Quantitative Meta-barcoding for Rapid and Accurate Assessment of Species Diversity: Application to Community and eDNA samples

2020 Fiscal Year Annual Research Report

• Project/Area Number: 19H02276
• Research Institution: Ehime University
• Principal Investigator: 渡辺 幸三 愛媛大学, 沿岸環境科学研究センター, 教授 (80634435)
• Co-Investigator(Kenkyū-buntansha): 加藤 幹男 大阪府立大学, 高等教育推進機構, 教授 (30204499) ; 竹門 康弘 京都大学, 防災研究所, 准教授 (50222104)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Keywords: メタバーコーディング / 河川 / 底生動物 / 環境DNA

Outline of Annual Research Achievements

【項目１　DNAデータベースの超高速拡充】申請者らが保管する約200種の水生昆虫標本を使ってCOIマーカーおよびヒストンH3の配列を解読した。また，新たに採取した昆虫標本から未記載種（新種）が双翅目から多く見つかったため，種の記載とDNA情報のDNAデータベースへの登録も行った。
【項目２ 定量DNAメタバーコーディング法の開発】まず，サンガーシークエンシングでCOI配列を解読した4種20個体（ハプロタイプ）を段階的濃度（0.01, 0.05, 0.10 ng/μL）で混合した人工群集サンプルを準備した。メタバーコーディング解析の結果，低い濃度で混合したハプロタイプはPCRサイクル数を高めても検出されないことがわかった。また，４種とも検出されたことに加えて，濃度を高めに混合した14ハプロタイプも正確に検出できることがわかった。
【項目３ 砂州を活用した効率的な環境DNA回収技術の開発】天竜川の中流域に分布する３つの砂州を選び，各砂州の上流端，中央部，下流端で河床間隙水を採取した。間隙水から環境DNA（河床，表層水）を採取した。環境DNAのフィルタリング効果に対する河川流量の影響を見るために，調査流程の上流からのダム放流量が高い状態と低い状態で同様のサンプリングを行った。間隙水中に大型無脊椎動物がほとんど採取できなかったため，細菌を対象生物として，メタバーコーディング解析を行った。
【項目４　迅速・正確な種多様性の評価】事例研究として高知県の四万十川と仁淀川，奈良県の高見川の種多様性を評価した。河川水（環境DNA）と底生動物群集（群集DNA）を採取して，COI領域を対象にメタバーコーディング解析を行った。概ね両者から同じ分類群が検出されたが，環境DNAの方がより幅広い種が検出された。高見川では，系統学的種分類法（GMYC法）を適用して369GMYC種が検出された。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

