Abstract
Freshwater biodiversity has been severely threatened in recent years, and to conserve endangered species, their distribution and breeding habitats need to be clarified. However, identifying breeding sites in a large area is generally difficult. Here, by combining the emerging environmental DNA (eDNA) analysis with subsequent traditional collection surveys, we successfully identified a breeding habitat for the critically endangered freshwater fish Acheilognathus typus in the mainstream of Omono River in Akita Prefecture, Japan, which is one of the original habitats of this species. Based on DNA cytochrome B sequences of A. typus and closely related species, we developed species-specific primers and a probe that were used in real-time PCR for detecting A. typus eDNA. After verifying the specificity and applicability of the primers and probe on water samples from known artificial habitats, eDNA analysis was applied to water samples collected at 99 sites along Omono River. Two of the samples were positive for A. typus eDNA, and thus, small fixed nets and bottle traps were set out to capture adult fish and verify egg deposition in bivalves (the preferred breeding substrate for A. typus) in the corresponding regions. Mature female and male individuals and bivalves containing laid eggs were collected at one of the eDNA-positive sites. This was the first record of adult A. typus in Omono River in 11 years. This study highlights the value of eDNA analysis to guide conventional monitoring surveys and shows that combining both methods can provide important information on breeding sites that is essential for species’ conservation.
Similar content being viewed by others
References
Adrian-Kalchhauser I, Burkhardt-Holm P (2016) An eDNA assay to monitor a globally invasive fish species from flowing. PLoS One 11:e0147558. https://doi.org/10.1371/journal.pone.0147558
Alberdi A, Aizpurua O, Gilbert MTP, Bohmann K (2017) Scrutinizing key steps for reliable metabarcoding of environmental samples. Methods Ecol Evol. https://doi.org/10.1111/2041-210X.12849
Allan JD, Flecker AS (1993) Biodiversity conservation in running waters. Biol Sci 43:32–43. https://doi.org/10.2307/1312104
Burnes MA, Turner CR (2016) The ecology of environmental DNA and implications for conservation genetics. Conserv Genet 17:1–17. https://doi.org/10.1007/s10592-015-0775-4
Butchart SHM, Walpole M, Collen B et al (2010) Global biodiversity: indicators of recent declines. Sci 328:1164–1168. https://doi.org/10.1126/science.1187512
Buxton AS, Groombridge JJ, Zakaria NB, Griffiths RA (2017) Seasonal variation in environmental DNA in relation to population size and environmental factors. Sci Rep 7:46294. https://doi.org/10.1038/srep46294
Bylemans J, Furlan EM, Hardy CM, McGuffie P, Lintermans M, Gleeson DM (2017) An environmental DNA (eDNA) based method for monitoring spawning activity: a case study, using the endangered Macquarie perch (Macquaria australasica). Methods Ecol Evol 8:646–655. https://doi.org/10.1111/2041-210X.12709
Davy CM, Kidd AG, Wilson CC (2015) Development and validation of environmental DNA (eDNA) markers for detection of freshwater turtles. PLoS One 7:e0130965. https://doi.org/10.1371/journal. pone.0130965
Deiner K, Altermatt F (2014) Transport distance of invertebrate environmental DNA in a natural river. PLoS One 9:e88786. https://doi.org/10.1371/ journal.pone.0088786
