Affiliations:
Hinano Mizuno, Kouji Nakayama
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo, Kyoto 606-8502, Japan
Tetsuya Akita
National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan
Yasuyuki Hashiguchi
Department of Biology, Faculty of Medicine, Osaka Medical and Pharmaceutical University, 2-7 Daigaku-machi, Takatsuki, Osaka 569-0801, Japan
Tomonori Osugi
Water Resources Environment Center, Kojimachi, Chiyoda-ku, Tokyo 102-0083,Japan
Hirohiko Takeshima
Research Center for Marine Bioresources, Faculty of Marine Bioscience, Fukui Prefectural University, 49-8-2 Katsumi, Obama, Fukui 917-0003, Japan
Address corresponding to:
Kouji Nakayama
nakayama.kouji.8z@kyoto-u.ac.jp
Abstract
In the context of initiatives focused on captive breeding and reintroduction of endangered animal species, it is crucial to minimize any bias in reproductive success during the reintroduction phase in order to preserve genetic diversity. One population of Tachysurus ichikawai , a critically endangered bagrid catfish endemic to Japan, faces a threat from the construction of a dam. To address this, a captive breeding program followed by translocation is being implemented. Multiple breeding families are involved in this process; however, if there is a bias in reproductive success among them after release, it will result in a decline in genetic diversity. To identify potential biases in breeding lineages, we conducted kinship analysis between individuals born at the release site and breeding individuals. Because there were no samples available from the released individuals themselves, we examined distant kinship relationships, such as grandparent–grandchild and uncle–aunt–nephew–niece relationships, using a substantial number of single-nucleotide polymorphisms obtained from the whole genome re-sequence. Our findings indicate no bias between lineages in the first year after reintroduction, but a significant bias in the second year, emphasizing the need for continuous management and monitoring of reintroduced populations. This study demonstrates that monitoring kinship after reintroduction can correct lineage bias, which is critical for the prompt restoration of genetic diversity.
Keywords: endangered species, kinships estimation, reintroduction, reproductive success bias, whole-genome resequencing
INTRODUCTION
Captive breeding is one of the effective measures for conserving threatened populations. Conservation of endangered species must consider genetic diversity, not just population growth (Frankham et al., 2002). However, captive breeding of endangered species often involves only a few individuals, which can lead to inbreeding (Wajiki et al., 2015) and a decrease in genetic diversity (Philippart, 1995). Because low genetic diversity can negatively affect the future viability of a population (Charlesworth and Charlesworth, 1987; Keller and Waller, 2002), captive breeding programs are designed to conserve genetic diversity as much as possible (Fraser, 2008). Moreover, even with many individuals released or used as breeding parents within the reintroduction program, biased breeding after release can lead to a decline in genetic diversity. Nevertheless, there have been only a few investigations into reproductive bias after reintroduction (e.g., Jamieson, 2010), and as far as our knowledge goes, there are no documented instances in fishes. In stock enhancement programs of fishes, there have been instances of substantial reproductive bias in the seed production process (e.g., Sekino et al., 2003), and it is plausible that a comparable phenomenon is occurring after the reintroduction of endangered species.
To investigate whether any bias has occurred after reintroduction of endangered fish species, it is necessary to conduct a kinship analysis in the wild for 1–2 generations after release. The most basic scheme for captive breeding and subsequent reintroduction of fish involves capturing wild fish (F0), breeding them to produce offspring (F1), and releasing these offspring into the wild. The released F1 individuals breed in the field and produce the next generation (F2). To evaluate the reproductive contribution of each F0 individual, it is sufficient to observe the proportion of F2 individuals with a grandparent–grandchild (GG) relationship with the F0 individual. However, as released fish are typically small, it is not feasible to conduct genetic analyses of the released F1 individuals themselves, so it is difficult to examine the parent–offspring relationships of the F0–F1 or F1–F2 generations. It is therefore necessary to directly examine the GG relationships between F0 and F2 generations. Additionally, it would be valuable to investigate the uncle–aunt–nephew–niece (UANN) relationships among the siblings of the released F1individuals and the wild-bred F2 individuals , particularly in cases where genetic analysis of the siblings of F1 is feasible, for instance, when they are preserved for subsequent breeding purposes. Typically, more genetic markers are required to elucidate more distant relationships such as GG or UANN, compared to those required for examining parent–offspring relationships (Snedecor et al., 2022). However, this is difficult in populations with low genetic diversity because there are few genetic differences among individuals; general methods of genetic analysis often do not yield a sufficient number of polymorphisms to estimate kinship (Collevatti et al., 2007).
