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Feb.  2022
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Evolutionary dynamics and conserved function of the Tudor domain-containing (TDRD) proteins in teleost fish

  • Corresponding author: Jie Cheng, jiecheng@ouc.edu.cn
  • Received Date: 2021-01-07
    Accepted Date: 2021-07-15
    Published online: 2021-10-08
  • Edited by Jiamei Li.
  • Tudor domain-containing (TDRD) proteins, the germline enriched protein family, play essential roles in the process of gametogenesis and genome stability through their interaction with the PIWI-interacting RNA (piRNA) pathway. Several studies have suggested the rapid evolution of the piRNA pathway in teleost lineages with striking reproductive diversity. However, there is still limited information about the function and evolution of Tdrd genes in teleost species. In this study, through genome wide screening, 13 Tdrd family genes were identified in economically important aquaculture fish, including spotted sea bass (Lateolabrax maculatus), Asian sea bass (Lates calcarifer), and tongue sole (Cynoglossus semilaevis). With copy number, structure, phylogeny, and synteny analysis, duplication of Tdrd6 and Tdrd7, as well as loss of Stk31 and Tdrd10, were characterized in teleost lineages. Codon based molecular evolution analysis indicated faster evolution of teleost Tdrd genes than that in mammals, potentially associated with the accelerated evolution of the piRNA pathway in teleost lineages. The evolutionary diversity of Tdrd genes was also detected between different teleost lineages. RNA-seq analysis showed that most teleost Tdrd genes were dominantly expressed in gonads, particularly highly expressed in testis, such as Tdrd6, Tdrd7a, Tdrd9, Ecat8, and Tdrd15. The varied expression and evolutionary pattern between the duplicated Tdrd6 and Tdrd7 in teleosts may indicate their functional diversification. All these results suggest a conserved function of teleost Tdrd family in gametogenesis and the piRNA pathway, which could lay a foundation for the evolution of Tdrd genes and be helpful for further deciphering of Tdrd functions in teleosts.
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Evolutionary dynamics and conserved function of the Tudor domain-containing (TDRD) proteins in teleost fish

    Corresponding author: Jie Cheng, jiecheng@ouc.edu.cn
  • 1. Key Laboratory of Marine Genetics and Breeding (Ocean University of China), Ministry of Education, Qingdao 266003, China
  • 2. Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266237, China
  • 3. Laboratory of Tropical Marine Germplasm Resources and Breeding Engineering, Sanya Oceanographic Institution, Ocean University of China, Sanya 572000, China

Abstract: Tudor domain-containing (TDRD) proteins, the germline enriched protein family, play essential roles in the process of gametogenesis and genome stability through their interaction with the PIWI-interacting RNA (piRNA) pathway. Several studies have suggested the rapid evolution of the piRNA pathway in teleost lineages with striking reproductive diversity. However, there is still limited information about the function and evolution of Tdrd genes in teleost species. In this study, through genome wide screening, 13 Tdrd family genes were identified in economically important aquaculture fish, including spotted sea bass (Lateolabrax maculatus), Asian sea bass (Lates calcarifer), and tongue sole (Cynoglossus semilaevis). With copy number, structure, phylogeny, and synteny analysis, duplication of Tdrd6 and Tdrd7, as well as loss of Stk31 and Tdrd10, were characterized in teleost lineages. Codon based molecular evolution analysis indicated faster evolution of teleost Tdrd genes than that in mammals, potentially associated with the accelerated evolution of the piRNA pathway in teleost lineages. The evolutionary diversity of Tdrd genes was also detected between different teleost lineages. RNA-seq analysis showed that most teleost Tdrd genes were dominantly expressed in gonads, particularly highly expressed in testis, such as Tdrd6, Tdrd7a, Tdrd9, Ecat8, and Tdrd15. The varied expression and evolutionary pattern between the duplicated Tdrd6 and Tdrd7 in teleosts may indicate their functional diversification. All these results suggest a conserved function of teleost Tdrd family in gametogenesis and the piRNA pathway, which could lay a foundation for the evolution of Tdrd genes and be helpful for further deciphering of Tdrd functions in teleosts.

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Introduction
  • Tudor domain-containing (TDRD) proteins, namely, with multiple repeated Tudor domains, are members of a highly conserved protein family which are abundantly expressed in germline cells and involved in germ cell development. The Tdrd family is widely recognized across diverse organisms, with 13 members including Tdrd1, Tdrkh (Tdrd2), Tdrd3, Rnf17 (Tdrd4), Tdrd5, Tdrd6, Tdrd7, Stk31 (Tdrd8), Tdrd9, Tdrd10, Snd1 (Tdrd11), Ecat8 (Tdrd12), and Tdrd15 (Chen et al. 2017; Wang et al. 2019b). The Tdrd family genes have functions associated with many fundamental biological processes, including histone modification, RNA metabolism, as well as the response of DNA damage (Chen et al. 2011; Lasko 2010; Pek et al. 2012; Siomi et al. 2010; Thomson and Lasko 2005). More specifically, Tdrd genes participate mainly and regulate the course of gonadal development and spermatogenesis through the recognition of the N-terminal arginine-rich repeats of PIWI proteins with their conserved Tudor domains. TDRDs could recruit the PIWI-interacting RNA (piRNA)-induced silencing complexes to inhibit transposable elements (TEs) (Siomi et al. 2010), which are essential in the ping-pong cycle of the piRNA pathway (Chuma and Nakano 2013; Shoji et al. 2009; Vagin et al. 2009). The biological and biochemical functions of Tdrd family genes have been extensively studied, especially in model organisms. For example, knockout and mutagenesis of several Tdrd genes in mice led to male infertility ascribed to abnormal spermiogenesis, further supporting their important roles in germ cell development (Gan et al. 2019).