Dejean T, Valentini A, Miquel C, Taberlet P, Bellemain E, Miaud C (2012) Improved detection of an alien invasive species through environmental DNA barcoding: the example of the American bullfrog Lithobates catesbeianus. J Appl Ecol 49:953–959. https://doi.org/10.1111/j.1365-2664.2012.02171.x
Dudgeon D, Arthington AH, Gessner MO, Kawabata Z, Knowler DJ, Lévêque C, Naiman RJ, Prieur-Richard AH, Soto D, Stiassny MLJ, Sullivan CA (2006) Freshwater biodiversity: importance, threats, status and conservation challenges. Biol Rev 81:163–182. https://doi.org/10.1017/S1464793105006950
Erickson RA, Rees CB, Coulter AA, Merkes CM, McCalla SG, Touzinsky KF, Walleser L, Goforth RR, Amberg JJ (2016) Detecting the movement and spawning activity of bigheaded carps with environmental DNA. Mol Ecol Resour 16:957–965. https://doi.org/10.1111/1755-0998.12533
Erickson RA, Merkes CM, Jackson CA, Goforth RR, Amberg JJ (2017) Seasonal trends in eDNA detection and occupancy of bigheaded carps. J Gt Lakes Res 43:762–770. https://doi.org/10.1016/j.jglr.2017.06.003
Eva B, Harmony P, Thomas G, Francois G, Alice V, Claude M, Tony D (2016) Trails of river monsters: detecting critically endangered Mekong giant catfish Pangasianodon gigas using environmental DNA. Glob Ecol Conserv 7:148–156. https://doi.org/10.1016/j.gecco.2016.06.007
Evans NT, Shirey PD, Wieringa JG, Mahon AR, Lamberti GA (2017) Comparative cost and effort of fish distribution Detection via environmental DNA analysis and electrofishing. Fisheries 47:90–99. https://doi.org/10.1080/03632415.2017.1276329
Ficetola GF, Miaud C, Pompanon F, Taberlet P (2008) Species detection using environmental DNA from water samples. Biol Lett 4:423–425. https://doi.org/10.1098/rsbl.2008.0118
Ficetola GF, Pansu J, Bonin A, Coissac E, Giguet-Covex C, Barba MD, Gielly L, Lopes CM, Boyer F, Raye FPG, Taberlet P (2015) Replication levels, false presences and the estimation of the presence/absence from eDNA metabarcoding data. Mol Ecol Resour 15:543–556. https://doi.org/10.1111/1755-0998.12338
Ficetola GF, Taberlet P, Coissac E (2016) How to limit false positives in environmental DNA and metabarcoding? Mol Ecol Resour 16:604–607. https://doi.org/10.1111/1755-0998.12508
Fukumoto S, Ushimaru A, Minamoto T (2015) A basin-scale application of environmental DNA assessment for rare endemic species and closely related exotic species in rivers: a case study of giant salamanders in Japan. J Appl Ecol 52:358–365. https://doi.org/10.1111/1365-2664.12392
Furlan EM, Gleeson D, Hardy CM, Duncan RP (2016) A framework for estimating the sensitivity of eDNA surveys. Mol Ecol Resour 16:641–654. https://doi.org/10.1111/1755-0998.12483
Guillera-Arroita G, Lahoz-Monfort JJ, van Rooyen AR, Weeks AR, Tingley R (2017) Dealing with false-positive and false-negative errors about species occurrence at multiple levels. Methods Ecol Evol 8:1081–1091. https://doi.org/10.1111/2041-210X.12743
Jane SF, Wilcox TM, Mckelvey KS, Young MK, Schwartz MK, Lowe WH, Letcher BH, Whiteley AR (2015) Distance, flow and PCR inhibition: eDNA dynamics in two headwater streams. Mol Ecol Resour 15:216–227. https://doi.org/10.1111/1755-0998.12285
Japanese Ministry of Environment (2017) Japanese Ministry of Environment Red List 2017. http://www.env.go.jp/index.html Accessed 1 Sept 2017 [In Japanese]
Japanese Ministry of the Environment (2015) Red data book 2014—threatened wildlife of Japan—vol. 4 Pisces—brackishwater & freshwater fishes. Gyosei, Tokyo, Japan. xxx+414. [In Japanese]
