Appendix 4-5 Flueggea Neowawraea 5-Year Genetic Storage Plan

Appendix 4-5 Flueggea neowawraea: Five Year Genetic Storage Management Plan OIP Year 13-17, Oct. 2019 – Sept. 2024 MIP Year 16-20, Oct. 2019- Sept. 2024 Scientific name: Flueggea neowawraea W. Hayden Hawaiian name: Mehamehame Family: Euphorbiaceae (Spurge family) Federal status: Listed endangered 10 November 19941 Requirements for Stability – 4 Population Units (PU) – 50 reproducing individuals in each PU (long-lived perennial, dioecious, low to no reproduction, all senescent, major pest problems) – Stable population structure – Threats controlled – Complete genetic representation of all PUs in storage

Description and biology: Flueggea neowawraea is a tree growing up to 30 m (98 ft) tall, with a trunk up to 2 m (6.6 ft) in diameter. The trees are often multi-trunked. The species’ bark is rough and reddish-brown, and its wood is brown and often has a wavy grain. The leaves are 4-14 cm (1.6-5.5 in) long, and are arranged alternately along the stems. The flowers of an individual plant are usually all female or all male. They are borne in axillary clusters of 2-6. The fruits are globose, measure 3-6 mm (0.12-0.24 in) in diameter, are juicy, usually contain 6 seeds, and are reddish brown to black when ripe.

Flueggea neowawraea is dioecious, bearing either all male flowers or all female flowers. However, the species apparently is not completely dioecious, as a cultivated plant isolated from others has been observed to produce viable seeds (Chung pers. comm. 2000). Flowering occurs over a brief period sometime in the late summer through the fall. The timing of the flowering in a given area is likely dependent on the area's weather patterns and the distribution of rainfall in that particular year. The flowering of the different trees in a given area is normally well synchronized (Lau pers. comm. 2000). The pollination biology of F. neowawraea has not been studied. The species’ juicy fruits are suggestive of seed dispersal by fruit-eating birds. Flueggea neowawraea are often the most massive trees in the forests in which they are found. Many of the remaining live trees are partially dead, with a strip or strips of bark extending up the trunks to crowns that have died back. The remaining living branches are often relatively healthy (Lau pers. comm. 2000). For this species, dying back may be a means of coping with environmental stresses. Flueggea neowawraea's wood is very hard and lasts a long time after the death of the tree. It rots in a very distinctive fashion, and as a result, the decayed trunks and limbs of the species are readily identified. Old logs on the ground and pieces of wood in gulch bottoms and in streambeds document the former occurrence of the species throughout the Waianae Mountains.

Known distribution: Flueggea neowawraea has been documented from Kauai, the Waianae Mountain Range of Oahu, Molokai, East Maui, and the leeward side of the island of Hawaii. In the Waianae Mountains it has been found throughout the mountain range. The species has been recorded from 305-732 m (1,000-2,400 ft) in elevation. Population trends: The remaining living trees and the dead remains of F. neowawraea indicate that the species was formerly not uncommon in at least some parts of the Hawaiian Islands (Lau pers. comm. 2000). The recorded history of F. neowawraea is relatively short for a native Hawaiian tree, as it was not discovered until 1912. Reports of the species in the first half of the 1900's indicate that it had already been declining in numbers and health for a considerable time prior to its discovery. There were many reports of large mature trees, portions of which were already long dead; there were no reports of younger trees and immature plants. There are few records of immature plants to date. A pair of plants in Pahole Gulch was reported in the 1970’s (Nagata 1980). One plant was reportedly a tree 6.1 m (20 ft) tall, with a main trunk measuring 5.1 cm (2 in) in diameter The other plant a sapling about 1.5 m (5 ft) tall with a trunk measuring 2.5 cm (1 in) in diameter. A small immature tree measuring about 4 m was observed in Kaaikukai around 2000 (Sailer pers. comm. 2010). The decline of F. neowawraea has undoubtedly been greatly accelerated by the introduction of the black twig borer (BTB) Xylosandrus compactus in 1961. Of the individuals observed alive in the 1980’s, more than half are now dead (Lau pers. comm. 2000). Current status: Flueggea neowawraea is extant throughout its recorded range except on Molokai, where only a single tree has ever been found. That individual was documented with a voucher specimen in 1931 and it died sometime prior to 1939. Only two trees are known to persist on the southern flank of Haleakala, East Maui. Five to nine trees are known on the island of Hawaii. The species is most common on Kauai where an estimated 60-80 trees are known. Currently on Oahu, a total of 29 in situ trees are known alive.