    Teleost fish exhibit the most dramatic reproductive diversity among vertebrates (Smith and Wootton 2016). Most teleost species are gonochorists, whose gonad development could be differentiated or undifferentiated (Strüssmann and Nakamura 2002). Teleost fish can also show synchronous or sequential hermaphroditism, with the latter type including protandrous (e.g., black porgy, Wu et al. 2010), protogynous (e.g., swamp eel, Liem 1963), or serial hermaphroditism (e.g., Gobiidae, Avise and Mank 2009). Moreover, environmental factors, such as temperature, pH, and salinity, can also have great impact on teleost sex determination and gonadal differentiation. Adaptive evolution could actuate accelerated sequence evolution, and therefore, many reproduction-related genes, including those functioning in gametogenesis and sex determination, may evolve quickly due to evolutionary adaptation (Swanson et al. 2001; Whitfield et al. 1993; Zhang 2004). Moreover, whole-genome duplication (WGD) events are important in generating novel genes contributing to diversification within vertebrates. After two rounds of WGD in vertebrates, there is a third round in the evolution of the teleost lineage, termed as teleost-specific (TS)-WGD, which could provide a large amount of raw information for adaptive evolution and innovation (Glasauer and Neuhauss 2014; Sato and Nishida 2010). In this manner, several studies have reported that adaptive evolution could influence the piRNA pathway in teleost lineages consistent with the diversity of TEs in teleost genomes (Parhad and Theurkauf 2019), which supported the association of TE diversity with the piRNA pathway evolution in teleost fish, such as in Japanese flounder (Paralichthys olivaceus) (Song et al. 2019) and swamp eel (Monopterus albus) (Yi et al. 2014).

    However, as the key components participating in the piRNA pathway, studies about the evolutionary dynamics and function of Tdrd genes in teleosts, especially in nonmodel fish, are limited with only a few reports in zebrafish (Danio rerio) (Huang 2012), swamp eel (M. albus) (Chen et al. 2017), and Japanese flounder (P. olivaceus) (Wang et al. 2019b). Therefore, first, we characterized the Tdrd family members in teleost species with diverse sexual development patterns, including spotted sea bass (Lateolabrax maculatus) and Asian sea bass (Lates calcarifer). These two fish may both grow in a wide range of salinity values from fresh to sea water, but they are only able to complete sexual maturation and gametogenesis in sea water. Then, the evolutionary dynamics and expression profiles of these Tdrd genes were compared with that of flatfish, i.e., Japanese flounder (P. olivaceus) and tongue sole (Cynoglossus semilaevis), both of which have a sex reversal scenario from female to male during their sex differentiation under environmental stress (including temperature and salinity). All these fish species are economically important in aquaculture of Asian countries, and the understanding of their sexual development and gametogenesis is important to improve their breeding. Furthermore, the copy number, structure, phylogeny, and synteny of Tdrd genes were analyzed. Moreover, a comprehensive codon-based molecular evolution analysis was performed for the Tdrd genes between mammal and teleost lineages, which have different reproduction strategies. Finally, the expression profiles of the teleost Tdrd members were investigated through RNA-seq analysis. All these findings will generate a comprehensive understanding about the function and evolution of the Tdrd family in gametogenesis of teleost fish.

Results and discussion

    Conserved Tdrd family among teleost species

  • We identified 10 and 14 Tdrd family genes through the genome assembly and transcriptome of spotted sea bass (LmTdrd) and Asian sea bass (LcTdrd), respectively. In addition, 13 Tdrd genes were recognized in tongue sole (CsTdrd). Thirteen Tdrd members from Japanese flounder (PoTdrd) were retrieved from Wang et al. (2019b). Together with other represented vertebrate species (teleosts, birds, amphibians and mammals, Supplementary Table S1), the copy number of Tdrd genes was identified. As shown in Fig. 1, human (Homo sapiens), frog (Xenopus tropicalis) and coelacanth (Latimeria chalumnae) conserved all 13 Tdrd genes, whereas chicken (Gallus gallus) and spotted gar (Lepisosteus oculatus) conserved 12 Tdrd genes but without Tdrd10. Tdrd10 was absent from all selected teleost species, and Stk31 (Tdrd8) was found only in common carp (Cyprinus carpio) and zebrafish (D. rerio) (Fig. 1). Moreover, both Tdrd6 and Tdrd7 contained duplicated members in most teleost species, whereas only a single copy was found in tetrapod and spotted gar, which indicated the duplication of Tdrd6 and Tdrd7 through the third-round TS-WGD (Glasauer and Neuhauss 2014; Wang et al. 2019a). Furthermore, Tdrd3, Tdrd6, and Tdrd9 were lost in the spotted sea bass genome compared with other teleost species. Conversely the common carp genome, which went through a fourth WGD event during evolution, contained 21 Tdrd members that were mostly twice of the Tdrd number in other teleost species. For example, two Tdrd1 and four Tdrd7 genes were identified in common carp genome compared to one Tdrd1 and two Tdrd7 genes in other teleost species (Fig. 1).

