Genetic Diversity of Gold-Spotted Pond Frog, Rana chosenica in Korea

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1 Genetic Diversity of Gold-Spotted Pond Frog, Rana chosenica in Korea Mi-Sook Min, Sun Kyung Park, Kelly Lasater, Hae Jun Baek, Dae Sik Park2, and Hang Lee 4 Seoul National University College of Veterinary Medicine Kangwon National University Department of Science Education

2 Rana chosenica Gold-spotted pond frog, Korean Golden frog, Seoul frog Endemic to western Korean peninsula

3 Related Species Rana plancyi Part of the Pelophylax species complex R chosenica separated from this species Rana nigromaculata Part of the Pelophylax species complex Possibly Hybridizes with R chosenica in the wild Near Threatened in majority of range, common in Korea

4 Taxonomic Status Rana chosenica is very similar to R plancyi, considered the same species in the past Classified as a separate species in the early 0's (Kuramoto 93) Frost et al (26) proposed the separation of all species in the Pelophylax group to a new genus of the same name, thus Pelophylax chosenicus

5 Current Population Status Found at 6 sites in Gyeonggi-do, Chungcheongbuk-do, Chungcheongnamdo, Jeollabuk-do, Incheon, and Jeollanamdo Prefers small ponds, rapid declines due to habitat loss Vulnerable IUCN Red List Endangered Species Ⅱ Korean MOE

6 Threats to Wild Populations Endemic with limited range 6 sites have a total area of 0 km² Rice cultivation & urbanization reduce and fragment available habitat

7 Habitat Fragmentation

8 What needs to be done? For effective conservation strategies, it is important to maintain genetic diversity and to corroborate taxonomic status

9 Population Diversity Compared mitochondrial DNA cytochrome b gene and control region to examine the genetic diversity of R chosenica populations within Korea

10 Methods Genomic DNA was extracted Partial cytochrome b, control region, 2S rrna, and 6S rrna genes amplified from mtdna using PCR PCR products were purified and directly sequenced using 4 primers primers were designed based on a sequence of R nigromaculata & R plancyi and the new sequence generated in the present study The universal primers of F9, L2, and ControlP-H were also used Only cytochrome b and control region had polymorphic loci and were used for building trees

11 Primers Used in the Present Study 6 Cytochrome b (,43bp) F9 L2 24 Control Region (243bp) ControlP-H L-inner KGfinner S rrna (932bp) 2L S rrna (,9bp) 2SR KGf-R3 2L3 KGf-i4 KGf-inner2 KGf-inner3 Primer Name F9 ControlP-H L29 L-inner KGf-i4 KGf-R3 KGf-inner KGf-inner2 KGf-inner3 2sZ-H 2L20 2SR 2L3 6H2 32 2sZ-R3 Sequence ( 3 ) CAT CGA CCT TCC AGC CCC ATC AAA CAT GTC CAT AGA TTC AST TCC GTC AG GAC AAA GCA TCC CTC ACC CG CTC ACA TGG ATC GGG GGA CAA C ACA ATC AAC ACG ACG C AAG CCT TGG GCA GGG TTT AG CTT GAT CGC ACC TTC CCT TG AGG AGG TAT CTG ACG GAA CTG CCT GCC AGA CCT CAA CCT T AAG GCT AGG ACC AAA CCT TT AAA GTA HAG CAC TGA ARA CGC DAA G TTT CAT GTT TCC TTG CGG TAC AAA GAA GAG GAA AGT CGT AAC ATG GTA TTA GGG AGA GGA TTT GAA CCT CTG 6H2

12 Sample Locations R chosenica 9 individuals sampled across four provinces in South Korea R plancyi sample from Chongqing, China sample from Genbank R nigromaculata sample from Genbank

13 Sample Locations Collection Locality No of Samples Cheongju, Chungcheonbuk-do 2 Taean, Chungcheongnam-do 3 Ongjin-A, Incheon 4 Ongjin-B, Incheon 6 Gangwha, Incheon 9 6 Pyeongtaek, Kyeonggi-do 2 Goyang, Kyeonggi-do 4 Ansan, Kyeonggi-do 9 Paju, Kyeonggi-do Total 9

14 Samples Information and Number of Haplotypes Locality No of samples (N) Cyt b CR Yeoungheung-do A Yeoungheung-do B Taean Cheongju Goyang Ansan 4 3 Gangwha 9 9 Paju Pyeongtaek N No of Haplotypes Cyt b CR

15 PAJ (4) 6 Cytochrome b Haplotypes Paju (PAJ) Taean (TAE) Ansan (ANS) 9 Gangwha (GAN) 0 R plancyi R plancyi (EF9669) 2 R nigromaculata (NC20) PAJ () ONG A (2) 2 TAE () GOY (4) PAJ (2) ONG A () 66 ONG B () Hap 2 Hap 3 Hap 4 Hap Hap 6 ONG A (2) Hap ANS () Hap PYE () Hap 9 PYE (3) Hap 0 PYE (2) Hap PYE () Hap 2 PYE (3) Hap 3 PYE () Hap 4 69 ONG A (2) 96 Hap PYE () 0 6 ONG B () 0 Cheongju (CHE) 2 Pyeongtaek (PYE) 3 Ongjin A (ONG A) 4 Ongjin B (ONG B) Goyang (GOY) GAN (9) Hap PYE (4) 69 CHE () 34 Hap 6 PYE (2) 2 ONG A () 4 PYE (2) Hap Rana plancyi - EF9669 Rana plancyi Rana nigromaculata - NC20 Cytb Haplotype UPGMA tree 0 0

16 PT () Hap OJ B () 9 OJ A (2) 29 Hap 2 OJ A () 43 Hap 3 OJ B () 2 OJ B () Hap 4 Hap PT () Hap 6 OJ A () Hap OJ B () Hap OJ B () 9 Hap 9 2 OJ B () 23OJ A () Hap 0 4OJ B () Hap 2 PT () Hap 2 PJ (2) Hap 3 43 GH () Hap 4 GH () Hap 44 GH () 3 Hap 6 GH () Hap TA () Hap PT () Hap 9 3 GH () Hap PJ () Hap 2 PT () Hap 22 6 PT () 2 6 PT () Hap 23 Hap 24 OJ A () Hap 2 AS () Hap AS () Hap 2 AS (2) Hap 2 GY (4) Hap 29 6 PJ () Hap 30 OJ A () Hap 3 PT (2) Hap PT () Hap 33 CJ () 4 40OJ A (2) Hap 34 3PT () Rana plancyi (EF9669) Rana nigromaculata (NC20) 23 D-loop Haplotypes Cheongju (CJ) 2 Pyeongtaek (PT) 3 Ongjin-A (OJA) 4 Ongjin-B (OJB) Goyang (GY) 6 Paju (PJ) Taean (TA) Ansan (AS) 9 Gangwha (GH) 0 R plancyi (EF9669) R nigromaculata (NC20) 02 D-loop Haplotype UPGMA tree

17 Summery Corroborate the division of R chosenica & R plancyi as distinct species R chosenica has relatively high genetic diversity for its population size Has no regionally distinct haplotypes indicating well connected populations in the past

18 Future Research Further comparison using microsatellite markers find more recent population isolations Begin more systematic monitoring find population trends and disease outbreaks Population rescues as needed In progress in Pyeongtaek

19 Thank You