Habitat: Flueggea neowawraea’s center of abundance is in the drier parts of the mesic forests, which are often dominated by lama (Diospyros sandwicensis) or dominated by lama and ohia (Metrosideros polymorpha). Only a few live trees remain in the dry forests. The species was formerly more common in the dry forest than today, as evidenced by numerous old logs and standing dead trunks. Most trees occur either in gulch bottoms or on north facing lower to mid-gulch slopes.

Taxonomic background: Flueggea neowawraea is the only member of the genus occurring in Hawaii. There are no obvious morphological differences between plants on the different islands (Lau pers. comm. 2000). Threats: The primary threat to F. neowawraea is the introduced black twig borer (X. compactus), which has affected all populations of F. neowawraea. The female black twig borer tunnels into the center of living twigs and lays its eggs in the hollowed twig. Physical damage, accompanied by the introduction of pathogens, often contributes to the death of the twig. Chronic infestation leads to a gradual weakening of the tree, and its eventual premature death (Hara and Beardesly 1979). Another threat to F. neowawraea is the Chinese rose beetle (Adoretus sinicus), which arrived in Hawaii before 1896 (Koebele 1897). This beetle feeds on the leaves of the tree, sometimes reducing them to skeletons. Other major threats include feral pigs and goats, alien plant species, cattle grazing, and fire. On the island of Hawaii much of the species' habitat in Kona and Kau has been destroyed or severely degraded by farming, ranching, and residential development. The species is further endangered by the need for cross-pollination between male and female trees in populations whose numbers have decreased greatly and are now comprised of widely separated trees, which in some cases, may be too far apart to be effectively cross-pollinated.

Reproductive Biology Table

Observed Phenology Reproductive Biology Seeds

Population Flower Immature Mature Breeding Suspected Average # Dormancy Unit Fruit Fruit System Pollinator Per Fruit (viable)

All Oct-Dec Nov-Feb Nov-Apr Dioecious Wind * 1.78 ± 1.52 Non- Dormant (ND) / Physiological Dormancy (PD)

*Presumed wind pollinated based on floral morphology, breeding system, and pollen dispersal at anthesis Comments: The trees in the wild appear to flower once a year. Large clones in the living collection often flower more than once a year, typically in Oct-Nov and then again in Jan-Feb. Some plants will produce a small number of flowers at any time throughout the year. Seeds of this species were initially thought to be non-dormant; however based on current data some seeds and/or accessions of seeds to not germinate within 45 days. It is possible that seeds of F. neowawraea exhibit some type of physiological dormancy.

Genetic Storage Plan from the 2010 Five Year Plan

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

Seeds + Living Ex situ living Seed banking (-18C / 10 yrs. (seeds) and Yes Collections Collection + collection 20% RH) & pollen 2+ yrs. (pollen) will be made Pollen storage (-18C / 20% from ex situ RH) & clones of in hand- situ stock pollinations and reintroductions

Updated Genetic Storage Plan

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

Seeds + Pollen + Ex situ living Seed banking (-18C / 10 yrs. (seeds) Yes. Next Seed Clones of in situ collection and 20% RH) & pollen and 2+ yrs. tests 2020- Collections stock inter situ plantings storage (-18C / 20% (pollen) 02-09 (10 yr. will primarily RH) & living test) and be made from collection (including 2022-01-09 ex situ and inter situ (20 yr. test) inter situ hand- representation) pollinations and cuttings collected from ex situ and inter situ collections

*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: Pollen stored for over 2 years was used to produce viable seed. Males are, or will be, represented as clones in ex situ and inter situ living collections. Pollen will be collected from these males. It will be used in the breeding program to produce stock for reintroductions and seed for genetic storage. As pollen ages or it is used up, it will be continually replaced from ex situ and/or inter situ collections. The current seed recollection interval is based on viability tests from one accession of seed. To determine a more reliable recollection interval, further testing on multiple accessions is necessary.

