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.