Stoneflies (Plecoptera) are known for being sensitive to water pollution and are used as bioindicators for evaluating water quality. Among them, Nemouridae, especially the genus Nemoura, which are commonly referred to as winter stoneflies, can be found around streams even during the cold winter months. Nemoura geei Wu, 1929, among them, was originally described from Beijing and is widely distributed in Korea, China, Japan, and the Russian Far East. Here, we report the development and characterization of new functional microsatellite markers of N. geei using high-throughput sequencing technology. A total of 80,661 microsatellite loci were identified with a total length of 1,801,591 bp. The average length was 22.34 bp, and microsatellites occupied 0.42% of the entire sequence. The novel 20 microsatellite markers developed in this study can be usefully applied to the population genetics analyses as important genetic resources for understanding the ecological and evolutionary characteristics of a stonefly species at the population level in Korea.

The invasive red swamp crayfish, Procambarus clarkii, is native to southcentral United States and northeastern Mexico. Recently, it has been being spreading in the wild in South Korea. However, its primary sources, introduction routes, establishment, and expansion in South Korea remain unclear. Here, we analyzed genetic diversity and population genetic structures of its domestic natural populations during early invasion, commercial stock from local aquaria (a suspected introduction source), and original United States population using mitochondrial COI gene sequences for 267 individuals and eight microsatellite markers for 158 individuals. Natural and commercial populations of P. clarkii showed reduced genetic diversity (e.g., haplotype diversity and allelic richness). The highest genetic diversity was observed in one original source population based on both genetic markers. Despite a large number of individuals in commercial aquaria, we detected remarkably low genetic diversity and only three haplotypes among 226 individuals, suggesting an inbred population likely originating from a small founder group. Additionally, the low genetic diversity in the natural population indicates a small effective population size during early establishment of P. clarkii in South Korea. Interestingly, genetic differentiation between natural populations and the United States population was lower than that between natural populations and aquarium populations. This suggests that various genetic types from the United States likely have entered different domestic aquariums, leading to distinct natural populations through separate pathways. Results of our study will provide an insight on the level of genetic divergence and population differentiation during the initial stage of invasion of non-indigenous species into new environments.

Brachymystax lenok tsinlingensis (family Salmonidae), cold freshwater fish, is endemic to Asia. This species is currently distributed throughout Russia, Mongolia, China and the Korean Peninsula. B. lenok tsinlingensis in South Korea was severely affected by anthropogenic activities such as habitat destruction, agricultural run-off and water pollution, and hence this fish has recently been dramatically decreased in its population sizes and become now critically endangered. To recover the number of individuals of B. lenok tsinlingensis, stocking or translocation programs have been conducted continuously by local governments since 1970s. However, these programs made little effort to clarify populations that may have originated from stocked, translocated or introduced fish. An understanding of genetic characteristics of endangered populations is critical to develop effective conservation and restoration plans especially because genetic diversity ensues their future fate. Therefore, we assessed the “conservation status” of this species by estimating the level of genetic diversity and genetic structure among ten geographic populations including restored populations via reinforcement and supplementation. Also, we aimed