Summary

Introduction
The National Agriculture and Food Research Organization (NARO) Genebank has been conducting frequent collection trips for the ex-situ conservation of crops and their wild relatives in Japan (see Annual Report on Exploration and Introduction of Plant Genetic Resources, https://www.gene.affrc.go.jp/publications. php#plant report). With regards to leguminous crops, many beans belong to the genus Vigna, such as cowpea, mung bean, and azuki bean, and at present, three wild relatives of the azuki bean inhabit Japan. One particular species, Vigna angularis var. nipponensis (Ohwi) Ohwi & Ohashi, are widely distributed from the Kagoshima Prefecture to Akita Prefecture, whereas two other species, Vigna nakashimae (Ohwi) Ohwi & Ohashi and Vigna riukiuensis (Ohwi) Ohwi & Ohashi, inhabit a limited area in southern Japan. V. nakashimae inhabits Nagasaki and Kumamoto Prefectures, and V. riukiuensis inhabits the Okinawa Prefecture (see the data of the Genebank database). Similarly, Vigna marina (Burm.) Merr. is one of the wild relatives of cowpea inhabiting Japan. It is a pantropical plant with sea-drifted seeds that is widely distributed in tropical and subtropical coastal areas of the world and is also known as the beach pea. In Japan, our field surveys have shown that V. marina grows well on the coral sand beaches of Okinawa and Sakishima Island, with distinctive yellow flowers and brown pods (Egawa et al. 1990, 1991; Tomooka et al. 2000, 2005, 2012, 2013; Takahashi et al. 2014, 2018; Muto et al. 2015), In the past, I have been involved in seven field surveys of leguminous plants and their symbiotic rhizobia (Muto et al. 2015; Baba-Kasai et al. 2016, 2017, 2018, 2019; Takahashi et al. 2017, 2018). Through these field surveys, I confirmed that V. marina is the best host plant for identifying salt-tolerant rhizobia. Since V. marina can grow in sunny beach front areas and possesses high tolerance to salt stress and drought (Chankaew et al. 2014), it is likely that their symbiotic rhizobia also possess high salt tolerance (Elanchezian et al. 2009). Another important factor is that V. marina prefers sandy beaches as habitats; therefore, it is very easy to collect root nodules without special tools. Although many collection trips have been previously performed and many habitats of V. marina have been recorded on Okinawa Island and Sakishima Island, there have been few surveys conducted on Amami Island and Osumi Island. Therefore, I decided to conduct a field survey of V. marina and their symbiotic rhizobia in 2019 on Tokunoshima Island, Okinoerabu Island, and Yoron Island, which collectively belong to the Amami Islands.
Methods
A field survey of the Tokunoshima, Okinoerabu, and Yoron islands in Kagoshima Prefecture, Japan, was conducted by car from November 18 to 22, 2019. The survey began at Tokunoshima Airport and ended at Yoron Airport (the itinerary is shown in Table 1). When naturally growing V. marina or a habitat with conditions suitable for V. marina was found, the area was searched for natural populations. Bulk seed samples were collected from each population. Seeds of each morphotype were collected separately when the population contained plants with different traits. The passport data recorded included the location of collection sites, such as latitude, longitude and altitude. Maps was sketched for each habitat and any special characteristics of sampled plants were noted, as shown in Table 3. This information was stored in the Genebank database after the sampled plants were registered as accessions. The latitude and longitude were measured using the WGS84 world geodetic system and a Garmin GPSMAP 60sc handheld GPS device (Germin International, Inc., Olathe, KS ). Root nodules were collected from V. marina's roots at nine specific habitats, and they were placed in a screw bottle with desiccant silica gel.
Results and Discussion
For all islands
Individuals of V. marina were often found in areas slightly inland from the beach, such as parking lots near beaches, at a parking space near Tokunoshima Airport (Photo 1), and along roadsides leading to the ocean, for example, at a roadside leading to Hakibina Beach on Yoron Island (Photo 2). This is characteristic of the parking space near the Tokunoshima Airport. The location is marked as r1  on the map (Fig. 1).
