Full paper genetics

Genetic Assessment of Reinforcement of the Japanese Brown （ japonica）

Mitsuaki OGATA1 ）＊ and Hiroshi SICHIRI2）

1）Preservation and Research center, City of Yokohama, Kanagawa, 241-0804, Japan 2）Yokohama Environmental Science Research Institution, Kanagawa, 221-0024, Japan

［Received 1 March 2018; accepted 7 January 2019］

ABSTRACT To assess the genetic suitability of reinforcement of two populations of the Japanese brown frog (Rana japonica) in Yokohama City, we attempted to clarify their genetic relationship based on sequence variation of mitochondrial DNA control region and polymorphism of 10 microsatellite DNA loci. The results showed that these two populations are genetically related, though they are presently isolated. This appears to conform to the conditions for reinforcement of the two populations. Key words: genetic variation, habitat fragmentation, reinforcement － Jpn J Zoo Wildl Med 24（1）：1-7, 2019

second most populous city and is highly urbanized [12]. Despite INTRODUCTION this high urbanization, Yokohama City is the main habitat of The worldwide decline of populations has been the frog in Kanagawa Prefecture [13]. However, in Yokohama previously reported [1]. Several factors have been proposed City, marshes, rice fields and other green spaces have as the cause of the decline, such as pollution, habitat loss, drastically reduced over the past 40 years (46.4% of the total ultraviolet radiation, and pathogens (e.g. [1]). Of these, habitat area in 1975 compared with 28.8% in 2015) [14], reducing loss and fragmentation are major factors [2], as they reduce the frog’ s available habitat. We predict a concurrent reduction population size, and lead to detrimental genetic effects such in the population. Indeed, the number of spawned egg masses as genetic load, inbreeding depression and reduced genetic reduced drastically during the last decade in the northern variation (e.g. [3-5]). To prevent such detrimental effect in small population in Yokohama City (P5 in Table 1). According to a populations, reinforcement (also known as augmentation), previous study, 757 egg masses were spawned in 2001 [13], defined as introduction of new individuals into a population of but in 2015, this number was estimated to be less than 50 conspecifics, is effective for supplying a new genetic resource [15]. Such drastic reduction led us to predict extinction of the [6-7]. Several translocations of amphibian species have already local population would occur unless conservation efforts were been reported [8]. applied to the population. Another large R. japonica population The Japanese brown frog, Rana japonica, is endemic to the was known to exist nearby (P6 in Table 1), although the two Japanese archipelago. This frog lives in hillside areas and populations are isolated by residential areas [13, 15]. In fact, spawns egg masses in water in marshes or rice fields during the these two populations are the largest (P6) and second largest early spring [9-10]. This frog has been assigned a vulnerable (P5) R. japonica populations in Kanagawa Prefecture [13]. status in several areas, including Kanagawa Prefecture [11]. Therefore, local extinction of P5 would greatly negatively Yokohama City, located in Kanagawa Prefecture, is Japan’s impact overall R. japonica levels in the prefecture. To prevent local extinction of P5, reinforcement from P6 was expected to be an effective approach. The genetic relationship between the * Corresponding author：Mitsuaki OGATA source population and the recipient population is important （E-mail: [email protected]）

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Table 1 Locations and haplotypes in control region of mitochondrial DNA. Population No. of Sample Location Haplotype P1 4 egg 35.566583, 139.495604 CR1(3/4),CR5(1/4) P2 2 tadpole 35.55615, 139.584683 CR6 (2/2) P3 4 egg 35.548467,139.568515 CR5 (4/4) P4 3 tadpole 35.539967, 139.579324 CR6 (3/3) P5 14 egg, finger 35.51336, 139.51572 CR2 (14/14) P6 11 egg, finger 35.503494, 139.534341 CR3 (7/11),CR4 (4/11) P7 2 tadpole 35.40828, 139.52122 CR7 (2/2) Total 40 Numbers in parentheses indicate the ratio corresponding to each haplotype.

factor in the effectiveness of reinforcement, so as to prevent using ABI 310 genetic analyzer (Applied BioSystems) according outbreeding depression and encourage genetic rescue (e.g. [7, to the manufacturer’s instruction. DNA alignment was 16]). However, no information on the genetic relationship of performed with MEGA6 [18]. For estimating DNA divergence, the two populations has been reported so far and there is no we excluded sites where gaps or insertions were observed. information on the genetic relationships among the various R. Nucleotide diversity and haplotype diversity of two populations japonica populations in Yokohama City, which is necessary for (P5 and P6) was estimated using DnaSP ver. 5.1 [19]. The assessing the genetic suitability of reinforcement from P6 to effective number of migrants per generation (Nm) and fixation P5. Therefore, in this study, we investigated genetic relationships among the populations of R. japonica in Yokohama City, focusing in particular on the two local populations (P5 and P6), to assess the genetic suitability of reinforcement from P6 to P5.