to trace the genetic origins of the newly translocated population (Chiak) through a restoration practice program. Moreover, we inferred the phylogenetic relationships among Korean lenok populations as well as across the Northeast Asia. Two hundred eighteen individuals of B. lenok tsinlingensis were sampled from ten localities (Yanggu, Injae, Seorak, Bangtae and Hongcheon: North Han River basin; Pyeongchang, Chiak and Jeongseon: South Han River basin; Taebaek and Bonghwa: Nakdong River basin in South Korea). Based on mitochondrial DNA (mtDNA) control region and eight nuclear microsatellite loci, we found extremely low levels of within-population genetic diversity, which suggests small effective population sizes (Ne) within populations. For mtDNA control region, each population housed one, or at most, two haplotypes that are restricted to the respective localities, meaning that these ‘genetically unique’ lineages will be lost permanently if the local populations undergo extinction. The overall values of haplotype diversity (h) and nucleotide diversity (π) for the entire Korean population were 0.703 ± 0.024 and 0.021 ± 0.010, respectively. In the case of microsatellites, average number of alleles across the eight loci for the entire population was 9.1 and allelic richness (AR) per population ranged from 2.375 to 4.144 (mean = 3.104). The values of observed heterozygosity (HO) and expected heterozygosity (HE) were similar to each other [HO: 0.400 ~ 0.590 (mean = 0.518); HE: 0.407 ~ 0.608 (mean = 0.504)]. The inbreeding coefficient (FIS) values were generally low, ranging from 0.048 to 0.279. Consequently, the majority of the populations (except Yanggu and Pyeongchang) were not significantly deviated from Hardy-Weinberg equilibrium (HWE), suggesting random mating at these loci tested. In addition, we found that Korean lenok populations were significantly genetically isolated from each other, with private mtDNA haplotypes and microsatellite alleles, indicating limited gene flow among populations, strong effects of genetic drift due to small Ne, or a combination of both. The Mantel test of microsatellites revealed a significant correlation (r = 0.414, P = 0.04) between genetic and geographic distances for pairwise comparisons among the ten populations, while that of mtDNA showed a lack of correlation. Given the shared identical mtDNA haplotype and similar microsatellite allelic distributions between Chiak and Hongcheon populations, we suggest that the restored (introduced) Chiak population would be inferred to be genetically originated from Hongcheon population. Phylogenetic relationships among Northeast Asian populations showed that South Korean lineages have more recently diverged from China (Yellow River), than between North Korea and Russia. Although the phylogenetic relationship would be expected to be associated with geography, South-North Korea and China populations with a similar latitude was more phylogenetically closely related. These findings may suggest a possible scenario for the historical movements of B. lenok tsinlingensis in Northeast Asia during Last Glacial Maximum (LGM). It would be supported by the line of evidence that most lenok populations migrated to southward from Northern Asia such as Russia and Mongolia during LGM because the Korean Peninsula was landlocked as inland epoch and functioned as a southern shelter with Yellow River. For this reason, the Korean Peninsula is suggested to be an important geographical region for better understanding phylogenetic relationships and evolutionary histories of B. lenok tsinlingensis across the Northeast Asia. Despite large efforts made to develop several restoration programs in South Korea for B. lenok tsinlingensis, it is still unknown whether these past restoration efforts were successful or fruitless, mainly because of little attention paid to post-restoration monitoring research. Hence, there was a lack of their published official records. In the future, conservation and restoration projects of the Korean lenok populations should consider the genetic data for a better understanding of their ecological and evolutionary trajectories. And finally, we hope that our findings here can help inform on the future effective conservation and restoration plans for B. lenok tsinlingensis populatio ns in South Korea.