    Figure 1.  Tdrd family copy number among represented vertebrate genomes according to their WGD through evolution. 2R, 3R, and 4R refer to the number of WGD events in vertebrate evolution. The number of boxes indicates the number of Tdrd genes in the species, green box means common to teleosts and mammals with one copy in teleosts, brown box means common to teleosts and mammals with two copies in teleosts, blue box means unique to mammals, and dashed line box indicates the loss of this gene in certain lineages. Species names were abbreviated as Cca: Cyprinus carpio; Cse: Cynoglossus semilaevis; Dre: Danio rerio; Gga: Gallus gallus; Hsa: Homo sapiens; Lca: Lates calcarifer; Lch: Latimeria chalumnae; Lma: Lateolabrax maculatus; Loc: Lepisosteus oculatus; Oni: Oreochromis niloticus; Pol: Paralichthys olivaceus; Tru: Takifugu rubripes; Xtr: Xenopus tropicalis

    TDRD proteins normally contain multiple tandem repeated Tudor domains, which are essential to recognize multiple arginine sites of PIWI proteins in the piRNA pathway (Gan et al. 2019). To further explore the conservation of Tdrd family among vertebrates, the Tudor domains of each TDRD protein were collected (Table 1, Fig. 2). In general, TDRD members have similar amounts of Tudor domains between mammals and teleosts with minor exceptions. For example, TDRKH, TDRD3, TDRD5, TDRD9 and SND1 contain only one Tudor domain in both mammals and teleosts, whereas other TDRDs contain multiple Tudor domains from 2 (ECAT8) to 8 (TDRD15), respectively (Table 1, Fig. 2A). Moreover, multiple Tudor domains in a single protein might also have different biological functions other than as PIWI binders (Gan et al. 2019), and multiple sequence alignment among Tudor domains of Asian sea bass revealed only partial conservation, which was found also in that of human Tudor domains (Fig. 2B, Gan et al. 2019).

    Human Mouse Zebrafsh Spotted sea bass Asian sea bass Japanese founder Tongue sole Swamp eel
    TDRD1 4 4 4 4 4 4 4 4
    TDRKH 1 1 1 1 1 1 1 1
    TDRD3 1 1 1 1 1 1 1
    RNF17 5 5 5 4 5 5 5 4
    TDRD5 1 1 1 1 1 1 1 1
    TDRD6 7 7 7 7 7 7 7
    TDRD6L 7 7 7 7 7 6
    TDRD7A 3 3 3 3 3 3 3 3
    TDRD7B 3 3 3 3 3 3
    STK31 1 1
    TDRD9 1 1 1 1 1 1 1
    TDRD10 1
    SND1 1 1 1 1 1 1 1 1
    ECAT8 2 2 2 2 2 1 2 2
    TDRD15 7 7 5 8 8 7 8
    – indicates the missing of this TDRD member in certain species

    Table 1.  Number of Tudor domains in different TDRD members of selected vertebrates

    Figure 2.  Tudor domains in Asian sea bass TDRD proteins. A Schematic diagram of Tudor domains. Other confidently predicted domains are noted by their official names, and the length of each protein sequence is shown at the end. TUDOR domains are highlighted in blue, zf-MYND: zinc finger MYND domain, KH: K homology RNA-binding domain, LOTUS: domain on Tudor-containing protein 7, SNc: staphylococcal nuclease homologs, SrmB: Superfamily Ⅱ DNA and RNA helicase; B Multiple sequence alignment of the Asian sea bass TDRD Tudor domains. Sites showing over 80% conservation are highlighted in blue

  • Phylogenetic relationship of vertebrate Tdrd genes

  • Amino acid sequences of represented vertebrates were used to construct the phylogeny of the Tdrd family, which illustrated the conserved evolutionary relationship of each Tdrd gene (Fig. 3). Most of the vertebrate Tdrd genes were clustered into 12 sub-family clades compatible with their phylogenetic expectations, with the exception of Tdrd10 clustering in the Tdrd9 clade, probably due to the loss of Tdrd10 in most vertebrates (Fig. 3). Most of the Tdrd sub-family clades contained a clear division between mammal and teleost lineages, with the exception of Stk31 being lost in most teleost species (Fig. 3). Moreover, due to the TS-WGD event, there was duplication of Tdrd6 in the ancestral teleost branch after their divergence from mammals, whereas in Tdrd7, the teleost Tdrd7b were first clustered with mammalian Tdrd7, and then together clustered with teleost Tdrd7a. This was probably due to the artefacts of the phylogeny construction, and the synteny analysis below provided more evidence for the Tdrd7 duplication in teleost lineages.