Prefecture. habitat distribution of V. marina on Tokunoshima Island, Okinoerabu Island, and Yoron Island, and it seems to be a very common seaweed on these three islands. In fact, I observed park maintenance workers weeding V. marina at the beach near Tetehama Seaside Park in Tokunoshima due to heavy overgrowth (Photo 3). V. marina was generally found growing on clean coral sand on all three islands (Photo 4: Tokunoshima, Photo 5: Okinoerabu, and Photo 6: Yoron) even though it was also found in a variety of non-beach soil environments as noted above. On all three islands, Cassytha filiformis L., also referred to as love-vine, was often found growing parasitically on V. marina and flowering (Photos 7–9) on sandy beaches. As summarized in Table 2, V. marina seeds were collected from 19 sites on three islands, and root nodules were collected from 9 accessions of V. marina. Soil samples were also collected from 6 of the 19 habitat sites. The weather was not favorable, with heavy squalls on Tokunoshima and Yoron Island; therefore, some parts other weeds, were found near Tetehama Seaside Park. The park maintenance workers were busy weeding. The location is marked as TOY8 on the map (Fig. 1). map (Fig. 1). Tokunoshima Island. The location is marked as TOY5 on the map (Fig. 1). parasitically on Vigna marina's pods. The location is marked as TOY 1 on the map (Fig. 1).
marina. The location is marked as TOY14 on the map (Fig. 1). of the coastlines on the three islands were not explored. Because one of the aims of this survey was to visit as many sites as possible, root nodules were not collected from all of the identified V. marina habitats. This is because the collection of root nodules, in addition to the collection of seeds, is an extra time-consuming process. Therefore, the number of rhizoids collected was equal to approximately half of the number of seeds collected.
Tokunoshima Island
Tokunoshima Island was started to survey on November 18. On this particular day, it was raining heavily; therefore, the southern half of the island could not be properly surveyed. Nevertheless, V. marina was often found on the side of the road in areas near the beach (Photos 1 and 10). Overall, 9 accessions (JP286186 −286194) of V. marina and 4 samples of V. marina root nodules (at TOY-01, 04, 05, and 08) were collected from Tokunoshima Island (detail are shown in Table 3). Root nodules were dug up from the beach with a shovel (Photos 11 and 12) and stored in sealed sample jars filled with silica gel (Photo 13). vacant ground on the road at the Omonawa fishing port. The location is marked as r2 on the map (Fig. 1). beach with shovels. The location is marked as TOY10 on the map (Fig. 1).
Okinoerabu Island
Okinoerabu Island was formed by uplifted coral reefs, and the map gave the impression of a flat island surrounded by many sandy beaches. However, the erosion of uplifted coral reefs has created a complex coastline with strange rocky beaches such as Ujiji location is marked as TOY 1 on the map (Fig. 1). Beach, spouting caves such as Fucha, and cliffs such as Cape Tamina. In addition, some beaches, such as Wanjo Beach, have been so well maintained that there are hardly any wild plants due to recent tourism. Hence, the number of collection points for V. marina was less than expected. Overall, 6 accessions (JP286195–286200) of V. marina and 3 samples of V. marina root nodules (at TOY- 10, 11, and 15) were collected from Okinoerabu Island (details are shown in Table 3). Lablab purpureus (L.) was observed at a roadside near Yakomo, China-cho (Photo 14). Unfortunately, all of the pods were too young to collect seeds. Glycine tabacina (labill.) Benth grows in Tamina Cape, which is the northern limit of the G. tabacina habitat in Japan (Ooya 2013; Ryukyu Plant Research Group 2018 onward). Similarly, several studies have reported that habitats of Vigna reflexo-pilosa Hayata were confirmed marked as r6 on the map (Fig. 1). in Okinoerabu, along with Tokunoshima and Yoron (Tomooka et al. 1992; Ryukyu Plant Research Group 2018 onward). In the future, I believe that it would be valuable to resurvey the inland areas to collect their seeds.
