Conclusion
With eDNA assay, this study revealed the fine-grained and deep phylogeographic structure of a primary freshwater fish, B. toni , over its large distribution area. Combining the eDNA assay with conventional approaches and a molecular clock analysis, novel phylogeographic events originating from the glacial period over one million years ago were uncovered. Conventional surveys in phylogeographic studies are prone to sampling biases due to the difficulty of directly collecting many individuals from different sites. The eDNA-based method provides an unbiased understanding of population genetic structures over a wide geographic range. When combined with a sufficient number of appropriate capture-based analyses, eDNA-based analyses can considerably reduce the effort and time required for acquiring high-resolution phylogeographic information. In fact, approximately 300 river samples were collected for this study, covering the entire Hokkaido region with high geographic density in 32 days. One of the advantages of this approach is that the same eDNA samples can be reused for detecting many other species in the same river systems, facilitating comparative phylogeographic studies. At present, extensive eDNA projects are being executed on national and global scales. For example, the All Nippon eDNA Monitoring Network includes more than 800 monitoring sites across the Japanese archipelago (https://db.anemone.bio/), whereas the eDNA expedition project of UNESCO World Heritage marine sites covers more than 15 countries (https://www.unesco.org/en/edna-expeditions). The integration of broad-scale eDNA biomonitoring with phylogeographic analyses has the potential to largely advance our understanding of global biodiversity. Our findings demonstrate the huge advantages of the eDNA technique as an innovative population genetic method that can rapidly and extensively detect biodiversity patterns.
References
1. J. C. Avise, J. Arnold, R. M. Ball, E. Bermingham, T. Lamb, J. E. Neigel, C. A. Reeb, N. C. Saunders, INTRASPECIFIC PHYLOGEOGRAPHY: The Mitochondrial DNA Bridge Between Population Genetics and Systematics.Annu. Rev. Ecol. Syst. 18 , 489–522 (1987).
2. R. J. Petit, I. Aguinagalde, J. L. De Beaulieu, C. Bittkau, S. Brewer, R. Cheddadi, R. Ennos, S. Fineschi, D. Grivet, M. Lascoux, A. Mohanty, G. Müller-Starck, B. Demesure-Musch, A. Palmé, J. P. Martín, S. Rendell, G. G. Vendramin, Glacial refugia: Hotspots but not melting pots of genetic diversity. Science 300 , 1563–1565 (2003).
3. E. K. F. Chan, A. Timmermann, B. F. Baldi, A. E. Moore, R. J. Lyons, S. S. Lee, A. M. F. Kalsbeek, D. C. Petersen, H. Rautenbach, H. E. A. Förtsch, M. S. R. Bornman, V. M. Hayes, Human origins in a southern African palaeo-wetland and first migrations. Nature 575 , 185–189 (2019).
4. G. Larson, K. Dobney, U. Albarella, M. Fang, E. Matisoo-Smith, J. Robins, S. Lowden, H. Finlayson, T. Brand, E. Willerslev, F. Rowley-Conwy, L. Andersson, A. Cooper, Worldwide phylogeography of wild boar reveals multiple centers of pig domestication. Science.307 , 1618–1621 (2005).
5. A. R. Templeton, Out of Africa again and again. Nature .416 , 45–51 (2002).
6. K. H. Kjaer, M. W. Pedersen, B. De Sanctis, B. De Cahsan, C. S. Michelsen, K. K. Sand, S. Jelavić, A. H. Ruter, A. M. Z Bonde, K. K. Kjeldsen, A. S. Tesakov, I. Snowball, J. C. Gosse, I. G. Alsos, Y. Wang, C. Dockter, M. Rasmussen, B. Skadhauge, A. Prohaska, J. Å Kristensen, M. Bjerager, E. Allentoft, E. Coissac, P. Consortium, A. Rouillard, A. Simakova, A. Fernandez-Guerra, C. Bowler, M. Macias-Fauria, J. J. Welch, A. J. Hidy, M. Sikora, M. J. Collins, R. Durbin, N. K. Larsen, E. Willerslev, A 2-Million-year-old ecosystem in Greenland uncovered by environmental DNA. Nature . 612 (2022).
7. D. M. Leigh, C. B. van Rees, K. L. Millette, M. F. Breed, C. Schmidt, L. D. Bertola, B. K. Hand, M. E. Hunter, E. L. Jensen, F. Kershaw, L. Liggins, G. Luikart, S. Manel, J. Mergeay, J. M. Miller, G. Segelbacher, S. Hoban, I. Paz-Vinas, Opportunities and challenges of macrogenetic studies. Nat. Rev. Genet. 22 , 791–807 (2021).