    Figure 3.  Phylogeny of the Tdrd family among represented vertebrate species. The amino acid sequences of TDRD were aligned by MEGA7.0 and the ML phylogenetic tree was built with IQ Tree. Ame: Astyanax mexicanus; Cse: Cynoglossus semilaevis; Dre: Danio rerio; Gga: Gallus gallus; Hsa: Homo sapiens; Lca: Lates calcarifer; Lch: Latimeria chalumnae; Lma: Lateolabrax maculatus; Loc: Lepisosteus oculatus; Pol: Paralichthys olivaceus; Xtr: Xenopus tropicalis

  • Duplication and reduction of Tdrd genes in teleost lineages

  • Synteny analysis was conducted to verify the duplication and reduction of teleost Tdrd genes after the TS-WGD event. Consequently, three possible evolutionary directions of the duplicated Tdrd genes were found in teleost lineages (Fig. 4, Supplementary Fig. S1). For example, one single Tdrd copy was retained (Tdrd1, Fig. 4A), both duplicates were retained (Tdrd6 and Tdrd7, Fig. 4B, 4C) or both duplicates were lost (Stk31, Fig. 4D). First, Tdrd1 was well conserved between selected mammal and teleost lineages. Most of its neighboring genes were in synteny in one chromosome of teleosts, but less conserved in the other duplicated chromosome fragment probably reflecting the loss of the duplicated Tdrd1 copy in teleosts (Fig. 4A). Second, there were two Tdrd6 copies (Tdrd6 and Tdrd6l) in teleosts after the WGD. Their neighboring genes were highly conserved among teleosts, although they were quite different from mammals in both chromosome fragments (Fig. 4B). Similarly, Tdrd7 contained two copies in teleosts (Tdrd7a and Tdrd7b), and the synteny of Tdrd7a and Tdrd7b was similar as Tdrd6 with less conservation between mammals and teleosts. Moreover, the neighboring genes of Tdrd7a and Tdrd7b in spotted sea bass were also quite different from other teleosts (Fig. 4C), probably indicating their structure variation in spotted sea bass genome. As for Stk31, which was lost in most teleost species, the duplicated copies of its neighboring genes could be identified in teleost lineages, with both copies conserved between teleosts and mammals (Fig. 4D).

    Figure 4.  Synteny analysis of Tdrd genes with teleosts and other vertebrates according to the TS-WGD, as Tdrd1 (A), Tdrd6 (B), Tdrd7 (C), and Stk31 (D). Genes were shown as colored pentagons with their names on top. The pentagons with the same color were orthologous among different species. The pentagon's direction illustrated the gene orientation. The dashed boxes indicated Tdrd gene loss from the origin loci after TS-WGD

  • Rapid evolution of Tdrd genes in teleosts than in mammals

  • To have more insight into the evolutionary dynamics of Tdrd genes, we employed the codon-based molecular evolution analysis from the PAML package (site, branch, and branchsite models) with coding nucleotide sequences of each Tdrd gene. Thus, the evolutionary pattern of Tdrd genes was compared between teleosts, which was adapted to live in water, with sexual development diversity and external fertilization, and mammals, adapted to live on land with internal fertilization.

    The site models assume the dN/dS ratio (nonsynonymous to synonymous substitution ratio, ω ratio) could change among codons. For mammal and teleost Tdrd genes, site model tests with three pairs (M0 vs. M3, M1a vs. M2a, and M7 vs. M8) were performed. In summary, M0 and M3, testing for variation of ω among codons, showed M3 (discrete) all significantly differed from M0 (oneratio) (P < 0.05), suggesting striking variations in selection pressure among codons of Tdrd genes (Supplementary Table S2). Further comparisons of M1a (neutral) and M2a (selection), as well as M7 (β) and M8 (β and ω), testing whether or not the analyzed codon was evolving under positive selection, revealed significant differences between the M7 and M8 models of all mammal and teleost Tdrd genes (P < 0.05, Supplementary Table S2). This indicated elevated evolution in the vertebrate Tdrd family. The dN/dS ratio of teleost Tdrd genes was higher than that of mammals (Fig. 5) indicating increased rates of non-synonymous substitution in teleost.

    Figure 5.  dN/dS (ω) ratio of Tdrd genes between teleost and mammal lineages under site model tests

    The branch model assumes the dN/dS ratio could change among branches of the phylogeny and is used to identify positive selection affecting particular lineages. To validate the evolution pattern, branch model tests were employed for the Tdrd genes between mammal and teleost lineages (Fig. 6, Supplementary Table S2). Thus, except for Tdrd3, the tworatio models all showed significantly different ω1 values for the teleost clade compared to ω0 in the one-ratio model (P < 0.05), while ω1 was higher in the teleost clade than ω0 for mammals (Fig. 6). These results confirmed the elevated evolution of Tdrd genes in teleosts compared to mammals, which was consistent with the results of site model tests.

