Appendix ES-11

OAHU ARMY NATURAL RESOURCES PROGRAM MONITORING PROGRAM

RESULTS OF A LABORATORY SEED SOW TRIAL FOR SUPERBA SUBSP. SUPERBA

A preliminary trial to assess germination rates of seeds from senesced versus fresh Cyanea superba subsp. superba fruit

Introduction

Limited dispersal and recruitment of Cyanea superba subsp. superba occurs at reintroduced populations, with the majority of fruits either depredated by (seeds are destroyed), or rotting on the and falling to the ground with limited subsequent seed germination and seedling survival, despite having typically high seed germination rates in fresh mature fruit (Pender et al. 2013, OANRP 2015a, 2015b, pers. obs.). Several factors may limit successful recruitment, including microsite specificity, predation of seedlings by slugs, soil moisture, light availability and fruit senescence. In order for the Oahu Army Natural Resources Program (OANRP) to achieve goals of long term self-sustaining C. superba subsp. superba populations, these issues must be taken into consideration. Should self-sustainment be ineffective, populations will require on-going replacement via outplanting or seed sowing. Greenhouse production and outplanting has been successful; however, efforts to determine if seed sowing is a feasible and more economical approach have been limited. This trial explores two questions to gain a preliminary understanding of recruitment limitations and factors affecting seed sow success. Do seeds from senesced C. superba subsp. superba fruit have reduced viability as compared with those from fresh mature fruit? If seed sowing is used to sustain populations, does total removal of fruit pulp promote higher germination rates? A laboratory trial was conducted by OANRP to examine 1) C. superba subsp. superba seed germination rates of senesced fruit in comparison with fresh material as a means of exploring the ability of seeds from senesced fruit to germinate upon falling on the ground vs. those from fresh fruit; and 2) germination rates of C. superba subsp. superba seeds with and without pulp extract to examine the effects of fruit pulp on germination during seed sow efforts.

Methods

Senesced and fresh mature C. superba subsp. superba fruits were collected from Makaha, Pahole and Kahanahaiki Management Units in December 2014 and January 2015 (Figures 1 and 2). All fruits were collected from infructescences (not from the ground). Fruits from 10 individuals were used for each of four treatments (with a minimum of 1 fruit per plant per treatment): untreated seeds of senesced fruits, seeds of senesced fruits with pulp extract, untreated seeds of fresh fruits, and seeds of fresh fruits with pulp extract. The degree of senescence was not quantified, but was estimated to be less than 1 week following peak maturation. Number of seeds sown per sample ranged from 22 to 200 (mean = 88.4, SD = 37.4, 3534 total seeds sown). Seeds were sown on filter paper in petri dishes. Filter paper with untreated seeds was moistened with deionized water, while those with pulp extract were moistened with decanted liquid from a solution of water and smashed fruit. Petri dishes were stored in a Percival Controlled Environment Chamber (with diurnal light and temperature settings matching average monthly temperatures for the Nike missile installation at Pahole, at approximately 2100 feet elevation), and examined weekly for germination for a total of 10 weeks. Germination rates were compared using t-tests, performed in IBM SPSS Statistics Version 20. Appendix ES-11

Figure 1. Locations of Cyanea superba subsp. superba fruit collections on Oahu.

a b

c d

Figure 2. Cyanea superba subsp. superba. a) fruiting at the Makaha reintroduction site, b) fresh mature fruit, c) seeds embedded in fruit pulp, and d) seeds germinating on filter paper. Appendix ES-11

Results and Discussion

Seeds were approximately 50% less viable from senesced vs. fresh C. superba subsp. superba fruit regardless of treatment (untreated seeds: t = 6.659, p < 0.001; seeds with pulp extract: t = 5.077, p < 0.001) (Figure 3). The reduced germination in seeds from senesced fruit limits recruitment potential in the absence of dispersers, as fresh mature fruits that are not consumed by dispersers will senesce and fall to the ground, and subsequently have reduced potential for germination. The rate and extent of reduction of seed viability of senesced fruit over time remains unexplored. Dispersers that cache or drop whole fruit likely do not facilitate recruitment. However, whole fresh mature fruits of C. superba subsp. superba are not easily removed from infructescences (pers. obs.), and are more likely to be consumed directly from the plant.

Figure 3. Seed viability from senesced and fresh mature fruits, with and without fruit pulp extract (n = 10).

Fruit extract had no effect on germination of seeds for either senesced (t = 0.022, p = 0.982) or fresh mature fruit (t = 1.075, p = 0.296). Seeds used for sowing in the field will not necessarily require laboratory processing and cleaning, but rather may be processed in the field by gently smashing fruit pulp in water to extract seeds. The seeds will sink to the bottom of the container, and the floating smashed pulp may be poured off. The remaining seed slurry may then be sown, without concern for reduced viability resulting from pulp chemicals in the slurry. Because seed viability is reduced by fruit senescence, only fresh mature fruit should be used for seed sowing, and whole fruits should not be scattered as a means to enhance recruitment.

The results of this laboratory trial may help partially explain some of the population dynamics observed at reintroduction sites. The limited recruitment observed at Kahanahaiki (despite large numbers of mature plants, extensive control, and prolific production of viable seed), may be influenced by a lack of dispersers. Recruitment at this location is primarily located below mature plants. Fruit that is not Appendix ES-11

predated by rats is likely senescing, falling to the ground and having little to no recruitment. By comparison, at Palikea, recruitment occurs distant from the few mature plants present. This area has a more diverse avifauna, which may be effectively dispersing seeds.

Future Directions

To examine the rate and extent of reduction in C. superba subsp. superba seed viability over time during fruit senescence, fresh mature fruit may be collected and stored at room temperature over a period of time to allow for total senescence, during which viability assays may be made at pre-determined intervals. E.g., seeds from fruit samples may be sown on the date of collection, then again every three days for three weeks. Similar trials may be considered for other OANRP managed species thought to be dispersal dependent.

To explore bird dispersal at C. superba subsp. superba reintroduction sites, fruit consumption by birds at these locations may be quantified by game cameras. Additionally, the number and height of seedlings as well as distance from the nearest plant may be quantified, excluding those in slug controlled areas and seedlings known to occur as a result of prior seed sow efforts. Seedling surveys are currently underway.

References

Pender, R. J., A. B. Shiels, L. Bialic-Murphy, S. M. Mosher. 2013. Large-scale rodent control reduces pre- and post-dispersal seed predation of the endangered Hawaiian lobeliad, Cyanea superba subsp. superba (). Biological Invasions 15:213-223.

Oahu Army Natural Resources Program. 2015a. Oahu Army Natural Resources Program Rare Plant Database.

-----. 2015b. Oahu Army Natural Resources Program Seedbank Database.