Study sites and sampling
All animal collections and experiments were approved by the Committee on
the Ethics of Animal Experiments of School of Life Sciences of Lanzhou
University. Adult male tree sparrows were caught by mist nets in our
study sites, Baiyin (BY) and Liujiaxia (LJX), during their breeding
season (April-July) from 2020 to 2022 (Table S1). The polluted site BY
has a long history of mining non-ferrous metals since 1956, in contrast
with LJX, a tourism city and important water resource protection area
that is relatively unpolluted and chosen as control site. The tree
sparrow reaches sexual maturity within a year after hatching and breeds
every single year in our study sites. The sexes of individuals were
determined based on the presence of an incubation patch (Bailey, 1952).
Afterward, females were ringed with a uniquely numbered metal band and
released, while males were kept from light and transported alive to the
laboratory for further experiments. In the laboratory, males were
euthanized and dissected immediately. Their tissues were collected and
frozen in liquid nitrogen, and then stored at -80℃ until subjected to
DNA or RNA extraction.