    Figure 6.  Branch model tests of Tdrd genes between mammal and teleost lineages. Red columns represented ω1 values in teleost lineages (foreground branch), whereas blue columns represented ω0 values in mammalian lineages (background branch). *P < 0.05; **P < 0.01

    The branch-site model assumes the dN/dS ratio could change among codons as well as across branches of the phylogeny, with the intention of identifying positive selection acting on a few sites in particular lineages. We employed the branch-site model to investigate whether positively selected sites (PSSs) could be detected in the ancestral branch of teleost lineages. As a result, significant PSSs were detected in most Tdrd genes, except for Tdrd5, Tdrd9, and Tdrd15 (Table 2, Supplementary Table S2). Five PSSs from Rnf17 (one site), Tdrd6 (one site), Tdrd7a (two sites) and Ecat8 (one site) were located in their Tudor domains (Fig. 7). TDRD proteins could recruit PIWI proteins through the interaction between the Tudor domains and the arginine rich motifs of PIWI, which is vital in the piRNA pathway and germ cell functions (Gan et al. 2019). Therefore, these PSSs in the Tudor domains may suggest their significant role in functional evolution related to the piRNA biogenesis.

    gene 2∆L P value Positively selected sites
    Tdrd1 30.04799 0 361 Y 0.974*, 724 C 0.986*
    Tdrkh 33.06893 0 42 K 0.991**, 100 N 0.980*, 236 M 0.962*, 253 G 0.989*
    Tdrd3 33.25806 0 24 L 0.998**, 276 D 0.993**, 282 E 0.985*, 291 N 0.996**, 338 G 0.993**, 635 E 0.971*
    Rnf17 34.03305 0 1621 S 0.963*
    Tdrd5 29.49923 0
    Tdrd6 31.92239 0 280 P 0.962*, 1828 T 0.952*, 2368 E 0.962*
    Tdrd6l 42.98522 0 1753 K 0.970*
    Tdrd7a 38.71650 0 43 C 0.988*, 974 A 0.962*, 1028 V 0.986*, 1044 S 0.991**, 1048 K 0.956*, 1110 Q 0.973*,
    Tdrd7b 39.69649 0 150 T 0.997**, 320 G 0.994**, 574 R 0.963*, 1066 F 0.984*, 1070 P 0.994**
    Tdrd9 17.29876 0
    Snd1 4.91260 0.0267 769 C 0.969*
    Ecat8 29.62452 0 370 L 0.964*, 985 T 0.972*, 1157 S 0.985*, 1292 S 0.995**, 1384 F 0.955*
    Tdrd15 17.64851 0
    The ancestral branch leading to the teleost lineage was set as the foreground branch. Sites with the BEB posterior probabilities higher than 90% were presented, with those higher than 95% marked with * and higher than 99% marked with **. Positively selected sites from Tudor domains were in bold. P values < 0.05 were in bold

    Table 2.  Branch-site model tests among teleost and mammal lineages

    Figure 7.  Examples of positively selected sites (PSSs) in Tdrd7a (A) and Tdrd7b (B) Tudor domains from branch-site model tests. The partial amino acid sequence alignments of the selected vertebrates with their phylogeny are presented. Arrows indicate PSSs detected with their location at the bar above. …… represents sequences omitted between the two segments

    To test the evolutionary dynamics of Tdrd genes in specific teleost lineage, branch model tests were also performed for spotted sea bass and Japanese flounder lineages, respectively, which present specific reproductive characteristics. Spotted sea bass or Japanese flounder were set as the foreground branch (ω1) independently within the phylogeny of teleost species. As a result, the ω1 values of Tdrd1, Snd1, and Tdrd15 with spotted sea bass labeled as foreground, and Tdrd3, Rnf17, Tdrd5, Tdrd6, Tdrd6l, and Tdrd7a with Japanese flounder labeled as foreground, were significantly higher than ω0 in the rest teleosts (Fig. 8). The fast evolving Tdrd genes were different between the two fish suggesting potentially diverse functions for these Tdrd members linked to specific scenarios of sexual development and gametogenesis.

    Figure 8.  Branch model tests of Tdrd genes with spotted sea bass (A) or Japanese flounder (B) labeled as foreground, respectively. Red columns represented ω1 in selected teleosts (foreground branch), and blue columns represented ω0 from the rest teleost lineages (background branch). *P < 0.05; **P < 0.01

    The piRNA pathway is functionally vital in germline development, gametogenesis, TE silencing, and genome integrity (Ozata et al. 2019). Many piRNA pathway genes may have undergone evolutionary adaptation reported dominantly in human (e.g., Piwil1-4), Drosophila (e.g., Piwi, Ago3, and Aubergene) and C. elegans (e.g., PRG1-2). They are all under evolutionary pressure with piRNA-dependent transposon silencing (Gan et al. 2019; Parhad and Theurkauf 2019; Simkin et al. 2013; Song et al. 2019; Wynant et al. 2017). With high reproductive diversity in teleosts, which are subject to challenges from dynamic aquatic environments, the reproduction-related genes have previously been deemed to be under stronger adaptive evolution in teleosts than their orthologous counterparts in mammals (Yi et al. 2014). Moreover, gametogenesis in teleosts could be very active to fill sustained reproductive capacity giving the stronger activity of TE translocating along genomes (Yi et al. 2014). Therefore, the piRNA pathway in teleosts, as a protector of the genome in silencing TEs, is suggested to be under intensive evolutionary selection, probably coping with the higher abundance and diversity of transposons (Chalopin et al. 2015). For example, the rapid evolution of PIWI proteins in several fish, such as in swamp eel (Yi et al. 2014), Japanese flounder (Song et al. 2019) and tilapia (Tao et al. 2016) was reported. With the molecular evolution analysis, we highlighted that adaptive evolution was likely also to be present in the Tdrd family genes of teleost lineages, which could interact with PIWI proteins during the pingpong cycle.