Yoron Island
On November 21, Yoron Island was started to survey. Due to heavy rain, only 4 beaches on the southern side of the island were explored on that day. Furthermore, 2 of the 4 beaches were undergoing construction of seawalls or breakwaters, making the beaches inaccessible. Nevertheless, it was confirmed that V. marina grew at 3 sites; however, only young plants without mature pods were found at 2 of the 3 sites. On the north side of the island, mature pods of V. marina could be collected on 3 beaches of 6 site searched in the morning of November 22. On the remaining 2 beaches, V. marina plants were too young to collect seeds. The last beach, Chabana Beach, was over- maintained similarly to Wanjo Beach on Okinoerabu Island; therefore, no V. marina plants were found. As a result, four accessions (JP286201–286204) of V. marina and two samples of V. marina's root nodule (at TOY-16 and TOY-18) were collected on Yoron Island (details are shown in Table 3). In general, I found that the V. marina plants growing on Yoron Island were substantially younger than those on the other two islands. As indicated by the construction of seawalls and breakwaters, the sandy beaches of Yoron Island are narrow and are easily affected by high waves. Thus, I believe that salt damage caused by exposure to the tides is one of the reasons for the high proportion of young individuals on Yoron Island. Baba-Kasai A, Akiba M, Hirashima S, Ohmizu T and Tomooka N (2016) Field survey and collection of Glycine and Vigna genetic resources in Nagasaki Prefecture, Japan, 2015. APEIPGR 32: 1-27. [Genebank, NARO], [JaLC] Baba-Kasai A, Akiba M, Iizumi T and Ito Y (2017) Field survey and collection of leguminous genetic resources in Tanegashima and Yakushima islands of Japan in 2016. AREIPGR 33: 29-47. [Genebank, NARO], [JaLC] Baba-Kasai A, Akiba M and Iizumi T (2018) Field survey and collection of leguminous genetic resources in Kagoshima and Kumamoto Prefectures of Japan in 2017. AREIPGR 34: 17-33. [Genebank, NARO], [JaLC] Baba-Kasai A, Akiba M and Iizumi T (2019) Field survey and collection of leguminous genetic resources in Tokushima and Kochi Prefectures of Japan in 2018. AREIPGR 35: 16-32. [Genebank, NARO], [JaLC] Chankaew S, Isemura T, Naito K, Ogiso-Tanaka E, Tomooka N, Somta P, Kaga A, Vaughan DA and Srinives P (2014) QTL mapping for salt tolerance and domestication-related traits in Vigna marina subsp. oblonga, a halophytic species. Theor Appl Genet 127 (3): 691-702. [CrossRef] Egawa Y, Siriwardhane D, Horikoshi H, Tomooka N, Tomita K and Nakano H (1990) Collection of Vigna riukiuensis and V. reflexo-pilosa, wild relatives of adzuki bean on Iriomote Island, Okinawa Prefecture. AREIPGR 6: 23-30. [Genebank, NARO], [JaLC] Egawa Y, Horikoshi H, Tomooka N and Nakano H (1991) Collection of wild relatives of adzuki bean, Vigna riukiuensis and V. reflexo-pilosa on Yonaguni Island, Okinawa Prefecture. AREIPGR 7: 13-21. [Genebank, NARO], [JaLC] Elanchezian R, Rajalakshmi S and Jayakumar V (2009) Salt tolerance characteristics of rhizobium species