Discussion: Genetic Storage and Five Year Action Plan Flueggea neowawraea is a large, dioecious, wind pollinated tree. Seeds of this species exhibit orthodox seed storage behavior, tolerate desiccation to low moisture contents and store well at -18°C, making it a prime candidate for conventional seed banking. The seed recollection interval is set at ten years. Declines in viability below 70% of the tested accession’s highest viability were detected in 10 and 15 year tests, suggesting that seeds of F. neowawraea only store viably for 10 years. However, this interval is based on test results from only one accession. Continued testing of multiple accessions is necessary to adequately determine a more representative recollection interval for this species. Clones of in situ founders can be propagated by cuttings using commercially available rooting hormones, Clonex, Hormodin 3, and Dippin Grow (1:8 or 1:5); however, success rates are generally rather low. Preliminary results from an ongoing propagation trial at OANRP suggest that softwood and semi-hardwood cuttings have the highest rooting success as compared to hardwood cuttings. The main challenge to maintaining this living collection is managing damage caused by black twig borer (BTB). Staff currently use a combination of systemic insecticide, pheromone traps, and kaolin clay to control this pest, but is difficult to assess how effective these treatments are. Due to the decline of the remaining founders in the wild, accelerated by BTB, cuttings taken from in situ founders to establish a living collection has been our main approach to meet genetic storage goals for this taxon. Seed storage currently only accounts for 11% of the total genetic storage goal for this taxon with only five of the 45 founders (alive or dead and represented) in storage with ≥ 50 viable seeds. Collection of viable seeds from the living collection housed at the Nike greenhouse/nursery is possible with supplemental hand pollination; however, it is limited by the size individual plants can reach in a container. The current living collection of F. neowawraea has an average age of 8.15 years with 90% of the collection ≥ 5 years old and the oldest plant 16 years old. 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, we are proposing a five year genetic storage plan that will change the age structure of the collection housed at the Nike greenhouse/nursery facility and increase opportunities for seed collection to meet genetic storage goals. This plan combines living collection, seed storage and inter situ plantings to achieve genetic storage requirements, with the ultimate goal of realizing genetic storage goals for maternal lines through seed storage, and male founders via living collection. Based on the program’s experience managing this species ex situ, staff have identified five years as the maximum age of a plant maintained at the Nike greenhouse and nursery. At this point plants tend to become root bound in their containers and begin to decline. At five years staff recommend that plants are either culled, outplanted, or established at an inter situ site. The establishment of a breeding population (in this case all Oahu founders secured ex situ) at an inter situ site, such as Koko Crater Botanic Garden, Wahiawa Botanic Garden, Waimea Valley Botanic Garden, or Kahua, will significantly increase opportunities for pollen and seed collection. Establishing plants in the ground at an inter situ site will result in increased plant health, enabling them to grow larger and flower and fruit more prolifically. In addition, planting multiple founders at an inter situ site may increase the potential for successful open, wind pollination between individuals. These plantings will serve as sources of propagules to meet genetic storage goals, for reintroductions, and to maintain the living collection at the Nike greenhouse/nursery facility. With this five year plan OANRP also proposes that a maximum of two 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 inter situ plantings expand. This is a notable change to the 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 Nike greenhouse and two at an inter situ site, two plants in the Nike greenhouse and one at an inter situ site, or all three plants in the Nike greenhouse. To ensure the survivorship of plants establish 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 plan, OANRP will identify and secure one appropriate inter situ site where all founders currently in the living collection can be established, begin cloning founders with less than three plants, and start planting with the oldest stock first. OANRP has already identified Koko Crater Botanic Garden as a suitable site, though staff may consider Wahiawa Botanic Garden, Waimea Valley or Kahua as alternative sites. A small collection (12 plants representing 9 founders) of Alectryon macrococcus var. micrococcus, also susceptible to BTB, was establish at Koko Crater in December of 2016. Survivorship is 83% and those plants that died were in moderate health to begin with. BTB appears to have little impact on the overall health of the plants, with one plant surpassing 12 feet in height. Staff anticipate the same result for Flueggea. In year two of this plan cloning will continue and plantings will be completed. In years three through five staff will continue to maintain the living collection, collect pollen, hand pollinate flowers, and collect seed for storage at the OANRP Seed Conservation Laboratory.

Current Genetic Storage Status

Population Unit and # Founders in Average Age of Founders (>3) in Founders with ≥ 10 Genetic Storage Living Collection by Living Collection by Microprop. Viable Seeds in Complete PopRef. PopRef Storage Central and East LEH-B (1) 7.5 yrs 0 0 Makaleha (57%) LEH-C (3) 8.25 yrs 0 1 LEH-D (1) 4 yrs 0 0 LEH-I (1) 4 yrs 0 0 LEH-J (1) 5 yrs 0 0 Halona (50%) HAL-A (1) 10 yrs 0 0 Kahanahaiki to MMR-A (1) 16 yrs 0 1 Kapuna (29%) MMR-B (1) 10.33 yrs 0 1 MFS MMR-E (1) 2 yrs 0 0 PAH-C (1) 2 yrs 0 0 Kauhiuhi (0%) KAU-A (1) 9.5 yrs 0 0 Makaha (36%) MAK-A (2) 8.75 yrs 0 0 MFS MAK-B (1) 3 yrs 0 0 MAK-C (1) 10.5 yrs 0 0 MAK-D (3) 8.33 yrs 0 1 MAK-E (1) 13 yrs 0 0 MAK-F (2) 9.5 yrs 0 0 MAK-J (1) 7 yrs 0 0 Mt. Kaala NAR AAW-C (1) 11.67 yrs 0 1 (50%) ALI-B (1) 9 yrs 0 1 Nanakuli, south NAN-A (1) 7.33 yrs 0 0 branch (100%) Ohikilolo (50%) MMR-C (1) 9.5 yrs 0 0 MFS West Makaleha LEH-A (3) 5.2 yrs 0 1 (29%) LEH-E (2) 12.5 yrs 0 0 Total 33 ------0 7