  • Germline enriched expression of Tdrd genes in teleost species

  • To further understand the Tdrd functions in teleosts, we analyzed the Tdrd expression patterns with RNA-seq data for adult tissues of spotted sea bass and Japanese flounder (Fig. 9). As a result, the expression of Tdrd genes was conserved between the two species, which were both dominantly expressed in gonad tissues. For example, in spotted sea bass (Fig. 9A), except for Tdrd6l and Tdrd7b, all Tdrd genes were abundantly expressed in gonads indicating their potential role in germline and spermatogenesis. In Japanese flounder (Fig. 9B), all Tdrd genes were highly expressed in gonads, and most of them showed male-biased expression with the only exception of Tdrd3 and Snd1. Other than in gonads, some Tdrd genes were also highly expressed in somatic tissues. For example, in spotted sea bass, Tdrkh and Snd1 were widely expressed in all seven tissues. Furthermore, in Japanese flounder, Tdrd3 and Snd1 were broadly expressed in ten selected tissues, which suggested their diverse functions in somatic tissues.

    Figure 9.  Spatial expression profiles of Tdrd genes in adult tissues of spotted sea bass (A) and Japanese flounder (B) with RNA-seq data (n = 1)

    Considering the vital function of Tdrd family in gonadal differentiation and gametogenesis, we further invested the expression profiles of Tdrd genes in gonads and brain of four aquatic important teleost species through public transcriptome data. Brain was included, because it was important in the HPG axis (brain-pituitary-gonad) in the regulation of sexual development (Weltzien et al. 2004). In general, most Tdrd genes were expressed higher in testis than in ovaries (Fig. 10). For example, in spotted sea bass (Fig. 10A), which can grow with a wide range of salinities from fresh to sea water, but can only complete sexual maturation and gametogenesis in sea water, six Tdrd genes (Tdrd1, Tdrkh, Rnf17, Tdrd7a, Ecat8, and Tdrd15) were expressed higher in testis than in ovaries. Conversely, only two Tdrd genes (Tdrd5 and Snd1) were expressed higher in ovaries than in testis. In Asian sea bass, which needs sea water to develop mature gametes and represents serial male to female sex change, eight Tdrd genes (Tdrd1, Tdrkh, Rnf17, Tdrd6, Tdrd7a, Tdrd9, Ecat8, and Tdrd15) showed male-biased expression, whereas Tdrd3, Tdrd5, and Snd1 represented female-biased expression (Fig. 10B). In tongue sole and Japanese flounder, both of which have a sex reversal scenario from female to male under specific environments (including temperature and salinity) during their key sex differentiation period, 10 and 11 Tdrd genes had male-biased expression, with the only exception of Tdrkh and Tdrd6l in tongue sole, and Tdrd3 and Snd1 in gynogenetic Japanese flounder, showing biased expression in ovaries (Fig. 10C, D). Moreover, a few Tdrd genes were expressed also in teleost brains, for example, Tdrkh, Tdrd7b, and Snd1 in spotted sea bass, Tdrkh, Tdrd3, Tdrd7b, and Snd1 in Asian sea bass, Tdrkh, Tdrd3, Tdrd7b, and Snd1 in tongue sole, and Tdrd3, Tdrd6, and Snd1 in gynogenetic Japanese flounder. The conserved expression of Tdrkh, Tdrd3, Tdrd7b, and Snd1 in teleost brains may indicate their possible vital role in the piRNA pathway in brains.

    Figure 10.  Expression of Tdrd genes in brain and gonads of different teleost of (A) spotted sea bass, n = 1; (B) Asian sea bass, n = 1; (C) tongue sole, n = 3; (D) gynogenetic Japanese flounder, n = 3

    In model organisms, most Tdrd genes, except for Tdrd10 and Tdrd15, were reported to recruit PIWI protein both in vivo or in vitro. Moreover, all Tdrd-deficient mice of Tdrd1, Tdrkh, Rnf17, Tdrd5, Tdrd6, Tdrd7, Tdrd9 and Tdrd12 exhibited male infertility due to dysfunction in sperm development, but not with Stk31 (Gan et al. 2019). In teleosts, there is only limited information about the functional annotation of Tdrd genes. For example, Tdrd1 was verified to be germline-enriched and sexual dimorphically expressed in Japanese flounder (Zhao et al. 2018). In addition, it was testis-specifically expressed in large yellow croaker (Larimichthys crocea) (Luo et al. 2019). Tdrd12 was reported to be important in germ-cell development and maintenance in zebrafish (Dai et al. 2017). In this study, most Tdrd members were dominantly expressed in gonads, especially in the testis. More specifically, the expression of Tdrd genes was similar between spotted sea bass and Asian sea bass. For example, Tdrd1, Tdrkh, Rnf17, Tdrd7a, Ecat8 and Tdrd15 were highly expressed in testis, and Tdrd5 and Snd1 were highly expressed in ovaries. Moreover, the expression of Tdrd genes was similar between flatfish with Tdrd1, Rnf17, Tdrd5, Tdrd6, Tdrd7b, Tdrd9, Snd1, Ecat8, and Tdrd15 all highly expressed in testis of Japanese flounder and tongue sole. These results illustrate the conserved function of the Tdrd family in spermatogenesis and gonadal maturation of teleosts, and probably suggest functional divergence of certain Tdrd members (e.g., Tdrd5, Snd1) among teleost lineages with diverse reproductive manners.

