Appendix 4-4 koolauensis: Five Year Genetic Storage Management Plan OIP Year 13-17, Oct. 2019 – Sept. 2024 Scientific name: (Degener) Hawaiian name: Nioi Family: (Myrtle family) Federal status: Listed endangered March 28, 1994 Requirements for Stability: – 3 Population Units (PU) – 50 reproducing individuals – Factors for setting goal as >25 for a long-lived perennial: doubled due to threat from rust () – Stable population structure – Threats controlled – Complete genetic representation of all PUs in storage – Tier 1 stabilization priority Description and biology: Eugenia koolauensis is a small or 2-7 m tall. The oppositely arranged leaves are concave, 2-5 cm long, and 1-3.3 cm wide. The leaf margins are strongly revolute. The upper leaf surfaces have dense brown hairs when young. Older leaves are glossy and hairless, or bear short hairs near the veins. The lower leaf surfaces are densely covered by short brown hairs or rarely moderately covered. The flowers are white, borne 1-2 in the leaf axils, with four petals and about 150 stamens. The berries are yellow to red, ovoid in shape, measure 0.8-2.0 cm in length, and usually contain a single globose (though occasionally produces two ; OANRP Seed Laboratory observations). The species has been observed with flowers and year round, but peak production usually occurs in the summer. The flowers of E. koolauensis are presumably insect pollinated. The red and yellow fleshy berries suggest that fruit eating birds are the main dispersal agents for the species. Since the seeds are large and lacking a durable seed coat, they would not be expected to remain viable long after the fruit ripens. In fact, seeds are desiccation-sensitive and do not survive drying (OANRP Seed Laboratory observations), which may imply that seeds do not survive for long outside of the fleshy fruit. Both immature cultivated and wild plants are slow growing. Eugenia koolauensis is a long-lived species. A tree in Papali Gulch has been observed for 25 years, but it has not increased very much in size during that time (Lau pers. comm. 2005). Known distribution: Eugenia koolauensis has been found on the islands of Oahu and . On Oahu, the species has been recorded primarily from the northern Koolau Mountains, on both the windward and leeward sides of the mountain range, from 328-1,000 ft in elevation. The species has also been recorded from the Waianae Mountains, inland of Waialua. It was collected in this area by O. Degener in 1932 in the "gully having prominent dyke, north-northeast of Puu Kamaohanui" (Wilson 1957), known as Palikea Gulch. In 2000, a few plants were discovered in the same area in Palikea and Kaimuhole Gulches. Recorded elevations for the species in the Waianae Mountains are from 760-960 ft. Since the species grows in dry forests in the Waianae Range, it is possible that it formerly also occurred in the region between the two mountain ranges. If the species did indeed occur in that region, the now separated Koolau and Waianae plants would likely have been in genetic communication. On Molokai, the species is known from only two specimens collected by Joseph F. Rock. One of the specimens was collected in 1918, and the other in 1920 (Wilson 1957). Although elevations were not recorded for the West Molokai specimens, the or plants were likely located near the summit of Maunaloa, which is 1381 ft high in elevation. Little native vegetation remains on Maunaloa, and it is unlikely that any E. koolauensis plants survive there today. Population trends: The largest number of individuals of E. koolauensis occurs within the U.S. Army Kahuku Training Area (KTA) in the Northern end of the Koolau Mountains. Observations of these sites by OANRP from 1996-2006 showed that nearly all populations contained seedlings and saplings in addition to mature . During that period, the numbers of individuals were stable or slightly increasing. Most trees at Kaunala, Pahipahialua, Oio and Kaleleiki were fenced, heavy fuels were removed to reduce the risk of fire and weed control was ongoing. Since 2006, the species has been observed to be rapidly declining across its range due to an introduced pathogenic rust, A. psidii. The first sign of rust on E. koolauensis was observed in March 2006 in Kahuku. Stands of Syzigium jambos, an alien species which hosts A. psidii, are abundant in the KTA. Other hosts present in the area are the native Metrosideros polymorpha, and three alien species, Pimenta dioica, Eucalyptus robusta and Melaleuca quinquenervia. The rust affects plants of all sizes and ages. Some small, immature plants were quickly defoliated and all plants show some sign of infection. Few if any trees are considered to be in good health and nearly all new leaves are quickly infected and killed. Some trees are still able to produce flowers, but flowers and immature fruit are also infected and killed. Since 2006, there has been a 79% decline of all known plants. The decline in the number of plants has been observed in each of the MFS PUs: Pahipahialua (89%), Oio (93%), and Kaunala (63%). Monitoring of this species was last conducted in 2015. Current status: Eugenia koolauensis is still extant in the northern Koolau Mountains and a single tree remains in the northern Waianae Mountains. The center of abundance for the species is in the Action Areas of the Kahuku Training Area and the