MATERIALS AND METHODS

1）Sample We collected eggs or tadpoles of R. japonica from seven populations in Yokohama City (Table 1, Figure 1). Since all wild populations were in nature reserve areas, we minimized the sampling number. Tissue was collected from the tail of tadpoles, and one egg was collected per egg mass. We also collected fingers of male frogs found dead in water pools during breeding season. All samples were stored in 80% ethanol until DNA extraction. DNA was extracted using QIA DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instruction. 2）Mitochondrial DNA control region Partial DNA sequences of control region in mitochondrial DNA (CR) were amplified according to a previous study [17]. Fig. 1 Location of the seven populations examined in this We used 40 samples of the seven populations (Table 1). DNA study. P1–P7 correspond to the population identification sequences of the PCR product were analyzed in both directions number given in Table 1.

－ 2 － Genetic variation of Japanese brown frogs in Yokohama City

index (Fst) between P5 and P6 were estimated using DnaSP. Population differentiation between the two populations was tested using DnaSP. A median-joining network was constructed using Network [20]. 3）Microsatellite DNA We genotyped 10 types of microsatellite loci using a QIA multiple PCR kit (Qiagen) (Appendix). For genotyping, we used Fig. 2 Median-joining network based on the 563 bp two populations (P5 and P6). Of the 14 samples from P5, we sequences of the mitochondrial control region of the seven haplotypes observed in this study. CR1-CR7 corresponds to the eliminated 5 egg samples as they might have been descendants haplotype identification numbers given in Table 1. Numerals at of the eight adult males. Of the 11 samples from P6, we each node denote the number of differing sequences (one base eliminated one adult male sample as it might have been the if not otherwise specified). parent of 10 egg samples. PCR primers were used according to a previous study [21]. Annealing temperature was set at 57℃. Polymorphism analysis was conducted with the ABI 310 Table 2 Haplotype diversity and nucleotide diversity of genetic analyzer and GeneScan ver. 2.0 (Applied BioSystems) the two populations and the total of 7 populations of Rana japonica in Yokohama City, Kanagawa. according to the manufacturer’ s instructions. Linkage Population Haplotype diversity1） Nucleotide diversity1） disequilibrium and deviation from Hardy-Weinberg equilibrium in the wild populations were tested using Genepop ver. 4.2 [22]. P5 0 0 Expected heterozygosity (He), observed heterozygosity (Ho) P6 0.509 (0.101) 0.00181 (0.00036) and allelic richness were estimated using Fstat 2.9.3.2 [23]. Fst All 0.8 (0.039) 0.0052 (0.00089) between P5 and P6 was estimated using Genepop. Population (P1–P7) differentiation between the two populations was tested using 1） Numbers in parentheses indicate standard deviation. Genepop. To characterize genetic relationships between P5 and P6, STUCTURE (ver. 2.3) [24] was run on the 10 microsatellite DNA for 100,000 Markov chain Monte Carlo (MCMC) cycles 2）Microsatellite DNA polymorphism following 100,000 burn-in cycles, using an admixture model We stably amplified 10 microsatellite loci in 16 samples with independent allele frequencies. Ten replications were (Appendix). From the remaining three wild samples (one performed for each K, in the range K=1–5, and the optimal K sample of P5 and two samples of P6), several loci could not was estimated using STRUCTURE HARVEST [25]. Iterations be amplified in these DNA samples, possibly due to low DNA for the optimal K were analyzed using CLUMPP [26], and quality. No linkage disequilibrium was observed in this study. visualized using DISTRUCT [27]. Based on the polymorphism data of 10 microsatellite loci, Fst between P5 and P6 was estimated at 0.1489 (-0.0794 to RESULTS 0.3207), and they were significantly differentiated (exact G test, 1）Mitochondrial DNA control region df=20, p= 0.000). Structure analysis found that Delta K value Based on sequences of CR (563 bp), we found seven was largest in k=2: 15.45 (k=2), 2.13 (k=3) and 2.01 (k=4). The haplotypes from the 40 samples (Table 1). A median-joining distribution of the alleles in the structure map (k=2) clearly network is shown in Figure 2. From the network, we found two showed that P5 and P6 were differentiated (Figure 3). Number haplogroups, one contained five haplotypes (CR1–CR5) and the of alleles, He, Ho and allelic richness of each microsatellite loci other the remaining two haplotypes (CR6 and CR7). Haplotype were shown in Table 3. diversity and nucleotide diversity of two populations (P5 and DISCUSSION P6), and those of the 40 wild samples are shown in Table 2. P5 and P6 were significantly differentiated (Fst=0.74545, df=2, In this study, we tried to assess the genetic suitability of p=0.000). Nm between P5 and P6 was estimated at 0.3. reinforcement from the P6 population of R. japonica to the P5 population. Reinforcement can proceed when both