    Gene duplication is one of the most essential approaches to species differentiation and evolution (Cotton and Page 2005; Holland et al. 1994; Ohno 2013). In particular, there may well be functional diversification among the duplicated Tdrd genes in teleosts. For example, Tdrd7 was detected in the germline, and was important for germ cell development in zebrafish (Huang et al. 2011), Atlantic salmon (Kleppe et al. 2015) and Amphioxus species (Zhang et al. 2013). In Japanese flounder, Tdrd7a showed gonad-enriched expression among adult tissues and was also germ cell specifically distributed in embryo and gonads. Conversely, Tdrd7b was not expressed with the similar tissue and cell specificity (Wang et al. 2019b), which suggested that the duplicated Tdrd7a and Tdrd7b in Japanese flounder may have undergone functional divergence. Similarly, in spotted sea bass and Asian sea bass, Tdrd7a was more highly expressed in testis than in ovaries, whereas Tdrd7b was not highly expressed in gonads but with moderate expression in brain (Fig. 10). This result was consistent with the molecular evolution analysis that PSSs were only detected in Tudor domains of teleost Tdrd7a but not in Tdrd7b (Table 2, Fig. 7), which may suggest the functional diversification between the duplicated Tdrd genes.

Conclusion
  • In conclusion, Tdrd family genes were identified genomewide in teleost species, including 10 Tdrd genes in spotted sea bass (L. maculatus), 14 in Asian sea bass (L. calcarifer) and 13 in tongue sole (C. semilaevis). Molecular evolution analysis revealed a more rapid evolution of the Tdrd family genes in teleost lineages compared with mammals. Moreover, PSSs could be located in the functional Tudor domains, indicating that these amino acid substitutions were possibly adapting due to interaction with PIWI or to TE silencing in teleosts. RNA-seq analyses revealed significantly higher expression of Tdrd genes in teleost testis, suggesting their essential roles in spermatogenesis. All these results provide a comprehensive understanding of the teleost Tdrd family, and illustrate new insights into the evolutionary dynamics and functional differences of Tdrd genes in teleost lineages.

Materials and methods

    Tdrd family identification and characterization

  • The amino acid sequences of Tdrd genes across vertebrates, including mammals, human and mouse (Mus musculus); amphibians, African clawed frog; bird, chicken; and teleosts, fugu (T. rubripes), Japanese medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), tilapia (O. niloticus), and zebrafish were extracted from Ensembl (https://asia. ensembl.org) and the NCBI (https://www.ncbi.nlm.nih.gov) databases. The sequences were employed to search against the transcriptome of selected species with TBLASTN of an E value cutoff of e−10. BLASTN was also used to approve the Tdrd coding sequences by aligning them against the Nr and whole-genome database with an E value cutoff of e−10. Therefore, Tdrd genes were identified by screening the spotted sea bass genome (PRJNA408177) and transcriptome (SRP118961), the Asian sea bass genome (PRJNA294489) and transcriptome (SRP053272), as well as the tongue sole genome (PRJNA73987). Tdrd genes from Japanese flounder were retrieved from Wang et al. (2019b). Simple Modular Architecture Research Tool (SMART, http://smart.emblheidelberg.de) and NCBI batch CD-search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) were applied to detect the conserved Tudor domains. Multiple sequence alignment of Tudor domains was done using Clustal W by MEGA7.0 (Kumar et al. 2016) and the conservation was visualized by GeneDoc software (Nicholas 1997).

  • Phylogeny construction

  • The amino acid sequences of 13 Tdrd genes from representative vertebrates, including human, mouse, frog, chicken, coelacanth, spotted gar, spotted sea bass, Asian sea bass, Japanese flounder, tongue sole, zebrafish, and Mexican tetra (Astyanax mexicanus) were collected through Ensembl and NCBI databases. Multiple sequence alignment was performed with Clustal W from MEGA 7.0 (Kumar et al. 2016), and the maximum likelihood (ML) phylogenetic tree was built with a ultrafast bootstrap (Hoang et al. 2018) of 1000 replicates by IQ Tree web server (Trifinopoulos et al. 2016). The best model of Jones–Taylor–Thornton with Freqs. and Gamma 4 distributed sites (JTT + F + G4) was selected in IQ Tree.