northern end of the Kawailoa Training Area. All plants have been heavily impacted by rust. Habitat: Eugenia koolauensis occurs in dry to mesic forests, usually on gulch slopes. In the Koolau Mountains the plants occur in dry mesic forests with native trees such as ohia lehua (Metrosideros polymorpha) and/or lama (Diospyros sandwicensis), hoawa (Pittosporum glabrum), hao ( sandwicensis), alaa (Planchonella sandwicensis). These sites also have non-native areas with stands of strawberry guava (Psidium cattleianum), shoebutton ardesia (Ardesia eliptica), ironwood (Casuarina equisetifolia) and paperbark (Melaleuca quinquenervia). The known plants in the Waianae Mountains are located in dry forests with lama (Diospyros sp.), wiliwili (Erythrina sandwicensis), lonomea (Sapindus oahuensis). The trees are located along the margin where the remaining forest borders a highly degraded area with Urochloa maximum and are highly susceptible to fires. On Maunaloa, Molokai, the original dry forest vegetation has been largely destroyed, and there are no detailed descriptions of its original composition. Taxonomic background: Eugenia koolauensis is currently recognized as one of only two native Hawaiian species of Eugenia. The other species is the closely related E. reinwardtiana, whose range extends beyond Hawaii through much of the tropical Pacific Ocean and . Eugenia reinwardtiana had already been known as a rare plant in most regions of Hawaii except for the northern Waianae Mountains where it can be locally abundant, but there have not been recent surveys of this taxon since the introduction of A. psidii. There are certain populations of Eugenia in the Koolau Mountains with plants whose morphology is intermediate between the two Eugenia species. These intermediate population units have not been included among the population units included in this taxon summary. In the Waianae Mountains, E. reinwardtiana occurs in the same gulches containing known E. koolauensis trees, but in different parts of the gulches. There appears to be a zone of intergradation in these gulches between the typical plants of each of the two Eugenia species (Lau pers. comm. 2005). Threats: Feral pigs are a major threat to E. koolauensis in both the Koolau and Waianae Range. Feral goats also threaten the Waianae sites. The animals degrade the plants’ habitat by hastening the spread of invasive weeds. The PUs at Kaunala, Pahipahialua, Oio and some plants at Kaleleiki are now fenced and protected from pigs. Alien plants threaten E. koolauensis by altering the species’ habitat, competing with it for moisture, light, nutrients, growing space, and serve as a reservoir for A. psidii. Also, the spread of highly flammable alien grasses increases the incidence and destructiveness of wildfires. The single tree in Kaimuhole Gulch was killed by the Waialua fire in August 2007. The trees in the Palikea site were scorched, but were not immediately killed. Major threats in the action area at KTA due to army training are fire, trampling, and the introduction of competing non-native plant species. Fire in the action area has been documented to have affected populations of this species twice in the last 10 years, and the fuel load near some plants is high. Additionally, some of the onsite populations are threatened by habitat disturbance from motor cross bikes. In April 2005, the pathogenic fungus A. psidii was documented on cultivated ohia plants on Oahu. By August 2005, it was reported to be widespread across the state and considered to be a major threat to native ohia forests (Loope 2008). It was not observed during monitoring of E. koolauensis at Kahuku in February of 2005, but was reported to be present at all sites in May 2006. Damage to most trees has been severe and lethal. All trees appear to have been at least partially defoliated by infections of A. psidii and many smaller immature trees have since died. A. psidii has been observed to infect flowers and fruits and certainly affects overall health and fecundity reducing seed production and recruitment. No mature fruit has been collected from any of the trees since 2009. Collections of seed, cuttings, air layers and whole plants have been successfully propagated and established in the OANRP nursery. The threat from A. psidii to plants in the nursery is kept under control with a rotation of several fungicides and basic propagation and cultural techniques. Plants can be kept healthy and these are producing flowers and viable fruit. The pathogen appears to be thriving in the habitat for E. koolauensis in KTA where there are many other Myrtaceous host plants. Of the 80 known host plants worldwide, 42 occur in Hawaii. Control methods recommended by government agriculture and botanical gardens include: replanting with non-Myrtaceous species or using fungicides. There are currently no fungicides approved by the Hawaii Department of Agriculture for use in natural areas. While repeated fungicide applications have been shown to be effective in controlling the rust (Martins 2011), rotating bi-weekly applications of several different chemicals (to prevent resistance) onto wild trees in remote natural areas is not considered a sustainable effort at this time. Instead inter-situ sites with access to these management options will be established to manage a living collection. Reproductive Biology Table

