nations Convention on Biological Diversity with the aim to halt continuing loss of plant diversity. Within the GSPC, Target 8 calls for 75% of plant species to be preserved ex situ, preferably in their country of origin (
In addition, as only 43 out of 128 (34%) recognized species of Gardenia have been assessed under the IUCN Red List, a substantial information gap exists related to the conservation status of a majority of the members of this important genus. The remaining 66% of species that are Not Evaluated (NE) on the IUCN Red List require conservation assessments to determine which additional Gardenia species are threatened, and therefore prioritize which species should benefit from conservation activities in situ and ex situ.
Globally, at least 33% of IUCN Red List assessed threatened species (i.e. the three threatened categories; Critically Endangered, Endangered, Vulnerable) are estimated to produce desiccation intolerant seeds. Further, there is a positive relationship between the proportion of a species list that is likely to produce recalcitrant seeds and the proportion of the list made up by tree species from tropical moist forest and mangrove habitats (
Recently,
The results of the present study suggest that G. remyi and G. taitensis seeds exhibit non-deep physiological dormancy. We identified for the first time germination conditions for Critically Endangered G. remyi. Notably, there was no light requirement for germination indicating that the species is unlikely to form a persistent soil seed bank, which could, in part, explain some of the rarity of the species. Our results agree with those from other studies on Hawaiian species of Gardenia that generally seeds are “short lived”. We also found that seeds from immature seeds do not germinate, therefore fruits must reach full maturity prior to harvest for seed banking or propagation to be successful. Finally, with only 40% of the species of Gardenia represented at ex situ facilities, and 66% of the genus having not been evaluated for the IUCN Red List, we express the need for additional research and conservation assessments of more members of this iconic plant group.
The fact that the majority of the accessions used in the present study were never tested for initial germinability (nor any viability metric) as fresh seed highlights the need for initial germination testing especially, as well as subsequent longevity testing (
This project is the result of ‘laulima’ (lit. many hands). We thank Ben Nyberg for creating Figure
Summary of Gardenia accessions used in the experiment. The table presents accession provenance data, length of experiment, number of replicates, temperature, light, and media.
Gardenia data from
Global Gardenia distribution by region. The 128 Gardenia taxa recognized by Plants of the World Online (POWO) organized by taxonomic name, their IUCN status, and native range.