Chromosome‐Level Genome Assembly for the Largemouth Bass

Received: 1 April 2020 | Revised: 19 August 2020 | Accepted: 24 August 2020 DOI: 10.1111/1755-0998.13256

RESOURCE ARTICLE

Chromosome-level genome assembly for the largemouth bass Micropterus salmoides provides insights into adaptation to fresh and brackish water

1Key Laboratory of Tropical and Subtropical Fishery Resource Application and Abstract Cultivation, Ministry of Agriculture and Largemouth bass (LMB; Micropterus salmoides) has been an economically important Rural Affairs, Pearl River Fisheries Research Institute, Chinese Academy of Fishery fish in North America, Europe, and China. This study obtained a chromosome-level Sciences, Guangzhou, China genome assembly of LMB using PacBio and Hi-C sequencing. The final assem- 2 Shenzhen Key Laboratory of Marine bled genome is 964 Mb, with contig N50 and scaffold N50 values of 1.23 Mb and Genomics, Guangdong Provincial Key Laboratory of Molecular Breeding in Marine 36.48 Mb, respectively. Combining with RNA sequencing data, we annotated a total Economic Animals, BGI Academy of Marine of 23,701 genes. Chromosomal assembly and syntenic analysis proved that, unlike Sciences, BGI Marine, BGI, Shenzhen, China 3Biozeron Shenzhen Inc., Shenzhen, China most Perciformes with the popular haploid chromosome number of 24, LMB has only 23 chromosomes (Chr), among which the Chr1 seems to be resulted from a chro- Correspondence Xing Ye, Key Laboratory of Tropical and mosomal fusion event. LMB is phylogenetically closely related to European seabass Subtropical Fishery Resource Application and spotted seabass, diverging 64.1 million years ago (mya) from the two seabass and Cultivation, Ministry of Agriculture and Rural Affairs, Pearl River Fisheries Research species. Eight gene families comprising 294 genes associated with ionic regulation Institute, Chinese Academy of Fishery were identified through positive selection, transcriptome and genome comparisons. Sciences, Guangzhou, China. Email: [email protected] These genes involved in iron facilitated diffusion (such as claudin, aquaporins, sodium channel protein and so on) and others related to ion active transport (such as Qiong Shi, Shenzhen Key Laboratory of Marine Genomics, Guangdong Provincial sodium/potassium-transporting ATPase and sodium/calcium exchanger). The claudin Key Laboratory of Molecular Breeding in gene family, which is critical for regulating cell tight junctions and osmotic homeo- Marine Economic Animals, BGI Academy of Marine Sciences, BGI Marine, BGI, stasis, showed a significant expansion in LMB with 27 family members and 68 copies Shenzhen, China. for salinity adaptation. In summary, we reported the first high-quality LMB genome, Email: [email protected] and provided insights into the molecular mechanisms of LMB adaptation to fresh and Funding information brackish water. The chromosome-level LMB genome will also be a valuable genomic Special Fund for Scientific Research in Public Welfare and Capacity Building of resource for in-depth biological and evolutionary studies, germplasm conservation Guangdong Province, Grant/Award Number: and genetic breeding of LMB. 2017A030303002; Central Public-interest Scientific Institution Basal Research Fund of the Chines Academy of Fishery Sciences, KEYWORDS Grant/Award Number: 2017HY-XKQ0208; chromosomal fusion, chromosome-level genome, ion transport, largemouth bass (Micropterus Youth Program of National Natural Science salmoides), salinity adaptation Foundation of China, Grant/Award Number: 31902354; China Agriculture Research System, Grant/Award Number: CARS-46; Central Public-interest Scientific Institution Basal Research Fund，CAFS, Grant/Award Number: 2020TD23

Chengfei Sun, Jia Li and Junjian Dong contributed equally to this work.