8. X. P. Liu, G. A. Duffy, W. S. Pearman, L. R. Pertierra, C. I. Fraser, Meta-analysis of Antarctic phylogeography reveals strong sampling bias and critical knowledge gaps. Ecography. e06312 (2022).
9. J. D. Phillips, R. A. Gwiazdowski, D. Ashlock, R. Hanner, An exploration of sufficient sampling effort to describe intraspecific DNA barcode haplotype diversity: examples from the ray-finned fishes (Chordata: Actinopterygii). DNA Barcodes . 3 , 66–73 (2016).
10. C. I. M. Adams, C. Hepburn, G.-J. Jeunen, H. Cross, H. R. Taylor, N. J. Gemmell, M. Bunce, M. Knapp, Environmental DNA reflects common haplotypic variation. Environ. DNA . (2022).
11. S. Tsuji, N. Shibata, R. Inui, R. Nakao, Y. Akamatsu, K. Watanabe, Environmental DNA phylogeography: Successful reconstruction of phylogeographic patterns of multiple fish species from cups of water.Mol. Ecol. Resour. (2023).
12. K. Weitemier, B. E. Penaluna, L. L. Hauck, L. J. Longway, T. Garcia, R. Cronn, Estimating the genetic diversity of Pacific salmon and trout using multigene eDNA metabarcoding. Mol. Ecol. 30 , 4970–4990 (2021).
13. A. Goto, T. Nakanishi, H. Utoh, K. Hamada, A preliminary study of the freshwater fish fauna of rivers in southern Hokkaido. Bull. Fish. Sci. Hokkaido Univ. 29 , 118–130 (1978).
14. Japan Association for Quaternary Research, Quaternary Maps of Japan (with explanatory text). Univ. Tokyo Press (1987).
15. M. Nakagawa, M. Amma-Miyasaka, D. Miura, S. Uesawa, Tephrostratigraphy in Ishikari Lowland, Southwestern Hokkaido: Eruption history of the Shikotsu-Toya volcanic field. Jour. Geol. Soc. Japan . 124 , 473–489 (2018).
16. E. Bermingham, J. C. Avise, Molecular zoogeography of freshwater fishes in the southeastern United States. Genetics . 113 , 939–965 (1986).
17. A. Perdices, I. Doadrio, P. S. Economidis, J. Bohlen, P. Bǎnǎrescu, Pleistocene effects on the European freshwater fish fauna: Double origin of the cobitid genus Sabanejewia in the Danube basin (Osteichthyes: Cobitidae). Mol. Phylogenet. Evol. 26 , 289–299 (2003).
18. R. G. Reeves, E. Bermingham, Colonization, population expansion, and lineage turnover: Phylogeography of Mesoamerican characiform fish.Biol. J. Linn. Soc. 88 , 235–255 (2006).
19. L. E. MacConaill, R. T. Burns, A. Nag, H. A. Coleman, M. K. Slevin, K. Giorda, M. Light, K. Lai, M. Jarosz, M. S. McNeill, M. D. Ducar, M. Meyerson, A. R. Thorner, Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing. BMC Genomics .19 , 30 (2018).
20. D. W. Fadrosh, P. G. Bing Ma, N. Sengamalay, S. Ott, R. M. Brotman, J. Ravel, An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome .2 , (2014).
21. M. Miya, Y. Sato, T. Fukunaga, T. Sado, J. Y. Poulsen, K. Sato, T. Minamoto, S. Yamamoto, H. Yamanaka, H. Araki, M. Kondoh, W. Iwasaki, MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: Detection of more than 230 subtropical marine species.R. Soc. Open Sci. 2 , 150088 (2015),
22. S. Tsuji, M. Miya, M. Ushio, H. Sato, T. Minamoto, H. Yamanaka, Evaluating intraspecific genetic diversity using environmental DNA and denoising approach: A case study using tank water. Environ. DNA .2 , 42–52 (2020).
23. R. Saka, Y. Takehana, N. Suguro, M. Sakaizumi, Genetic population structure of Lefua echigonia inferred from allozymic and mitochondrial cytochrome b variations. Ichthyol. Res. 50 , 301–309 (2003).
24. D. M. Irwin, T. D. Kocher, A. C. Wilson, Evolution of the cytochromeb gene of mammals. J. Mol. Evol. 32 , 128–144 (1991).
25. G. Orti, M. A. Bell, T. E. Reimchen, A. Meyer, Global survey of mitochondrial DNA sequences in the threespine stickleback: Evidence for recent migrations. Evolution. 48 , 608–622 (1994).