Seagrasses, sea flowering plants, comprise approximately 60 species globally and are often called ‘ecosystem engineers’ because they create their own habitats by modifying the surrounding environments, which provide coastal zones with a number of crucial ecosystem services. Zostera marina (the common name ‘eelgrass’) is one of the seagrass beds-forming species distributed widely in northern hemisphere including the Korean coast, which plays a pivotal role in ecosystem as a primary producer and a nursery habitat or refuge for other marine organisms. However, due to global climate change and anthropogenic activities such as reclamation and dredging, there has recently been a drastic decline in population sizes of Z. marina in Korea. In order to develop effective conservation and restoration management programs of Z. marina populations, it would be helpful to consider all biological aspects of this species such as genetic characteristics as well as ecological and physiological features. This study first provides information on genetic diversity and genetic structure of Jeju Island and Namhae populations of Z. marina, which will contribute to the establishment of appropriate conservation and restoration management plans for future persistence of this species. Using six microsatellite markers, we investigated the level of genetic diversity and genetic structure among 10 geographic populations of Z. marina inhabiting Jeju Island (Hamdeok, Tokki-seom, Sungsan, Woljeong, Ojo) and Namhae (Gamak bay, Jindong bay, Nampo, Anggang bay, Geoje) on the southern coast of Korea. The level of genetic diversity within Jeju populations (mean allelic richness [AR]: 1.57 ~ 3.09) was found to be significantly lower than Namhae populations (AR: 3.09 ~ 4.29) (Mann-Whitney U-test, P < 0.05). These findings suggest that effective population sizes (Ne) of Jeju populations are generally smaller than those of Namehae populations. Within Jeju Island, Hamdeok population had the smallest population size (coverage: 138 m2) and the lowest genetic diversity (AR: 1.57), while Ojo population had the largest population size (coverage: 275,736 m2) and the greatest level of genetic diversity (AR: 3.09). Hamdeok population showed evidence of genetic bottleneck. These results again suggest that Ne of Jeju populations is generally low (except Ojo population). Among Jeju populations, all pair-wise comparisons of FST values (i.e., degree of genetic differentiation) were highly significant (FST = 0.0612 ~ 0.7168, P < 0.001) despite Jeju populations that were geographically closely located, indicating that these local populations are genetically divergent, probably due to a lack of gene flow among the populations. The observed strong population structure was substantiated by evidence that five genetic clusters are most likely, based on population assignment test (STRUCTURE). The Mantel test showed a positive relationship between genetic distance (FST) and geographic distance (km) across all the populations sampled (R2 = 0.4118, P < 0.05), suggesting that our data follow Isolation By Distance (IBD) model. Woljeong population revealed the highest level of FST values compared to other populations within Jeju Island in IBD. STRUCTURE and factorial correspondence analysis (FCA) further showed that some Woljeong individuals included genotypes of Namhae populations. Population size of Woljeong (coverage: 310m2) was approximately 50 % smaller than that of Sungsan (coverage: 841m2); however, extent of its genetic diversity (AR: 2.39) was even higher than that of Sungsan population (AR: 1.77). We speculated that Woljeong population underwent a transplantation from Namhae populations with relatively higher level of genetic diversity. FST values within Namhae populations were relatively lower (compared to within Jeju Island) despite the populations that were geographically more distant. It means that