MATERIALS AND METHODS
Target population and
samples
Tachysurus ichikawai is nocturnal species, resides in pools and
flats with slow currents. It does not exhibit significant migratory
patterns over its lifespan, and it is not uncommon for individuals to
reside in a single pool for multiple years (Watanabe, 1995). The average
lifespan for this species ranges from 3 to 6 y, with sexual maturity
typically attained at approximately 2 to 3 y (Watanabe, 1994a, 2008).
The sex ratio within the breeding population is skewed towards females
(Watanabe, 1994a), likely due to the higher mortality rate of adult
males. Breeding is concentrated during June and July, during which males
establish breeding territories and females visit them, thus
demonstrating continuous polygamy (Watanabe, 1994a, 1994b, 2008).
The area 70 km upstream from the mouth of the Toyokawa River, which
flows into Mikawa Bay, is an important habitat for a population of this
species. In this location, Shitara Dam is under construction (impounded
water surface area: approximately 3 km²), and when the dam is completed,
the habitat of the population will disappear. Therefore, surveys and
consideration of specific conservation measures have been ongoing for
about 20 years since 2003. The Shitara Dam Construction Office has
conducted an environmental impact assessment (EIA) of the dam project
based on the EIA Law; the assessment report was published in 2007. This
report recommends conservation measures for T. ichikawai because
of the impact of the project, and translocation experiments are being
conducted (Fig. 1).
Samples were collected from three successive generations in a single
breeding population (Fig. 1): the F0 generation,
collected near the dam construction site and used for breeding at the
facility; the F1 generation, produced by breeding
between F0 males and females at the facility; and the
F2 generation, produced by breeding between
F1 individuals under natural conditions at the
translocated site. F0 rearing and breeding at the
facility were conducted in multiple tanks containing multiple
F0 males and females or one F0 male and
one F0 female each, with each tank used for a single
breeding season. For this study, all F0 individuals used
for breeding in each tank will be referred to as a “family”. Some of
the F1 individuals born in each family were released to
the transfer sites in the spring and fall, resulting in
F1 individuals from 12 families having been released by
spring 2019 (families A1, A2, A3, A4, A5, A6A7A9, A10, A11, A12, A15,
A16 and A17). Table S1 contains a summary of information about the
F0 individuals in each family.
The reproductive contribution of individual families was estimated by
determining the number of F2 individuals derived from
each family. The simplest way to achieve this is to check the
parent–offspring relationships between F1 and
F2 generations. However, as previously stated, the
F1 individuals that were released were not sampled for
genetic analysis. Therefore, it was imperative to investigate GG
relationships between F0 and F2generations, as well as UANN relationships between siblings of the
released F1 individuals and wild-bred F2individuals (Fig. 1).
The selection criteria for individuals used in determining relationships
were as follows. First, if samples from all F0individuals from a particular family were available, all
F0 individuals from that family were included for
analysis. This applied to families A2, A3, A6A7A9, and A17. Second, if a
sample from at least one F0 individual in a family was
not obtainable, the F1 individuals (the siblings of the
released F1 individuals) from that family were analyzed
instead. This approach was applied to families A1, A4, A5, A12, A15, and
A16. For families where a single male and female pair were used for
breeding, a single F1 individual was selected, given
that all F1 individuals within the family were FS. For
families where breeding involved multiple F0individuals, two F1 individuals were analyzed. As for
the F2 generation, individuals born in the NN6 pool, one
of the release sites and the pool that showed the highest reproductive
success after release, were selected for analysis. Samples from the
F2 generation consisted of two year-groups: the 2018
year-group (F2 individuals born during the breeding
season around June–July 2018, sampled in June 2019 at approximately 1
year old) and the 2019 year-group (F2 individuals born
during the breeding season around June–July 2019, sampled in June
2020). A total of 70 individuals were chosen for analysis based on these
criteria: 15 F0, 8 F1, and 47
F2 individuals (35 from the 2018 year-group and 12 from
the 2019 year-group).