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[15]. Therefore, these two populations are considered to have similar environmental conditions. The two populations are also genetically related because they are of the same mitochondrial CR haplogroup (Figure 2 and Table 1). Based on the result, we supposed the risk of outbreeding depression or loss of local adaptation would not be large in reinforcement from P6 to P5. Nm between these two populations, however, was less than 1 (0.3), indicating that they are now genetically isolated. Based on these results, we supposed that the two populations might have previously been genetically connected but are presently isolated by physical barriers, such as traffic road and residential areas. Two of eight frogs in P5 shared a half or one-third of alleles of 10 microsatellite loci with P6 (Figrure3), although these two populations were significantly differentiated bases on the polymorphisms data of 10 microsatellite DNA loci. This well matched our assumption. Thus, our data appears to conform to the conditions for reinforcement from P6 to Fig. 3 Histograms of the STRUCTURE assignment test for 10 P5. One haplotype was observed in the recipient population microsatellite DNA markers. Value of K (No. of clusters) is 2. (P5) and both haplotype diversity and nucleotide diversity were lower than that of P6. Allelic richness and expected recipient and donor populations are in similar environmental heterozygosity in 10 microsatellite DNA loci were lower than condition, and are isolated recently and genetically related, that of P6. Thus, genetic diversity in P5 appears lower than in due to the lower risk of outbreeding depression or loss of local P6. These data indicate that reinforcement would be necessary adaptation [7]. The two populations of R. japonica assessed in for increasing genetic diversity in P5, and genetic diversity of this study (P5 and P6) are geographically adjacent (4.3 km) P5 would increase upon reinforcement from P6 to P5 as these

Table 3 Characterization of 10 microsatellite DNA loci of two populations of Rana japonica from Yokohama City, Kanagawa Prefecture. P5 (n=8) P6 (n=8) Locus No. of alleles Allelic richness He Ho No. of alleles Allelic richness He Ho Raja3 2 1.767 0.233 0 5 3.753 0.733 0.75 Raja4 3 2.763 0.633 0.5 3 2.499 0.592 0.375 Raja5 5 3.933 0.725 0.75 3 2.497 0.575 0.875 Raja6 4 3.228 0.641 0.5 5 3.8 0.717 0.625 Raja9 1 1 0 0 2 1.767 0.233 0.25 Raja10 3 2.971 0.708 0.375 5 4.33 0.808 0.625 Raja12 6 4.167 0.733 0.75 6 4.821 0.85 0.625* Raja13 5 3.533 0.608 0.375 4 3.262 0.692 0.625 Raja18 4 3.262 0.691 0.625 5 3.933 0.725 0.375* Raja20 4 3.686 0.767 0.5* 3 2.955 0.7 0.25* Average - 3.031 0.574 0.438 - 3.367 0.663 0.538 He indicates expected heterozygosity, Ho indicates observed heterozygosity. *indicates significant deviation from Hardy–Weinberg equilibrium (p < 0.05).