Jerde CL, Mahon AR, Chadderton WL, Lodge DM (2011) “Sight-unseen” detection of rare aquatic species using environmental DNA. Conserv Lett 4:150–157. https://doi.org/10.1111/j.1755-263X.2010.00158.x
Kano Y (2014) Everyone wants to give a gene to the next generation: reproductive strategy of rosy bitterlings. In: Nagata Y (ed) Introduction of research on freshwater fish. Tokai University Press, Kanagawa, pp 12–34 [In Japanese]
Kawagishi M, Fujimoto Y, Sindo K (2007) Rediscovery of Acheilognathus typus in Lake Izunuma-Uchinuma basin. Izunuma-Uchinuma Wetl Res 1:7–10 [In Japanese]
Kitajima J (2005) Present status of Acheilognathus typus in Tohoku district and symposium of Acheilognathus typus. Kasumigaura Study 15:21–24 [In Japanese]
Lefort MC, Boyer S, Barun A, Emami-Khoyi A, Ridden J, Smith VR, Sprague R, Waterhouse BR, Cruickshank RH (2015) Blood, sweat and tears: non-invasive vs. non-disruptive DNA sampling for experimental biology. PeerJ Prepr 3:e655v3. https://doi.org/10.7287/peerj.preprints.655v3
Minamoto T, Yamanaka H, Takahara T (2012) Surveillance of fish species composition using environmental DNA. Limnol 13:193–197. https://doi.org/10.1007/s10201-011-0362-4
Nakamura M (1969) Cyprinid fishes of Japan: studies on the life history of cyprinid fishes of Japan. Research Institute for Natural Resources, Tokyo
Otokita M, Kikuchi N, Suzuki C, Takahashi K, Saito C (2011) Summary of field survey on Acheilognathus melanogaster and educational activity for conservation on site. Res Bull Environ Educ Cent Miyagi Univ Educ 13:23–29 [In Japanese]
Pfleger MO, Rider SJ, Johnston CE, Janosik AM (2016) Saving the doomed: using eDNA to aid in detection of rare sturgeon for conservation (Acipenseridae). Glob Ecol Conserv 8:99–107. https://doi.org/10.1016/j.gecco.2016.08.008
Saitoh K, Shindo K, Fujimoto Y, Takahashi K, Shimada T (2016) Mitochondrial genotyping of an endangered bitterling Acheilognathus typus (Cyprinidae). ZooKeys 623:131-141
Schmidt BR, Kery M, Ursenbacher S, Hyman OJ, Collins JP (2013) Site occupancy models in the analysis of environmental DNA presence/absence surveys: a case study of an emerging amphibian pathogen. Methods Ecol Evol 4:646–653. https://doi.org/10.1111/2041-210X.12052
Schultz MT, Lance RF (2015) Modeling the sensitivity of field surveys for detection of environmental DNA (eDNA). PLoS One 10:e0141503. https://doi.org/10.1371/journal.pone.0141503
Shimizu A (2010) Environmental regulations of reproductive cycles in teleosts. Bull. Jpn Soc Fish Oceanogr 74:58–65 (In Japanese)
Shorgen AJ, Tank JL, Andruszkiewicz E, Olds B, Mahon AR, Jerde CL, Bolster D (2017) Controls on eDNA movement in streams: transport, retention, and resuspension. Sci Rep 7:5065. https://doi.org/10.1038/s41598-017-05223-1
Sigsgaard EE, Carl H, Møller PR, Thomsen PF (2015) Monitoring the near-extinct European weather loach in Denmark based on environmental DNA from water samples. Biol Conserv 183:46–52. https://doi.org/10.1016/j.biocon.2014.11.023
Simmons M, Tucker A, Chadderton WL, Jerde CL, Mahon AR (2015) Active and passive environmental DNA surveillance of aquatic invasive species. Can J Fish Aquat Sci 73:76–83. https://doi.org/10.1139/cjfas-2015-0262
Smart AS, Weeks AR, van Rooyen AR, Moore A, McCarthy MA, Tingley R (2016) Assessing the cost-efficiency of environmental DNA sampling. Methods Ecol Evol 7:1291–1298. https://doi.org/10.1111/2041-210X.12598