1 | INTRODUCTION three-spined stickleback Gasterosteus aculeatus [Jones et al., 2012]) are euryhaline fishes that can adapt to fresh, brackish, and seawa- Largemouth bass (LMB; Micropterus salmoides; order Perciformes, ter habitats (Hirai et al., 1999; Tine et al., 2014). European seabass suborder Percoidei, family Centrarchidae) is one of the most popular can even tolerate up to 60‰ salinity (Jensen et al., 1998; Varsamos game fishes in North America. It is often considered a freshwater fish. et al., 2002). Euryhaline fishes exhibit unique and complex osmo- However, this species is distributed not only in freshwater habitats regulatory functions, whereby the water-ion balance in the body is of North America from central and eastern USA to northern Mexico, maintained via osmoregulatory organs (such as gills, kidneys, and gas- but also in the brackish water and freshwater habitats affected by trointestinal tracts) to adapt to environmental salinity fluctuations tides along the Atlantic coasts of North America and the Gulf of (Boutet et al., 2006; Forsyth & Wallis, 2002; Hirai et al., 1999). The Mexico (Bailey et al., 1954; DeVries et al., 2015; Glover et al., 2012). complex and ever-changing chemical environment in coastal water Most freshwater fishes are hardly adaptable to salinities higher than due to salinity fluctuations is an arduous challenge for freshwater 10‰ (Peterson & Meador, 1994), but LMB exhibits a relatively high fishes like LMB. However, previous studies have shown that LMB liv- salinity tolerance. LMB is often caught in coastal marshes with 1‰– ing in coastal waters, like their inland counterparts with high location 12‰ salinity and in coastal waters with 16‰ salinity. Furthermore, loyalty and do not exhibit large-scale migrations from their original LMB can even be found in aquatic environments with up to 24‰ habitats in response to increased salinity, indicating that LMB can salinity (Bailey et al., 1954; Meador & Kelso, 1990; Page, 2008; tolerate certain levels of salinity and the related physiological effects Peterson & Ross, 1991; Renfro, 1959; Susanto & Peterson, 1996). (Bain & Boltz, 1992; Copeland & Noble, 1994; DeVries et al., 2015; LMB is particularly abundant and has been an important support Glover et al., 2012). In the present study, PacBio and Hi-C sequenc- of the recreational fishing industry in oligohaline areas of estuar- ing techniques were applied to obtain a chromosome-level genome ies throughout the USA Atlantic and Gulf of Mexico coasts (Bailey for LMB, and its evolutionary relationships with other fishes partic- et al., 1954; Glover et al., 2012; Guier et al., 1978; Nack et al., 1993). ularly spotted seabass, European seabass, and Asian seabass, were It has been widely translocated for many decades as a game fish and elucidated via comparative genome analysis. Furthermore, LMB was also as part of large-scale stocking efforts, making it a cosmopolitan exposed to various salinity treatments to compare the transcriptome species (Barthel et al., 2010; Carpenter & Kitchell, 1996). The aqua- changes between fresh and brackish water. Based on the results of culture industries in China, Italy, and Mexico produce approximately comparative genomics and transcriptome analysis, we explored the 458,503 tons of LMB per year (FAO, 2017). Therefore, LMB has be- molecular mechanism of the brackish water adaptation of LMB. Our come an important freshwater aquaculture fish in addition to being a data may provide important resources for extensive studies on ger- popular game fish, with ecological and economic significance. mplasm conservation, mapping of traits with economic importance, High-throughput sequencing has been used to obtain genome and genetic improvements, as well as facilitate in-depth understand- sequences of various fishes, with great significance in clarifying bi- ing of the adaptive mechanisms of freshwater fishes in ecological ological characteristics, evolutionary history, and genetic basis of environments of brackish water. important traits (Bian et al., 2019; Ravi & Venkatesh, 2018). PacBio, a third-generation sequencing technology, and the high-throughput chromosome conformation capture (Hi-C)-assisted genome 2 | MATERIALS AND METHODS assembly technique have recently been applied to obtain chromosome-level genome assemblies for many economically important 2.1 | Ethics approval and consent to participate fishes, such as spotted seabass (Lateolabrax maculatus), giant grouper (Epinephelus lanceolatus), Atlantic herring (Clupea harengus), and All the experiments on fishes were conducted in accordance with Alpine whitefish (Coregonus sp.) (Dekayne et al., 2020; Pettersson the specific guidelines on the care and use of animals for scien- et al., 2019; Shao et al., 2018; Zhou et al., 2019), and coral reef fishes, tific purposes as outlined by the Institutional Animal Care and Use such as barred knifejaw (Oplegnathus fasciatus), orange clownfish Committee (IACUC) of the Pearl River Fisheries Institute, Chinese (Amphiprion percula), and Triplophysa tibetana (Lehmann et al., 2019; Academy of Fishery Sciences (CAFS), China. The IACUC approved Xiao et al., 2019; Yang et al., 2019). High-quality genome sequences this study under the CAFS project “Breeding of LMB-2019”. are of great significance for analysing the evolutionary history, sex-determination mechanisms, and genetic mechanisms for fish growth and immunity, as well as the adaptability of fishes living in 2.2 | Genomic DNA extraction and unique habitats, such as coral reefs and extreme environments on genome sequencing plateaus. To date, the complete genome sequences of over 200 aquatic A one-year-old female LMB with a bodyweight of 0.948 kg and body organisms have been published, among which Perciformes (such as length of 32.5 cm was collected from our local aquaculture base at European seabass Dicentrarchus labrax [Tine et al., 2014], Asian sea- the Pearl River Fisheries Institute in Guangzhou City, Guangdong bass Lates calcarifer [Vij et al., 2016], and spotted seabass Lateolabrax Province, China. Genomic DNA was extracted from blood sam- maculatus [Shao et al., 2018]) and Gasterosteiformes (such as ples using a QIAamp DNA Mini Kit (Qiagen). DNA integrity was SUN et al. | 303 checked by an Agilent 4200 Bioanalyser (Agilent Technologies). Self-ligated, nonligated, and other invalid reads (such as PCR ampl- A total of 8 μg of high-quality genomic DNA was sheared using icons, random breakages, and extreme fragments) were discarded. g-Tubes (Covaris), and then concentrated with AMPurePB mag- (ii) Juicer V1.5 (Durand et al., 2016) and 3d-dna v170123 (Dudchenko netic beads (Pacific Biosciences) for library construction using the et al., 2017) were applied to cluster the genomic contig sequences