－ 4 － Genetic variation of Japanese brown frogs in Yokohama City

two populations were found to be genetically differentiated in 6. International Union for Conservation of Nature and Natural both mitochondrial DNA and nuclear DNA. Thus, P6 appears Resources/Species Survival Commission. 2013. IUCN valid as a new genetic resource for P5, and reinforcement from guidelines for reintroductions and other conservation P6 to P5 appears effective for increasing genetic diversity of translocations version 1.0. (http://data.iucn.org/dbtw-wpd/ P5. However, there is another risk of reinforcement, namely, edocs/2013-009.pdf) disease transmission (e.g. [6-7]). Therefore, we must also 7. Weeks AR, Sgro CM, Young AG, Frankham R, Mitchell NJ, assess the risk of disease transmission before conducting Miller KM, Byrne M, Coates DJ, Eldridge MDB, Sunnucks reinforcement. Population viability analysis would also be P, Breed MF, James EA, Hoffmann AA. 2011. Assessing necessary to evaluate the extinction risk of P5 more precisely the benefits and risks of translocations in changing [28] before reinforcement could be initiated. environments: a genetic perspective. Evol Appl 4: 709–725. Finally, regarding the total genetic variation for all 8. Germano J, Bishop P. 2009. Suitability of and populations in Yokohama City (435 km2 ), we found total reptiles for translocation. Conserv Biol 23 (1): 7-15. seven haplotypes in seven populations (Table 1), although 19 9. Maeda N, Matsui M. 1989. Frogs and Toads of Japan. pp. haplotypes were found in a smaller area in Chiba Prefecture 44-47. Bunichi-Sogo syuppan. Tokyo (in Japanese with (40 km2 ) [17]. Haplotype diversity in Yokohama was 0.8 (Table English abstract). 2), compared with 0.961 in this area in Chiba [17], indicating 10. Osawa S, Katsuno T. 2001. Dispersal of the Brown frogs, that the overall genetic variation of R. japonica in Yokohama Rana japonica and R. ornativentris in the forest of the Tama City is low. Since low genetic diversity is a possible cause of hills. Curr Herpetol 20 (1): 1-11. population extinction [29], both in-situ and ex-situ conservation 11. Kanagawa prefectural museum of natural history. 2006. activities for this frog in Yokohama City are necessary in order Red data book of Kanagawa Prefecture. (http://nh.kanagawa- to prevent such extinction. museum.jp/research/archives/reddata2006/ryosei.html) 12. Sadohara S, Koike F, Kada R, Sato Y. 2011. Satoyama ACKNOWLEDGEMENT revitalization, pp. 104-121. Soshinsha. Tokyo (in Japanese We thank the staff in the sampling area for cooperating in with English abstract). this study. 13. Osawa S, Katsuno T, Tsujimoto A. 2002. The survey of standing stock of brown frogs as effective environmental REFERENCES information: present status of brown frogs in hilly area in 1. Morten EA, O’Brien J. 2010. Global amphibian declines, loss Kanagawa prefecture Japan. Environ Inf Sci 31(1): 68-76 (in of genetic diversity and fitness: A review.Diversity 2(1): 47- Japanese with English abstract). 71. 14. City of Yokohama. 2015. The rate of green space in 2. Cushman SA. 2006. Effects of habitat loss and fragmentation Yokohama city. (http://www.city.yokohama.lg.jp/kankyo/ on amphibians: A review and prospectus. Biol Conserv data/ryokuhi/ryokuhi.html) 128(2): 231-240. 15. Ogata M, Sitiri H. 2018. Genetic diversity of Rana japonica 3. Hedrick PW. 1995. Gene flow and genetic restoration: The in Yokohama city. Bull Herpetol Soc Jpn. 2018(1): 126-127 Florida panther as a case study. Conserv Biol 9 (5): 996– (in Japanese). 1007. 16. Frankham R. 2015. Genetic rescue of small inbred 4. Westemeier RL, Brawn JD, Simpson SA, Esker TL, Jansen populations: meta-analysis reveals large and consistent RW, Walk JW, Kershner EL, Bouzat JL, Paige KN. 1998. benefits of gene flow.Mol Ecol 24: 2610–2618. Tracking the long-term decline and recovery of an isolated 17. Kobayashi S, Abe S, Matsuki R. 2012. Development population. Science 282 (27): 1695-1698. techniques biodiversity conservation in environment impact 5. Pickup M. Young AG. 2008. Population size, self- assessment. CRIEPI V11014 (in Japanese with English incompatibility and genetic rescue in diploid and tetraploid abstract). races of Rutidosis leptorrhynchoides (Asteraceae). Heredity 18. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. 100: 268–274. MEGA6: Molecular Evolutionary Genetics Analysis version

－ 5 － Mitsuaki OGATA et al.

6.0. Mol Biol Evol 30: 2725-2729. Version 2.3. Retrieved from http://pritch.bsd.uchicago.edu/ 19. Librado P, Rozas J. 2009. DnaSP v5 A software structure.html comprehensive analysis of DNA polymorphism data. 25. Earl DA, Vonholdt BM. 2012. STRUCTURE HARVESTER: a Bioinformatics 25: 1451-1452. website and program for visualizing STRUCTURE output and 20. Bandelt HJ, Forster P, Röhl A. 1999. Median-joining implementing the Evanno method. Conserv Genet Resour 4: networks for inferring intraspecific phylogenies. Mol Biol 359-361. Evol 16: 37-48. 26. Jakobsson M, Rosenberg NA. 2007. CLUMPP: a cluster 21. Koizumi N, Watabe K, Mori A, Takemura T. 2009. Isolation matching and permutation program for dealing with label and characterization of 19 polymorphic microsatellite DNA switching and multimodality in analysis of population markers in the Japanese brown frog (Rana japonica). Mol structure. Bioinformatics 14: 1801-1806. Ecol Res 9: 248–250. 27. Rosenberg NA. 2004. DISTRUCT: a program for the 22. Raymond M, Rousset F. 1995. GENEPOP (version graphical display of population structure. Mol Ecol Note 4: 1.2): population genetics software for exact tests and 137-138. ecumenicism. J Hered 86: 248-249. 28. Boyce MS. 1992. Population viability analysis. Annu Rev 23. Goudet J. 2001. FSTAT: A program to estimate and test Ecol Syst 23: 481-506. gene diversities and fixation indices (version 2.9.3) (http:// 29. Frankham R, Ballou JD, Briscoe DA. 2002. Introduction to www.unil.ch/izea/softwares/fstat.html). conservation genetics, pp. 23-42. Cambridge Univ. Press, 24. Prichard J. 2010. Doculentation for structure software: Cambridge.