Spear SF, Groves JD, Williams LA, Waits LP (2014) Using environmental DNA methods to improve detectability in a hellbender (Cryptobranchus alleganiensis) monitoring program. Biol Conserv 183:38–45. https://doi.org/10.1016/j.biocon.2014.11.016
Stoeckle MY, Soboleva L, Charlop-Powers Z (2017) Aquatic environmental DNA detects seasonal fish abundance and habitat preference in an urban estuary. PLoS One 12:e0175186. https://doi.org/10.1371/journal.pone.0175186
Sugiyama H (2015) Netted bittering. Red data book 2014—threatened wildlife of Japan—Vol. 4 Pisces—brackishwater & freshwater fishes. Gyosei, Tokyo, Japan. In Ministry of the Environment, Japan, pp. 26–27. xxx+414. [In Japanese]
Suguro N (1995) Inhabiting Acheilognathus typus in Yokohama City. Kanagawa Prefecture Freshwater Fish Propagation Test Site Report 31:60–64 [In Japanese]
Takahara T, Minamoto T, Yamanaka H, Doi H, Kawabata Z (2012) Estimation of fish biomass using environmental DNA. PLoS One 7:e35868. https://doi.org/10.1371/journal.pone.0035868
Takahashi K (2002) Effect of large-mouth bass on the fish population. In: The Ichthyological Society of Japan (ed) Invader to lakes and rivers: large-mouth bass. Kouseisha-kouseikaku, Tokyo, pp 47–59
Thomsen PF, Kielgast J, Iversen LL, Wiuf C, Rasmussen M, Gilbert MTP, Orlando L, Willerslev E (2012a) Monitoring endangered freshwater biodiversity using environmental DNA. Mol Ecol 21:2565–2573. https://doi.org/10.1111/j.1365-294X.2011.05418. x
Thomsen PF, Kielgast J, Iversen LL, Møller PR, Rasmussen M, Willerslev E (2012b) Detection of a diverse marine fish fauna using environmental DNA from seawater samples. PLoS One 7:e41732. https://doi.org/10.1371/journal. pone.0041732
Trèguier A, Paillisson JM, Dejean T, Valentini A, Schlaepfer MA, Roussel JM (2014) Environmental DNA surveillance for invertebrate species: advantages and technical limitations to detect invasive crayfish Procambarus clarkii in freshwater ponds. J Appl Ecol 51:871–879. https://doi.org/10.1111/1365- 2664.12262
Uchii K, Doi H, Minamoto T (2016) A novel environmental DNA approach to quantify the cryptic invasion of non-native genotypes. Mol Ecol 16:415–422. https://doi.org/10.1111/1755-0998.12460
Vitousek PM, Mooney HA, Lubchenco J, Melillo JM (1997) Human domination of earth’s ecosystems. Sci 277:494–499. https://doi.org/10.1126/science.277.5325.494
WWF (2016) Living Planet Report 2016. WWF Global. https://www.wnf.nl/custom/LPR_2016_fullreport/. Accessed 26 Apr 2017
Yamanaka H, Minamoto T, Matsuura J, Sakurai S, Tsuji S, Motozawa H, Hongo M, Sogo Y, Kakimi N, Teramura I, Sugita M, Baba M, Kondo A (2017) A simple method for preserving environmental DNA in water samples at ambient temperature by addition of cationic surfactant. Limnol 18:233–241. https://doi.org/10.1007/s10201-016-0508-5
Funding
This study was partly supported by JSPS KAKENHI Grant Number 17H03735 and by a donation from a private company to which one of the authors (NM) belongs. The funders had no role in designing and conducting this research.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by: Sven Thatje
Rights and permissions
About this article
Cite this article
Sakata, M.K., Maki, N., Sugiyama, H. et al. Identifying a breeding habitat of a critically endangered fish, Acheilognathus typus, in a natural river in Japan. Sci Nat 104, 100 (2017). https://doi.org/10.1007/s00114-017-1521-1
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00114-017-1521-1