Observed Phenology Reproductive Biology Seeds

Population Flower Immature Mature Fruit Breeding Suspected Ave. # Dormancy Unit Fruit System Pollinator Per Fruit

Physiological Hermaphr All Mar-Sep May-Oct May-Nov insect 1 dormancy oditic (PD)

*Mature fruit has been collected from the living collection in the greenhouse from January through December.

Genetic Storage Plan from 2014 Five Year Plan

What propagule What is the What is the Genetic What is the Is seed Plan for maintaining type is used to source for the Storage Method proposed re- storage genetic storage meet genetic propagules? used to meet the collection testing storage goals? goal? interval for ongoing? seed storage?

Collect seeds and maintain living Nursery living Collecting seeds and in situ N/A Yes collection in the nursery collection cuttings and an inter situ collection

Genetic Storage Plan Comments from 2014 Five Year Plan: “Seeds have some slight level of physiological dormancy, as seeds take longer than 45 days to germinate. Average germination, however, is high (91%). Seed storage would be the preferred genetic storage method, however, seeds are desiccation-sensitive. Seed banking must utilize cryopreservation techniques. Cryopreservation protocols will be researched at the National Center for Genetic Resources Preservation (NCGRP, Fort Collins, CO), once enough seeds can be harvested from the OANRP living collection. In lieu of this, a collection of Syzygium sandwicensis was sent to NCGRP to initiate protocol development (Myrtaceae; desiccation- sensitive) and living collections of plants are kept at the nursery to represent each of the PU. Once nursery plants are cloned, replicates will be planted at an inter situ site at Waimea Botanical Garden where they can continue to be treated with fungicides. The living collection at the nursery will expand to include new founders as they become available. If cryopreservation protocols cannot be established for seeds of E. koolauensis, it will be attempted for buds and other meristematic tissues at NCGRP. Cuttings will be collected from all sites to establish a living collection in the OANRP nursery. Currently, OANRP has clones of 33 individual founders. In some cases, when propagules from larger plants are not available, whole plants will be removed from wild sites. Protocols for determining when whole plants can be removed from the wild have been developed by the HRPRG. These methods will be used to dig up and transport small whole plants to the OANRP nursery for propagation as part of the living collection. The HRPRG Salvage decision tree (below), however, only allows for salvaging whole plants of species with less than 50 individuals (PEPP species). Given the severity of the P. psidii infections, which has ceased reproduction of E. koolauensis at all sites, made clonal propagation nearly impossible, and caused the rapid decline of this species, OANRP proposes applying the whole plant harvesting (salvaging) to E. koolauensis. Salvaging now with 428 plants (64mature/112immature/252seedling) remaining will allow for ex situ representation from remnant plants at most known sites. A total of 150 founders (117 additional founders) will be secured from across the known range. The collection strategy will be to represent every population site and sample from individuals growing across each site to maximize the chances of capturing the most genetic diversity. Both known founders from the Waianae Mountains are already secured and collections from the other sites will be a priority over the next few years.” Updated Genetic Storage Plan

What What is the What is the What is the Is seed Plan for maintaining propagule type source for the Genetic Storage proposed re- storage genetic storage is used to meet propagules? Method used to collection testing genetic storage meet the goal? interval for ongoing? goals? seed storage?