Appendix Genotypes of 10 microsatellite DNA loci and haplotypes of mitochondrial DNA control region (CR) of 16 individuals of Rana japonica. Locus Haplotype Individual Raja3 Raja4 Raja5 Raja6 Raja9 Raja10 Raja12 Raja13 Raja18 Raja20 of CR (CA) (CA) (TA),(CA) (CA) (CA) (CA) (CA) (CT) (CT) (TA),(CA) P5-1 95/95 157/157 166/176 163/163 231/231 182/182 162/166 280/324 189/189 185/191 CR2 P5-2 95/95 149/157 168/176 153/183 231/231 176/182 152/176 244/280 191/195 185/191 CR2 P5-3 97/97 149/157 176/176 171/171 231/231 176/182 166/166 280/280 191/191 185/191 CR2 P5-4 95/95 149/157 170/174 171/183 231/231 178/178 162/206 280/280 189/191 187/187 CR2 P5-5 95/95 177/177 176/176 171/171 231/231 176/176 162/166 264/324 187/187 187/187 CR2 P5-6 95/95 149/157 168/176 163/171 23/1231 176/176 166/180 280/280 189/191 185/185 CR2 P5-7 95/95 157/157 168/176 163/171 231/231 178/182 166/180 280/280 189/191 185/191 CR2 P5-8 95/95 149/149 166/174 171/171 231/231 178/178 166/166 248/248 189/191 193/193 CR2 P6-1 97/103 145/145 166/176 157/157 231/231 176/176 198/204 236/264 179/191 185/185 CR4 P6-2 103/107 145/155 166/176 155/181 231/239 176/182 152/152 236/252 179/191 185/185 CR4 P6-3 95/95 145/145 166/176 181/181 231/239 178/178 170/170 264/264 189/189 187/187 CR4 P6-4 95/103 157/157 166/176 181/181 231/231 176/182 166/198 252/264 189/189 185/185 CR4 P6-5 103/103 145/157 166/176 155/181 231/231 174/176 152/166 264/264 189/189 191/191 CR3 P6-6 95/103 145/157 166/166 155/181 231/231 174/176 164/204 240/252 187/191 187/191 CR3 P6-7 97/101 157/157 166/182 157/181 231/231 180/180 152/152 252/252 189/189 18/7187 CR4 P6-8 95/103 157/157 166/176 153/183 231/231 178/182 198/204 252/264 181/181 187/191 CR3 Repeat motifs of each locus are shown in parentheses.

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原著論文 動物遺伝学

ニホンアカガエルの補強に関する遺伝的評価

尾形光昭 1 ）＊ ，七里浩志 2）

1）横浜市繁殖センター 〒 241-0804 神奈川県横浜市旭区川井宿町 155-1 2）横浜市環境科学研究所 〒 221-0024 神奈川県横浜市神奈川区恵美須町 1

［2018 年 3 月 1 日受領，2019 年 1 月 7 採択］

要 約 横浜市内に生息するニホンアカガエル 2 個体群間の補強について，その遺伝的妥当性を評価するために，ミトコンドリア DNA の調節領域の塩基配列および 10 領域のマイクロサテライト DNA の遺伝的多型に基づき，両個体群間の遺伝的関係を解析した。 その結果，両個体群は遺伝的に近縁であるものの，有意な遺伝的分化を示した。本研究により示された2 個体群間の遺伝的関係から， 2 個体群間で補強を実施することに遺伝的な問題はないと考えられる。 キーワード：遺伝的変異，生息地分断，補強 －日本野生動物医学会誌 24（1）：1-7, 2019

＊ 責任著者：尾形光昭（ E-mail: [email protected]）

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