Discussion
Based on the phenotypic variation of sperm in tree sparrow lived in BY which has been detected in our previous researches, we uncover a dramatically expanded PIM gene family may play an important role in shaping the phenotypic change of sperm under environmental pollution. As the only gene overlapped between the PSGs and DEGs, PIM1 is identified as candidate gene for sperm evolution and targeted as the focus of our research. Although there is no documented example ofPIM1 enables rapid evolution of sperm, the genetic variation and differentially expression of cAMP-dependent protein kinase (PKA ), another serine/threonine protein kinase which have high sequence homology with PIM1 (Jacobs et al., 2005), is associated with rapid evolution of sperm in deer mouse (Peromyscus maniculatus ) has been reported (Fisher et al., 2016).
As soon as our focus are narrowed down, the unusual and large-scale expansion of PIM gene family mainly caused by the duplication ofPIM1 are noticed in the genome of tree sparrow. 449 PIM1genes were predicted from the tree sparrow genome assembly and 142 of them are complete for the kinase domain. Gene duplications provide a source of genetic material for mutation, drift and selection to act upon, making new evolutionary opportunities for rapid adaptation (Crow & Wagner 2006; Hu et al., 2022; Magadum et al., 2013). Duplicate genes have been proven to be important targets of positive selection in Arabidopsis (Moore & Purugganan, 2003). As a result, the large number of duplicate PIM1 gene copies may provide raw materials that facilitate rapid evolution of sperm when selective pressures shift in BY, which is demonstrated from the side by the fact that multiplePIM1 duplicates appear repeatedly in outlier regions.
Our analysis also finds that both tandem duplication and segmental duplication events are involved in the expansion of PIM gene family, and the segmental duplications cause simultaneous expansion of 6 other gene families and LTR retrotransposons adjacent to PIM on chromosomes. The adjacent chromosomal distribution and simultaneously expansion suggest that the 7 families co-evolved in genome, and it is noteworthy that the expansion of these 7 gene family is so dramatical that make it may become a strong driving force of the evolution of tree sparrow genome.
Among the 7 expanded gene families in tree sparrow, the duplications of the most two prolifically expanded gene familiesC2H2ZNF and OR are widespread and prevalent in other species (Freitag et al., 1998; Seetharam & Stuart 2013) relative to the other 5 gene families. The duplication of PIM1 has only been reported in zebra finch (Kong et al., 2010) whereas PIM1 is believed to be single-copy and conserved in other amniotic genomes, even in chicken. Our analysis ofPIM1 duplication events in other avian genome indicate thatPIM1 expansion is not peculiar to passerine but arose in several lineages during the evolution of bird. Fixation of duplicate genes occurs when natural selection favors function or expression of duplicates (Cardoso-Moreira et al., 2016; Chain et al., 2011). Although the protein activity of duplicate PIM1 has not confirmed yet, compared with the species with single-copy PIM1 , the retention of duplicates in genome and the increased gene dosage may suggest a more important and beneficial function of PIM1 in the evolution and adaptation of the species with PIM1 expansion. At the same time, there still much less PIM1 duplicates are found in any other avian genome than in tree sparrow which may due to the further expansion of PIM1 in tree sparrow than other species or the incomplete genome assemblies.
The expression pattern of PIM1 seems to be different in different species. PIM1 has been reported to be expressed mainly in testis and hematopoietic tissues including thymus, spleen, bone marrow and fetal liver in both mouse and man (Bachmann & Möröy, 2005; Eichmann et al., 2000) when PIM1 expressed in the brain and testis of zebra finch (Kong et al., 2010). Based on the current evidence, it seems that testis is only tissue in which PIM1 of mammal and passerine both expressed, which indicates that PIM1 may play an important role in male reproduction. But besides a few early researches implyPIM1 is implicated in the normal development of male germ cells (Sorrentino et al., 1988; Wingett et al., 1992), the functions related to male reproduction of PIM1 are fleetingly addressed in studies and people pay more attention to its role in cancer (Narlik-Grassow et al., 2014) and brain function of songbird (Kong et al., 2010). A large number of duplicates and the testis-specific expression pattern, as revealed by our analysis, as well as the involvement of mammalPIM1 in normal germ cell maturation published before (Sorrentino et al., 1988; Wingett et al., 1992) may indicate PIM1 play a strong and essential role in spermatogenesis of tree sparrow. We thus consider the role of PIM1 in male reproduction, an indispensable and inseparable part of PIM1 function, should never be neglected not only in the species with multiple PIM1 but in species with conserved single-copy.
Higher expression levels of PIM1 , wholly or individually, are detected in testis sampled from BY tree sparrow. Interestingly, the expression level of PIM3 , a member of PIM family as well as PIM1 but without duplication and testis-specific expression pattern, shows no difference between BY and LJX. The elevated expression level may be the consequence of shifts in selective pressures on duplicate PIM1 . Furthermore, we test if the elevated expression level of PIM1 in polluted site is due to larger scale of duplication and higher copy numbers of PIM1 . However, the results show that there is no increase in copy number of PIM1 in BY population. Therefore, the molecular mechanism of change in expression level of PIM1 still need to be further researched in the future.
When we take into account the link between transcriptional changes to sperm phenotypic effects, the higher expression levels of PIM1seem to be consistent with our earlier findings of longer and faster swimming sperm in BY (Yang et al., 2020a, 2020b). No research is found to explore the relationship between expression level of PIM1 and sperm traits variation, nonetheless, several studies have proven thatPIM1 can accelerate cancer cell motility and overexpression ofPIM1 result in increased cancer cell motility (Mou et al., 2016; Santio et al., 2020; Tanaka et al., 2009). PIM1 kinase normally accelerates cell motility through phosphorylating and activating target proteins. We thus speculate that PIM1 also accelerate sperm motility through phosphorylating downstream proteins in tree sparrow. However, there are so many proteins that can be phosphorylated by PIM1. In prostate cancer cells, PIM1 can accelerate cell motility through phosphorylating actin capping proteins (Sanito et al., 2020). PIM1 accelerate human oral squamous cell carcinoma cell motility through activating Ras-related C3 botulinum toxin substrate 1 (RAC1) instead (Tanaka et al., 2009). Therefore, it is hard to confirm the target protein and signaling pathway of PIM1 in tree sparrow. We attempt to use PIM1 inhibitor to verify the molecular function and preliminary explore the downstream protein of PIM1 kinase. However, there is no observed sperm trait change are found in the inhibitor treatment group. The unchanged sperm trait may due to the limitations of our inhibition experiment. We cannot ensure whether the inhibitor can pass through the blood-testis barrier and reach the target tissue, or whether the mammal inhibitor can work equally on tree sparrow.
In conclusion, our work utilizes population genomic and transcriptomic studies, exploring the genetic underpinning of phenotypic variation of sperm in tree sparrow lived in BY which is polluted by heavy metal. On the one hand, our results find PIM1 , a single-copy gene in most of amniotic genomes, remarkably expanded in tree sparrow and other avian species of three lineages including Passeriformes, Psittaciformes and Strigiformes. Duplication of PIM1 may be an important force driving speciation and phenotypic diversity in avian evolution. On the other hand, while the molecular functions and pathways of the expandedPIM1 remain to be defined by experimental verification, the signatures of selection and upregulated gene expression in polluted sites, indicating the expanded PIM1 may be important sources for rapid evolution of sperm in tree sparrow following the environmental change.