Micropropagation Seeds + F1 Ex situ living and living Collect seeds and seedlings of in collection and collection maintain living collection situ stock + N/A Yes inter situ (including inter in the nursery and inter clones from in plantings situ site situ collections situ stock representation)

*For the purpose of this document ex situ to refers to seed collections in storage and living collections managed in Army greenhouse/nursery facilities where full day to day management takes place, whereas inter situ refers to collections established at botanic gardens or other areas intermediate between ex situ and in situ.

Updated Genetic Storage plan Comments: As mentioned in the initial genetic storage plan comments, seeds of Eugenia koolauensis exhibit some type of physiological dormancy, however, average germination is relatively high (91%). Conventional seed banking is not an option for this species because its seeds are desiccation-sensitive, exhibiting recalcitrant seed storage behavior. Storage at ultra-low temperatures (-80°C) is also not an effective solution. Cryopreservation research is currently underway at the Cincinnati Zoo and Botanic Garden and Lyon Arboretum’s Micropropagation Laboratory to determine storage protocols in liquid nitrogen of “exceptional species”, including E. koolauensis. This research is focused on establishing protocols for buds and other meristematic tissues rather than seed. In 2018 and 2019 OANRP supplied Lyon Arboretum with substantial collections of seed and F1 seedlings from which in vitro establishment protocols have been developed. In combination with the ex situ living collection, micropropagation can serve as a mid-term genetic storage method until cryopreservation protocols are developed. Currently, OANRP maintains clones of 159 individual founders in its living collection meeting the goal of 150. It is unlikely that additional founders will be secured ex situ given the drastic decline in the wild. In addition to the living collection, two inter situ collections were establish at Koko Crater Botanic Garden (3/15/16) and Kahua (3/8/18). Both collections include a mix of founders representing multiple PU. There are no future plans to reintroduce this species in the wild due to A. psidii. It is recommended that additional inter situ plantings are established using founders from individual PU. Seed collected from these founders will be used to establish explants in micropropagation. These inter situ sites are beneficial because plants can be adequately treated with fungicides.