associated with Vigna marina. Indian J Agric Sci 79 (12): 980-985. [Google Scholar] Muto C, Iseki K, Sakai H, Naito K, Baba A, Maruyama Y, Hagita S, Umezawa T and Yokoyama T (2015) Collection and conservation of leguminous crop wild relatives on Ishigaki, Miyako, Kurima, Irabu and Ikema islands, Okinawa, Japan, 2014. AREIPGR 31: 99-119. [Genebank, NARO], [JaLC] Ooya S (2013) The report of the plant collection on Okinoerabu Island, Kagoshima Prefecture. Bulletin of the Kagoshima Prefectural Museum 32: 67-71 (in Japanese). [NDL DIGITAL COLLECTIONS] Ryukyu Plant Research Group (2018 onward) Database of Ryukyu plants. National Museum of Nature and Science, Tokyo. [https://www.kahaku.go.jp/research/activities/project/ hotspot japan/ryukyus/db/], [Accessed December 22, 2022]. Takahashi Y, Naito K, Ogiso-Tanaka E, Inoue J, Hirashima S and Tomooka N (2014) Collection and field survey of wild Vigna genetic resources in the Yaeyama Archipelago, Okinawa Prefecture, Japan, 8th to 14th July, 2013. AREIPGR 30: 29-51. [Genebank, NARO], [JaLC] Takahashi Y, Baba-Kasai A, Akiba M, Iizumi T and Tomooka N (2017) Collection and conservation of legume genetic resources in Oita and Miyazaki Prefectures of Japan in 2016. AREIPGR 33: 1-27. [Genebank, NARO], [JaLC] Takahashi Y, Baba-Kasai A, Abe A, Akai K, Akiba M, Hirashima S, Kaga A and Tomooka N (2018) Collection and conservation of legume genetic resources on Okinawa Island, Yagaji Island, Kouri Island and Hamahiga Island in 2017. AREIPGR 34: 1-16. [Genebank, NARO], [JaLC] Tomooka N, Egawa Y, Lairungreang C and Thavarasook C (1992) Collection of wild Ceratotropis species on the Nansei Archipelago, Japan and evaluation of bruchid resistance. JARQ 26 (3): 222-230. [JIRCAS] Tomooka N, Vaughan DA, Egawa Y, Tateishi Y and Yamashiro T (2000) Exploration and collection of wild Vigna species on the Nansei Archipelago, Japan. AREIPGR 16: 39-49 (in Japanese with English summary). [Genebank, NARO], [JaLC] Tomooka N, Kuroda Y, Yokoyama T, Kashiwaba K, Kaga A, Isemura T and Vaughan DA (2005) Ecological survey and conservation of legumes - symbiotic rhizobia genetic diversity from Ishigaki Island, Okinawa, Japan, 2004. AREIPGR 21: 97-103. [Genebank, NARO], [JaLC] Tomooka N, Yoshida Y, Naito K, Akatsu T and Yokoyama T (2012) Collection and conservation of wild leguminous crop relatives on Ishigaki-jima, Iriomote- jima and Kuro-shima islands, Okinawa, Japan, 2011. AREIPGR 28: 27-41. [Genebank, NARO], [JaLC] Tomooka N, Inoue J, Akiba M and Umezawa T (2013) Collection and conservation of wild leguminous crop relatives on Ishigaki, Iriomote, Miyako, Kurima, Irabu and Ikema islands, Okinawa, Japan, 2012. AREIPGR 29: 1-17. [Genebank, NARO], [JaLC]
馬場(笠井)晶子農業・食品産業技術総合研究機構遺伝資源研究センター和文摘要本報告は，2019年11月18日から22日にかけて，鹿児島県の徳之島，沖永良部島，与論島の海岸で行った耐塩性マメ科遺伝資源Vigna marinaとその根粒の探索収集報告である．現地調査の結果，３島からV. marina 19点を収集した．また，共生根粒菌を分離するために，V. marinaの９つのアクセッションから根粒を採取し，さらに6つのアクセッションの生息地からは土壌試料も採取した．収集した材料はすべて農業・食品産業技術総合研究機構（NARO）ジーンバンクに保存し，増殖した種子は，研究，育種，教育目的のために，要望に応じて提供する予定である.
Photos of Seed samples Photos of Seed samples