研究項目１，２，３，４のすべてで予定していた研究計画を概ね実施することができた。

Strategy for Future Research Activity

最終年度では，本研究の総括となる項目４「迅速・正確な種多様性の評価」を完了し，メタバーコーディング解析による河川の生物多様性の有効性を示す成果を出したい。

Research Products

• [Journal Article] Detailed Description and Illustration of Larva, Pupa, and Imago of Holorusia mikado (Westwood, 1876) (Diptera: Tipulidae) from Japan (2021)
  • Author(s): Kolcsar, L-P, D. T. Nakamura, Kato, and K. Watanabe
  • Journal Title: Biodiversity Data Journal Volume: 9 Pages: e58009
  • DOI: 10.3897/BDJ.9.e58009
  • Peer Reviewed / Open Access
• [Journal Article] Simulation Modeling Reveals the Evolutionary Role of Landscape Shape and Species Dispersal on Genetic Variation within a Metapopulation (2020)
  • Author(s): Chiu, M-C, K. Nukazawa, T. M. Carvajal, V. H. Resh, B. Li, and K. Watanabe
  • Journal Title: Ecography Volume: 43 (12) Pages: 1891-1901
  • DOI: 10.1111/ecog.05118
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Branching Networks Can Have Opposing Influences on Genetic Variation in Riverine Metapopulations (2020)
  • Author(s): Chiu, M-C, B. Li, K. Nukazawa, V. H. Resh, T. M. Carvajal, and K. Watanabe
  • Journal Title: Diversity and Distributions Volume: 26 (12) Pages: 1813-1824
  • DOI: 10.1111/ddi.13160
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Revision of Japanese species of Nipponomyia Alexander, 1924 (Diptera, Pediciidae) (2020)
  • Author(s): Kolcsar, L. P., D. Kato, M. Gamboa, and K. Watanabe
  • Journal Title: ZooKeys Volume: 1000 Pages: 71-105
  • DOI: 10.3897/zookeys.1000.55021
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Spatial Genetic Structure of the Invasive Riparian Tree Robinia pseudoacacia to Determine Migration Patterns to Inform Best Practices for Riparian Restoration (2020)
  • Author(s): Yaegashi, S., T. Omura, and K. Watanabe
  • Journal Title: AoB PLANTS Volume: 12(5) Pages: plaa043
  • DOI: 10.1093/aobpla/plaa043
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Synopsis of the genus Ulomorpha Osten Sacken, 1869 (Diptera, Limoniidae) in Japan (2020)
  • Author(s): Kato, D., K. Watanabe, and L-P Kolcs?r
  • Journal Title: ZooKeys Volume: 999 Pages: 147-163
  • DOI: 10.3897/zookeys.999.52831
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Remarkable Anoxia Tolerance by Stoneflies from a Floodplain Aquifer (2020)
  • Author(s): Malison, R. L., B. K. Ellis, A. G. DelVecchia, H. N. Jacobson, B. K. Hand, G. Luikart, H. A. Woods, M. Gamboa, K. Watanabe, and J. A. Stanford
  • Journal Title: Ecology Volume: 101 (10) Pages: e03127
  • DOI: 10.1002/ecy.3127
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Detection of Adaptive Divergence in Populations of the Stream Mayfly Ephemera strigata with Machine Learning (2020)
  • Author(s): Li, B., S. Yaegashi, T. M. Carvajal, M. Gamboa and K. Watanabe
  • Journal Title: Ecology and Evolution Volume: 10 (13) Pages: 6677-6687
  • DOI: 10.1002/ece3.6398
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Quorum Sensing-linked agrA Expression by Ethno-synthesized Gold Nanoparticles in Tilapia Streptococcus agalactiae Biofilm Formation (2020)
  • Author(s): Fernando, S. I. D., K. G. J. Cruz and K. Watanabe
  • Journal Title: BioNanoScience Volume: 10 (3) Pages: 696-704
  • DOI: 10.1007/s12668-020-00758-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Haplotype-level DNA metabarcoding from freshwater macroinvertebrate community samples (2021)
  • Author(s): Joeselle M. Serrana and Kozo Watanabe
  • Organizer: 1st DNAQUA International Conference: Biotic Indices & Metrics - New trends in bioassessment of aquatic ecosystems: from organisms to DNA-based metrics
  • Int'l Joint Research
• [Presentation] Taxonomic Revision of Japanese Snow Crane Flies (Chionea, limoniidae): A First Step to Understand the Effect of Climate Change on Winter Active Insect (2021)
  • Author(s): Kolcsar L.P., Kato D. Watanabe K.
  • Organizer: 5th International Conference on Climate Change 2021
  • Int'l Joint Research
• [Presentation] Contrasting Adaptive Genetic Consequences of Stream Insects under Changing Climate (2021)
  • Author(s): Nukazawa K., Chiu M-C., Kazama S., Watanabe K.
  • Organizer: 5th International Conference on Climate Change 2021 (ICCC 2021)
  • Int'l Joint Research
• [Presentation] Elevational Patterns of Genetic Diversity in Riverine Populations in Japan (2021)
  • Author(s): Chiu M. C, Watanabe K
  • Organizer: 5th International Conference on Climate Change 2021 (ICCC 2021)
  • Int'l Joint Research
• [Presentation] Taxonomic Revision of Japanese Snow Crane Flies (Chionea, limoniidae): A First Step to Understand the Effect of Climate Change on Winter Active Insects (2021)
  • Author(s): Kolcsar L.-P, Kato D., Watanabe K.
  • Organizer: 5th International Conference on Climate Change 2021 (ICCC 2021)
  • Int'l Joint Research
• [Presentation] 高見川(奈良県)で採取した水生昆虫の次世代シーケンサーによる種判別 (2021)
  • Author(s): 稲井公二，脇村圭，渡辺幸三，加藤幹男
  • Organizer: 陸水学会近畿支部会第32回研究発表
• [Presentation] Molecular Genetic Approaches to the Assessment of Dam Impact on Stream Macroinvertebrates and Dengue Virus Surveillance of Vector Mosquitoes (2020)
  • Author(s): Kozo Watanabe
  • Organizer: 47th Philippine Society of Biochemistry and Molecular Biology (PSBMB) Annual Convention
  • Int'l Joint Research / Invited
• [Presentation] Sediment-associated microbial community profiling: sample pre-processing through sequential membrane filtration for 16s rDNA amplicon sequencing (2020)
  • Author(s): Joeselle M. Serrana and Kozo Watanabe
  • Organizer: Joint Meeting of the 3rd Annual Meeting of The eDNA Society and the 36th Annual Meeting of the Society of Population Ecology (Online)
  • Int'l Joint Research
• [Presentation] Recurrent seasonal dynamics of the planktonic eukaryotic communities in the polymictic eutrophic Lake M?ggelsee (Germany). (2020)
  • Author(s): Joeselle M. Serrana, Kingsly Chuo Beng, Jusytna Wolinska, Kozo Watanabe and Michael T. Monaghan
  • Organizer: Water and Environment Technology Conference 2020 Online (WET2020-Online)
  • Int'l Joint Research
• [Presentation] Microbial community diversity across hyporheic zoes of gravel bars: taxonomic and fucntio nal metrics (2020)
  • Author(s): Arnelyn Doloiras-Larano,Maribet Gamboa, Shinji Takahashi, Joeselle Serrana Yasuhiro Tekemon, Paul R. Johnston, Michael T. Monaghan, and Kozo Watanabe
  • Organizer: Water and Environment Technology Conference 2020 Online (WET2020-Online)
  • Int'l Joint Research