Discussion: Genetic Storage and Five Year Action Plan Eugenia koolauensis is a hermaphroditic, insect pollinated small tree or shrub. Seeds are recalcitrant and therefore not suitable for conventional seed banking. Long-term genetic storage of this species must utilize cryopreservation techniques; research is currently under way to develop these protocols. In the meantime micropropagation can serve a mid-term genetic storage solution utilizing protocols recently developed at Lyon Arboretum in combination with the living collection. E. koolauensis is reliably propagated by seeds or cuttings. Seeds must be sown shortly after harvest for best germination results. Alternatively, seeds can be stored (without drying) in plastic zip lock bags at 5°C for 1-3 months, however, viability will decline through time (OANRP Seed lab experiments). Cuttings should be made with 2-3 nodes. Typically, cuttings of thicker diameters (woodier material) were seen to generate roots best, compared to thinner diameter cuttings (skinny tip material) which almost never produce roots. Clonex was the standard hormone used, however, Hormodin 3 was observed to encourage the most consistent rooting in 2017. Root establishment requires 3-15 months under mist and rooted cuttings and seedlings are relatively slow growing. The main challenge to managing this species in living collection is the pathogenic fungus A. psidii. If left untreated, plants will eventual succumb to the infection. The preferred fungicide treatment for A. psidii is a foliar application of Bayer Advanced Rose and Garden Spray Tebuconazole. Plants grown in a plastic covered greenhouse seem to be more protected from the rust as compared to plants in the open. On average plants are treated for rust four times throughout the year. Treatment with Tebuconazole is initiated after signs of the infection are observed, with a follow-up treatment two weeks later in the greenhouse and a month later at inter situ sites. Other pests include ceroplastes scale, mealy bugs, and root mealy bugs. A weekly rotation of M-Pede and a growth regulator (such as Kontos) is applied during heavy infestations (usually greenhouse wide). Due to the rapid decline of founders in the wild, OANRPs approach to genetic storage for E. koolauensis has been building and maintaining a living collection of clones and whole plants representing wild founders. To date we maintain 159 individual founders representing 10 PU and 19 Population Reference Sites (PRS) as containerized plants. Because effective control mechanisms for A. psidii in situ are lacking, there are no future plans to reintroduce this species. With limited in situ management options, OANRP should be considering more sustainable approaches to genetic storage. The recent development of protocols to establish plants in vitro makes micropropagation a viable replacement for the labor and resource intensive living collection. The current living collection has an average age of 4.13 years. Based on the program’s experience managing this species ex situ, staff identified five years as the maximum age of a plant maintained at the West Base greenhouse and nursery. At this point plants tend to become root bound in their containers and begin to decline. As the living collection ages, more greenhouse/nursery space, resources, and staff time are required to adequately manage it. In order to more efficiently manage this living collection, OANRP proposes a five year genetic storage plan that will transition genetic storage from living collection to micropropagation and eventually to cryostorage when reliable protocols are developed. This plan combines living collection, inter situ plantings, and micropropagation to achieve genetic storage requirements in the mid-term, with the ultimate goal of realizing genetic storage goals for ex situ founders through cryostorage. OANRP currently manages two inter situ plantings of mixed founder stock at Koko Crater Botanic Garden and Kahua. The Koko Crater collection was initiated in March 2016 and Kahua in March 2018, with 81% and 76% survivorship respectively. Survivability is relatively high and seed production is taking place at both sites. Overall plants at Koko Crater are healthier than those at Kahua. Both sites are irrigated, however, Koko Crater has partial shade whereas Kahua has full sun. Building on this experience we propose establishing three additional inter situ collections, each specific to one MFS PU, Kanunala, Oio, and Pahipahialua. All ex situ founders representing a PU will be established at the site. Isolating each PU at its own inter situ site will maintain the genetic integrity of each population, minimizing any negative genetic consequences, i.e. genetic swamping or outbreeding depression, that may result from mixing stock. These plantings will serve as sources of propagules to meet genetic storage goals and to maintain the living collection. For all other PU we propose isolating each PU either in an insect/pollen isolation structure or separate greenhouse. Resulting seed collections will be utilized to establish founders in micropropagation. In this five year plan we also propose that a maximum of 2 plants representing each founder established at inter situ sites count toward our living collection goal of three plants per founder. This will save staff time and resources in the greenhouse as we expand inter situ plantings. This is a notable change to our current approach of managing this living collection and will result in multiple scenarios under which our living collection goals are satisfied; for example one plant in the West Base greenhouse and two at an inter situ site, two plants in the West Base greenhouse and one at an inter situ site, or all three plants in the West Base greenhouse. To ensure the survivorship of plants established at inter situ sites attributing to living collection goals, management and maintenance trips will be scheduled on a monthly basis and irrigation will be installed at sites where deemed necessary. In year one of the five year action plan, we will identify and secure three appropriate inter situ sites and begin cloning founders with less than three plants. Cloning will continue into year two and we will begin planting starting with the oldest stock. We will finish planting in year three and focus efforts in year four and five on seed collection to establish plants in micropropagation.

Current Genetic Storage Status

Population Unit and # Founders in Average Age of Founders (≥1) in Founders with ≥ 10 Genetic Storage Living Collection by Living Collection by Microprop. Viable Seeds in Complete PopRef PopRef Storage Aimuu (100%) PAU-H (13) 4 yrs 2 0 Kaiwikoele & KAI-A (2) 4.5 yrs 0 0 Kamananui (100%) KAM-C (6) 3.5 yrs 1 0 NMH-A (0) N/A 0 0 NMH-B (23) 3 yrs 5 0 Kaleleiki (58%) PAU-C (14) 3 yrs 0 0 PAU-J (9) 3 yrs 0 0 PAU-K (0) N/A 0 0 Kaunala (78%) KNL-B (34) 3.6 yrs 3 0 MFS KNL-C (1) 4 yrs 0 0 Malaekahana MKK-A (5) 3.33 yrs 1 0 (100%) Ohiaa & East Oio OHI-I (0) N/A 0 0 (100%) OIO-D (3) 6 yrs 1 0 Oio (50%) OIO-C (1) 4 yrs 0 0 MFS OIO-F (12) 5 yrs 2 0 Pahipahialua (58%) PHI-A (31) 5.9 yrs 1 0 MFS Hanaimoa (67%) HAU-A (3) 3.5 yrs 0 0 Palikea & ALI-A (2) 8.67 yrs 0 0 Kaimuhole (100%) IMU-A (0) N/A 0 0 Total 159 ------17 0