26. Y. Ono, The Northern Landbridge of Japan. Quat. Res.29 , 183–192 (1990).
27. L. Excoffier, Patterns of DNA sequence diversity and genetic structure after a range expansion: Lessons from the infinite-island model. Mol. Ecol. 13 , 853–864 (2004).
28. L. Excoffier, H. E. L. Lischer, Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol. Ecol. Resour. 10 , 564–567 (2010).
29. N. Azuma, J. I. Hangui, M. Wakahara, H. Michimae, Population structure of the salamander hynobius retardatus inferred from a partial sequence of the mitochondrial DNA control region. Zoolog. Sci.30 , 7–14 (2013).
30. T. Matsuhashi, R. Masuda, T. Mano, M. C. Yoshida, Microevolution of the mitochondrial DNA control region in the Japanese brown bear (Ursus arctos) population. Mol. Biol. Evol. 16 , 676–684 (1999).
31. T. Oishi, K. Uraguchi, K. Takahashi, R. Masuda, Population structures of the red fox (Vulpes vulpes ) on the hokkaido Island, Japan, revealed by microsatellite analysis. J. Hered.102 , 38–46 (2011).
32. K. Kawai, F. Hailer, A. P. De Guia, H. Ichikawa, T. Saitoh, Refugia in glacial ages led to the current discontinuous distribution patterns of the dark red-backed vole Myodes rex on Hokkaido, Japan.Zoolog. Sci. 30 , 642–650 (2013).
33. A. Ooyagi, D. F. Mokodongan, J. Montenegro, I. F. Mandagi, N. Koizumi, Y. Machida, N. Inomata, S. V. Shedko, A. A. Hutama, R. K. Hadiaty, K. Yamahira, Phylogeography of the eight-barbel loachLefua nikkonis (Cypriniformes: Nemacheilidae): how important were straits in northern Japan as biogeographical barriers? Ichthyol. Res. 65 , 115–126 (2018).
34. H. Sakai, T. Ueda, R. Yokoyama, Genetic structure and phylogeography of northern Far Eastern pond minnows, Rhynchocypris perenurus sachalinensis and R. p. mantschuricus (Pisces, Cyprinidae), inferred from mitochondrial DNA sequences. Biogeography .16 , 87–109 (2014).
35. I. Koizumi, N. Usio, T. Kawai, N. Azuma, R. Masuda, Loss of genetic diversity means loss of geological information: The endangered Japanese crayfish exhibits remarkable historical footprints. PLoS One .7, e33986 (2012)
36. T. Yatsuyanagi, H. Araki, Understanding seasonal migration of Shishamo smelt in coastal regions using environmental DNA. PLoS One . 15 , e0239912 (2020).
37. T. Minamoto, M. Miya, T. Sado, S. Seino, H. Doi, M. Kondoh, K. Nakamura, T. Takahara, S. Yamamoto, H. Yamanaka, H. Araki, W. Iwasaki, A. Kasai, R. Masuda, K. Uchii, An illustrated manual for environmental DNA research: Water sampling guidelines and experimental protocols.Environ. DNA . 3 , 8–13 (2021).
38. T. A. Hall, BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41 , 95–98 (1999).
39. J. D. Thompson, D. G. Higgins, T. J. Gibson, CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22 , 4673–4680 (1994).
40. S. Tsuji, N. Shibata, H. Sawada, M. Ushio, Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis. Mol. Ecol. Resour. 20 , 1323–1332 (2020).
41. M. Martin, Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet . 17 , 10–12 (2011).
42. R. C. Edgar, Search and clustering orders of magnitude faster than BLAST. Bioinformatics . 26 , 2460–2461 (2010).
43. X. Turon, A. Antich, C. Palacín, K. Præbel, O. S. Wangensteen, From metabarcoding to metaphylogeography: separating the wheat from the chaff. Ecol. Appl. 30 , 1–19 (2020).
44. J. P. Huelsenbeck, F. Ronquist, MRBAYES: Bayesian inference of phylogenetic trees. Bioinforma. Appl. Note . 17 , 754–755 (2001).
45. S. Kumar, G. Stecher, M. Li, C. Knyaz, K. Tamura, MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35 , 1547–1549 (2018).
46. J. W. Leigh, D. Bryant, POPART: full-feature software for haplotype network construction. Methods Ecol. Evol. 6 , 1110–1116 (2015).
47. S. Harada, S. Jeon, I. Kinoshita, M. Tanaka, M. Nishida, Phylogenetic relationships of four species of floating gobies (Gymnogobius ) as inferred from partial mitochondrial cytochromeb gene sequences. Ichthyol. Res. 49 , 324–332 (2002).