level of gene flow is higher among Namhae populations than among Jeju populations. Z. marina is known to have different life histories by water depth. In subtidal zone (deep water depth) populations predominantly undertake sexual reproduction through seeds such as annual life history, whereas those of intertidal zone (shallow water depth) undertake both sexual and asexual reproductions through horizontal rhizomes i.e., perennial life history. STRUCTURE analysis showed no clear differences between shallow and deep populations at Namhae, but some FST values were statistically significantly different despite their low values. For Geoje population sampled in 2005, intertidal and subtidal populations were not significantly different (FST = 0.0045, P = 0.033), but these populations sampled in 2015 showed a significant difference (FST = 0.0328, P < 0.001). It means that genetic structure of Geoje has been changed over the 10 year period between shallow and deep populations. Overall, the Jeju and Namehae populations analyzed in the current study have relatively low levels of genetic diversity and distinct genetic compositions, which warns the message that this ecologically important species should be conserved separately in the local populations and with high priority. We propose that future conservation and restoration plans for seagrasses should consider genetic characteristics particularly because a close relationship between genetic diversity and ecological performance in marine species has been well documented.

계곡산개구리는 2000년 형태적, 유전형질 분석결과를 토 대로 Rana속 유사종과 비교한 결과 별종으로 확인되어 계 곡산개구리로 명명되었다. 계곡산개구리는 주로 산간 계곡 하천의 돌 밑에서 월동하며, 번식기가 되면 웅덩이로 이동 하거나 계곡 물속에 잠겨 있는 바위나 계곡 가장자리에 다 수의 끈적끈적한 알덩어리를 붙여 산란한다. 국내 산개구리 류는 북방산개구리, 한국산개구리, 계곡산개구리 3종이 있 으나 계곡산개구리의 번식울음에 대한 연구는 미흡한 실정 이다. 따라서 본 연구는 계곡산개구리 번식울음의 일주기 및 시간주기 특성을 밝히는데 그 목적이 있다. 연구대상지는 치악산국립공원 구룡계곡 본류의 암반위 물울덩이로 하였다. 계곡산개구리 종 동정은 수컷 성체의 외형적 특징, 알의 특징, 올챙이 DNA 유전적 분석 3단계에 걸쳐 확인하였다. 성체의 외형 및 알 특징은 기존 문헌을 참고하여 현장에서 동정하였으며, 올챙이 DNA 유전적 분 석은 물웅덩이 내 올챙이를 채집하여 DNeasy Blood & Tissue Kit (Qiagen)를 이용하여 genomic DNA를 추출한 후, 미토콘드리아 cytochrome oxidate subunit I (COI) 유전 자 (641bp)를 증폭하여 nucleotide 데이터베이스(NCBI Genbank)에서 기존에 알려진 Rana속의 COI 염기서열과 함께 DNA 분자계통수 분석 (Neighbor joining method)을 시행하였다. 또한, 번식울음 녹음은 Idam PRO U11 Digital voice recorder를 이용하여 번식울음이 시작하기 전부터 종 료시까지 24시간 녹음하였다. 연구기간은 2015년 2월부터 3월까지이었다. 계곡산개구리 울음의 음향학적 특성은 울음의 우점주파 수를 분석하여 북방산개구리와 차이가 있는지 비교하였다. 계곡산개구리 울음의 일주기 및 시간주기 특성은 Adobe Audition CC (version 6.0)을 이용하여 1시간을 10분 단위로 구분한 후 울음 유무에 따라 점수화한 뒤 기상요인(기상청 국가기후자료센터, sts.kma.go.kr)과 상관관계분석을 실시하 였다. 이를 위한 통계분석은 IBM SPSS Statistics(Version 23) 프로그램을 이용하였다. 계곡산개구리 종 동정 결과, 생체는 발가락 끝이 둥글지 않으며, 물갈퀴가 매우 발달되어 있음을 확인할 수 있었다. 또한 턱 밑에 울음주머니가 없었고 앞다리 첫 번째 발가락 에 포접돌기가 있어 북방산개구리와는 구별되었다. 알은 바 위나 계곡 가장자리에 있는 돌 등에 서로 엉겨 붙어 있고, 작고 단단한 것을 특징으로 동정하였다. 올챙이 DNA 유전 적 분석 결과 각각의 북방산개구리, 계곡산개구리, 한국산 개구리의 명확한 계통 분기(clade)를 확인하였으며, 기존 데 이터베이스에서 동정된 종과 DNA 염기서열이 일치함을 확 인함으로써 분자유전학적인 방법을 통해 계곡산개구리 종 임을 판별하였다. 계곡산개구리와 북방산개구리의 우점주파수 비교분석결 과, 계곡산개구리 제1우점주파수는 최소 474Hz에서 최대 775Hz였고, 평균 637Hz이었다. 북방산개구리 제 1우점주 파수는 최소 609Hz에서 최대 844Hz였고, 평균 713Hz이었 다. 계곡산개구리 제 2우점주파수는 최소 1119Hz에서 최대 1543Hz였고, 평균 1273Hz이었다. 북방산개구리 제 2우점 주파수는 최소 1219Hz에서 1688Hz였고, 평균 1430Hz이 었다. 이러한 결과를 비교해 보면 제1우점주파수, 제2우점 주파수 모두 계곡산개구리가 북방산개구리보다 낮게 나타 났음을 확인할 수 있었다. 계곡산개구리 번식울음 기간은 3월 20일 오전 10시에 시 작해서 4월 5일 오전 7시에 종료되어 총 기간은 17일이었30 기경석 ․ 김지연 ․ 강지현 ․ 이혁제 ․ 이재윤 한국환경생태학회 학술대회논문집 25(2) 2015 다. 울음기간 중 3월 24일, 3월 26일은 울지 않았고, 3월 23일, 3월 25일은 거의 울지 않았다. 가장 활발히 운 기간은 3월 21일, 4월 1일, 4월 3일, 4월 4일이었다. 국내 산개구리 류의 기존 연구에 의하면 북방산개구리는 13시에서 14시, 18시에서 23시까지 두 번의 울음 피크를 이룬다고 하였으 나, 본 연구결과에 의하면 계곡산개구리 울음은 09시를 기 점으로 급격히 상승하였으며 13시부터 16시까지 피크를 이 루다가 22시까지 점진적으로 감소하였다. 또한 23시부터 09시까지는 급격히 감소하는 추세를 보였다. 일 울음 누적점수와 기상요인간 상관관계 분석결과, 평 균기온, 최고기온과 일최저기온이 번식울음과 고도 양의 상 관관계를 나타내었고, 일강수량, 평균습도 및 일조량과는 상관관계를 나타내지 않았다. 계곡산개구리의 시간대별 누 적울음과 기상 요인간 상관관계 분석결과, 기온과 습도와는 양의 상관관계, 일조량과는 음의 상관관계를 나타냈으며 강 수량과는 상관관계가 나타내지 않았다. 이상의 내용을 종합해 보면 계곡산개구리는 겨울이 끝나 는 이른 봄철에 가장 먼저 번식울음을 시작하는 양서류이면 서 17일 정도의 짧은 기간에 폭발적으로 번식하는 Explosive breeder의 유형에 속하였다. 단 짧은 번식기간임에도 불구 하고 이른 봄철이기 때문에 영하로 내려가는 시기에는 울음 을 멈추어 기온이 번식울음에 영향을 미치는 것으로 나타났 다.