In recent years, kinships have begun to be analyzed using single-nucleotide polymorphisms (SNPs) obtained by next-generation sequencers. For example, in rainbow trout, a panel of SNPs at 95 loci has been used to estimate parent–offspring relationships and to examine life-history characteristics such as age of participation in reproduction (Abadía-Cardoso et al., 2013). In addition, SNPs at 6437 loci in red hammerhead sharks obtained by diversity array technology sequencing (DArTseq) (Melville et al., 2017), an SNP acquisition method similar to restriction site-associated DNA sequencing (RAD-seq), have been used to estimate full-sibling (FS) and half-sibling (HS) relationships and discuss reproductive patterns (Marie et al., 2019). Nevertheless, there are few previous studies estimating GG or UANN relationships to determine familial lineages in wild fish, except for that by Delomas and Campbell (2021).
Tachysurus ichikawai (Siluriformes: Bagridae), the subject species of this study, is endemic to Japan, exclusively inhabiting the rivers that flow into Ise Bay and Mikawa Bay (Nakamura, 1963; Niwa, 1967) . Human interference in river systems has resulted in the destruction of this species’ natural habitat, leading to a decline in population size and placing the species at risk of extinction (Watanabe, 1998). Specifically, the construction of dams and weirs has had a significant detrimental impact on their habitat, both directly and indirectly (Mie Prefecture, 2005). Consequently, this species has been classified as an endangered species with a high risk of near-future extinction in the wild (Ministry of the Environment, Japan, 2020).
Recently, a population of this species has encountered a threat due to dam construction. As one of the conservation measures, an attempt is underway to collect parental fish from the area slated to be submerged by dam construction, breed them into several groups in a facility, and release the young fish at another location to establish an alternative population (Fig. 1). The release of fish began in 2017, and since 2018 the successful reproduction of released individuals at the transfer sites has been confirmed (Shitara Dam Construction Office, 2023). If reproduction at the release site only involved individuals from a particular family, this would lead to a loss of genetic diversity and inbreeding depression. It is therefore important to estimate the reproductive contribution of released F1 individuals in the reintroduced population. However, it is almost impossible to identify family structure by actually observing reproduction under natural conditions, so it is necessary to use genetic information to identify the family to which each individual belongs. There have been only a few genetic studies of T. ichikawai ; one is a study of polymorphisms in the control region of mitochondrial DNA, and another uses microsatellite DNA. In the study of mitochondrial DNA, all 75 individuals from 8 rivers analyzed were found to have a single haplotype (Watanabe and Nishida, 2003). And by surveying microsatellite DNA, the population in the Toyokawa River system was reported to have lower genetic diversity than populations of the same species in other rivers (Mie Prefecture, 2006). This information suggests that the genetic diversity of the reintroduced population focused on in this study is expected to be low. Consequently, elucidating kinships within this population using reduced representation genome sequencing, such as RAD-seq, is deemed challenging due to the limited abundance of its polymorphic genetic markers.
In this study, we utilized whole-genome resequencing, the most informative method currently available in genetic research, to obtain numerous SNPs in the species with low genetic diversity. We attempted to estimate detailed kinship relationships within three generations in the reintroduced population to examine the reproductive contribution of each family based on more distant GG and UANN relationships. Our objective was to assess the potential occurrence of post-release reproductive bias in the captive breeding and reintroduction process of fishes.