Five Year Action Plan

Population Unit # of OIP Year 13 OIP Year 14 OIP Year 15 OIP Year 16 OIP Year 17 and Genetic Individual Oct. 2019- Oct. 2020- Oct. 2021- Oct. 2022- Oct. 2023- Storage Founders Sept. 2020 Sept. 2021 Sept. 2022 Sept. 2023 Sept. 2024 Complete

Kaunala (78%) 35 Clone in situ Clone in situ Finish planting Collect seed Collect seed founders. founders. at inter situ site for for Identify inter Begin planting establishment establishment situ site at inter situ in microprop. in microprop. site

Oio (50%) 13 Clone in situ Clone in situ Finish planting Collect seed Collect seed founders. founders. at inter situ site for for Identify inter Begin planting establishment establishment situ site at inter situ in microprop. in microprop. site

Pahipahialua 31 Clone in situ Clone in situ Finish planting Collect seed Collect seed (58%) founders. founders. at inter situ site for for Identify inter Begin planting establishment establishment situ site at inter situ in microprop. in microprop. site

Aimuu (100%) 13 Isolate PU in Isolate PU in greenhouse. greenhouse. Collect seed for Collect seed establishment for in microprop. establishment in microprop Kaiwikoele & 31 Isolate PU in Isolate PU in Kamananui greenhouse. greenhouse. (100%) Collect seed for Collect seed establishment for in microprop establishment in microprop Kaleleiki (58%) 23 Isolate PU in Isolate PU in greenhouse. greenhouse. Collect seed for Collect seed establishment for in microprop. establishment in microprop. Malaekahana 5 Isolate PU in Isolate PU in (100%) greenhouse. greenhouse. Collect seed for Collect seed establishment for in microprop. establishment in microprop. Ohiaa & East 3 Isolate PU in Oio (100%) greenhouse. Collect seed for establishment in microprop

(continued)

Population Unit # of OIP Year 13 OIP Year 14 OIP Year 15 OIP Year 16 OIP Year 17 and Genetic Individual Oct. 2019- Oct. 2020- Oct. 2021- Oct. 2022- Oct. 2023- Storage Founders Sept. 2020 Sept. 2021 Sept. 2022 Sept. 2023 Sept. 2024 Complete

Hanaimoa 3 Isolate PU in (67%) greenhouse. Collect seed for establishment in microprop Palikea & 2 Isolate PU in Kaimuhole greenhouse. (100%) Collect seed for establishment in microprop

References Keir, M. December 2014. Chapter 2, Section 2.2 Five Year Rare Plant Plan: Eugenia koolauensis in Status Report for the Makua and Oahu Implementation Plans. http://manoa.hawaii.edu/hpicesu/DPW/2014_YER/default.htm Loope, Lloyd and Anne Marie La Rosa. 2008. An Analysis of the Risk of Introduction of Additional Strains of the Rust Puccinia psidii Winter (`Ohi`a Rust) to Hawai`i. U.S. Geological Survey Open File Report 2008-1008, Reston, Virginia. Martins, M. V. V., S. F. Silveira, L. A. Maffia, J. M. A. Rocabado, V. Mussi-Dias. 2011. Chemical control of guava rust (Puccinia psidii) in the Northern Region of Rio de Janeiro State, Brazil. Australian Plant Pathology 40:48–54. OIP 2008. Oahu Implementation Plan. United States Army Garrison, Hawaii, Directorate of Public Works, Environmental Division, Schofield Barracks, HI. Wagner, W. L., D. R. Herbst, and S. H. Sohmer. 1999. Manual of the Flowering Plants of Hawai‘i. revised edition. University of Hawai‘i Press & Bishop Museum Press, Honolulu.