Five Year Action Plan

Population Unit MIP Year 16 MIP Year 17 MIP Year 18 MIP Year 19 MIP YEAR 20 and Genetic OIP Year 13 OIP Year 14 OIP Year 15 OIP Year 16 OIP Year 17 Storage Oct. 2019- Oct. 2020- Oct. 2021- Sept. Oct. 2022- Sept. Oct. 2023- Sept. Complete Sept. 2020 Sept. 2021 2022 2023 2024 Central and East Clone in situ Clone in situ Collect pollen, Collect pollen, Collect pollen, Makaleha (57%) founders (≥5 founders (≥5yrs); pollinate flowers pollinate flowers pollinate flowers and yrs); identify plant at inter situ and collect seed at and collect seed at collect seed at inter inter situ sites; sites. inter situ sites. inter situ sites. situ sites. Maintain begin planting at Maintain Maintain complete complete living inter situ sites. complete living living collection. collection until seed collection. storage can replace genetic storage for maternal lines. Halona (50%) Clone in situ Plant at inter situ Collect pollen, Collect pollen, Collect pollen, founders (≥5 sites. pollinate flowers pollinate flowers pollinate flowers and yrs); identify and collect seed at and collect seed at collect seed at inter inter situ sites; inter situ sites. inter situ sites. situ sites. Maintain begin planting at Maintain Maintain complete complete living inter situ sites. complete living living collection. collection until seed collection. storage can replace genetic storage for maternal lines. Kahanahaiki to Clone in situ Clone in situ Clone in situ Collect pollen, Collect pollen, Kapuna (29%) founders (≥5 founders (≥5yrs); founders (≥5yrs); pollinate flowers pollinate flowers and yrs); identify plant at inter situ plant at inter situ and collect seed at collect seed at inter inter situ sites; sites. sites. inter situ sites. situ sites. Maintain begin planting at Maintain complete complete living inter situ sites. living collection. collection until seed storage can replace genetic storage for maternal lines. Kauhiuhi (0%) Clone in situ Plant at inter situ Collect pollen, Collect pollen, Collect pollen, founders (≥5 sites. pollinate flowers pollinate flowers pollinate flowers and yrs); identify and collect seed at and collect seed at collect seed at inter inter situ sites; inter situ sites. inter situ sites. situ sites. Maintain begin planting at Maintain Maintain complete complete living inter situ sites. complete living living collection. collection until seed collection. storage can replace genetic storage for maternal lines. Makaha (36%) Clone in situ Clone in situ Collect pollen, Collect pollen, Collect pollen, founders (≥5 founders (≥5yrs); pollinate flowers pollinate flowers pollinate flowers and yrs); identify plant at inter situ and collect seed at and collect seed at collect seed at inter inter situ sites; sites. inter situ sites. inter situ sites. situ sites. Maintain begin planting at Maintain Maintain complete complete living inter situ sites. complete living living collection. collection until seed collection. storage can replace genetic storage for maternal lines. Mt. Kaala NAR Clone in situ Plant at inter situ Collect pollen, Collect pollen, Collect pollen, (50%) founders (≥5 sites. pollinate flowers pollinate flowers pollinate flowers and yrs); identify and collect seed at and collect seed at collect seed at inter inter situ sites; inter situ sites. inter situ sites. situ sites. Maintain begin planting at Maintain Maintain complete complete living inter situ sites. complete living living collection. collection until seed collection. storage can replace genetic storage for maternal lines.

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References Keir, M. December 2010. Chapter 2, Section 2.2 Five Year Rare Plant Plan: Flueggea neowawraea in Status Report for the Makua and Oahu Implementation Plans. http://manoa.hawaii.edu/hpicesu/DPW/2010_YER/default.htm MIT 2003. Makua Implementation Plan. United States Army Garrison, Hawaii, Directorate of Public Works, Environmental Division, Schofield Barracks, HI. 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.