  • Synteny analysis

  • Several mammal and teleost species were selected for synteny analysis, including human and mouse from mammals, spotted gar as a fish that diverged from teleosts before the third-round TS-WGD event, and Amazon molly, turbot (Scophthalmus maximus), fugu, as well as spotted sea bass from bony fish. Tdrd1, Tdrd6, Tdrd7, and Stk31 were included as they represented different evolutionary fate (lost, reserved, or duplicated) after the TS-WGD. Synteny analysis of other Tdrd genes with their neighboring genes is shown in Supplementary Fig. S1. Genomicus (https://www.genomicus.biologie.ens.fr) and Ensembl were used to search their neighboring genes with the gene direction recorded. The synteny of Stk31, which was lost in teleost species, was based on its neighboring gene Npy in mammals. The neighboring genes were analyzed by BLASTN to determine their gene names.

  • PAML analysis

  • The coding nucleotide sequences of 14 teleosts, including spotted sea bass, Asian sea bass, Amazon molly (Poecilia formosa), fugu, spotted green pufferfish (Tetraodon nigroviridis), Japanese medaka, stickleback, Mexican tetra, platyfish (Xiphophorus maculatus), tilapia, cod (Gadus morhua), tongue sole, Japanese flounder, and zebrafish, as well as 12 mammals, including human, rhesus monkey (Macaca mulatta tcheliensis), gray short-tailed opossum (Monodelphis domestica), mouse, Norway rat (Rattus norvegicus), cow (Bos taurus), dog (Canislupus familiaris), horse (Equus caballus), pig (Sus scrofa), rabbit (Oryctolagus cuniculus), sheep (Ovis aries), and African savanna elephant (Loxodonta africana) were used to explore the molecular evolution of Tdrd genes between mammal and teleost lineages. The coding nucleotide sequences of each Tdrd were aligned using Clustal W with option -codons to make a codon alignment, and the ML tree was built with MEGA 7.0 to provide their evolutionary relationships (Supplementary Material S1). The codon based model tests were performed with the package PAML v4.9 (Yang 2007; Yang et al. 2005) as explained in detail by Song et al. (2019).

    Briefly, site model pairs, as M0 vs. M3, M1a vs. M2a, and M7 vs. M8, were employed to test the positive selection in individual codon of each Tdrd gene, using ML estimation of the dN/dS (ω) ratio with likelihood ratio tests (LRTs) on the phylogenetic tree. Twofold difference of the ML values between paired models (2∆lnL) was determined with Chi-square (χ2) test. The Bayes empirical Bayes (BEB) was employed to determine the Bayesian posterior probability of the sites with positive selection. The dN/dS (ω) values between mammal and teleost lineages were compared to assess the substitution rate of each Tdrd. In branch model tests, one-ratio model was the null hypothesis, and the tworatio model was employed to test the varied ω ratio along labeled branch against the one-ratio model. The ancestral branch leading to the teleost, spotted sea bass, or Japanese flounder was labeled as foreground clades (ω1), respectively, and the rest was deemed as background clades (ω0). 2ΔlnL was calculated to determine the significant difference of the LRTs with χ2 test. In branch-site model tests, the ancestral branch leading to the teleost lineages was set as foreground clades, and the rest as background clades. The LRT significance was tested by χ2 analysis. If it suggested positive selection, the PSSs were further verified with high BEB posterior probabilities (0.95). The PSSs were then retrieved from the amino acid sequences with functional Tudor domains from the Pfam database.

  • Transcriptome analysis of Tdrd genes among teleost species

  • To invest the spatial expression patterns of Tdrd family, the RNA-seq data of adult tissues of spotted sea bass (n = 1) and Asian sea bass (n = 1) were downloaded from NCBI using NCBI's SRA Toolkit. The SRA files were transformed into fastq format by "Fastq-Dump" in SRA Toolkit (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc&f=fastq-dump). The fastq file was optimized by trimmomatic (Bolger et al. 2014) to delete adapters, low-quality reads, and the reads with less base numbers. Next, the read mapping and quantification were performed with Hisat (Kim et al. 2015) and StringTie (Pertea et al. 2015) tools, and the FPKM values were extracted for each Tdrd gene. The corresponding SRA files of spotted sea bass tissues are: brain: SRR7528887, gill: SRR7528883, liver: SRR7528886, spleen: SRR7528888, stomach: SRR7528884, testis: SRR7528885, ovary: SRR2937376, and Asian sea bass tissues: brain: SRR1791593, testis: SRR1791598, ovary: SRR1791597. The RNA-seq data of Japanese flounder (n = 3, Zhao 2017) and tongue sole (n = 3, Wang et al. 2018) were also retrieved from previously published data. Heatmaps were generated by MeV software (Saeed et al. 2003).

Supplementary Information
Author contributions
  • JC and QZ designed the study, ZL, SL, SG, and WL analyzed the data. JC and ZL conducted the manuscript writing. All the authors have read and approved the final manuscript.

Acknowledgements
  • This research was funded by National Natural Science Foundation of China (31702331), China Agriculture Research System (CARS-47-G06), and National Infrastructure of Fishery Germplasm Resources.

Declarations

    Conflict of interest

  • The authors declare that they have no conflict of interest.

  • Animal and human rights statement

  • This study was conducted in accordance with the Institutional Animal Care and Use Committee of Ocean University of China, and it does not contain any studies with human participants.

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