SMRT (Single Molecule Real-Time) bell template prep kit 2.1 (Pacific into potential chromosomal groups. (iii) JuiceBox v1.8.8 (Dudchenko Biosciences). The constructed libraries were size-selected (>20 kb) et al., 2017) was used to validate the contig orientation and to remove on a BluePippin system (Sage Science), followed by primer annealing ambiguous fragments with the assistance of manual correction. and binding of SMRT bell templates to polymerases using a DNA/ Polymerase Binding Kit (Pacific Biosciences). Sequencing was performed on a Pacific Bioscience (PacBio) Sequel platform at Annoroad 2.5 | Assessment of the final chromosome-level Gene Technology Company. A total of eight SMRT cells were run for genome assembly whole-genome sequencing. We used an Illumina platform to generate short high-quality se- Three routine methods were employed to evaluate the completeness quencing reads as we reported previously (Zhang et al., 2018). In of our final genome assembly as follows. (i) Benchmarking Universal brief, genomic DNA was extracted from pooled muscle samples of Single-Copy Orthologues (BUSCO; Simão et al., 2015) assessment: the same LMB using Qiagen GenomicTip100. Subsequently, we con- The actinopterygii_odb9 (Zdobnov et al., 2017) orthologues were structed three short-insert libraries (270, 500, and 800 bp). The final used as the BUSCO reference. (ii) Transcriptome (RNA-seq) data as- paired-end sequencing was performed on a HiSeq X-Ten sequencer sessment: The de novo assembled transcripts were mapped onto our (Illumina). final assembly to calculate the transcriptomic coverage. (iii) GC variation assessment: Variation in GC content in the final assembly was calculated within 50 kb nonoverlapping sliding windows for subse- 2.3 | Genome size estimation and quent comparisons with other vertebrate species. genome assembling

The distribution of k-mers presents a Poisson distribution (Liu 2.6 | Annotation of repetitive elements et al., 2013). We therefore calculated the genome size of LMB using the following equation: G = k-mer_number/k-mer_depth, where G Repetitive elements in the final assembly were annotated using represents the genome size, the k-mer_number represents the total the following two different strategies. (i) de novo annotation: number of k-mers, and the k-mer_depth is the peak of k-mer accumu- RepeatModeller v1.0.8 (Tarailo-Graovac & Chen, 2009) and ltr _ lation (Liu et al., 2013; Zhang et al., 2018). finder v1.0.6 (Xu & Wang, 2007) were used to build a local repeat

We employed Canu V1.7.1 (Koren et al., 2017) to assemble the reference. Subsequently, the genome assembly was aligned with LMB genome using the PacBio long reads. After the primary assem- this reference to annotate the de novo predicted repeat elements bly, we applied the Illumina short-insert reads to polish the genomic using RepeatMasker v4.0.6 (Chen, 2004). (ii) Homology annotation: data by operating Pilon V1.22 (Walker et al., 2014) for temporary Our genome assembly was searched in the RepBase v21.01 (Jurka assembly. Detailed software parameter settings were in Table S1. et al., 2005) using RepeatMasker v4.0.6 and RepeatProteinMask v4.0.6. Finally, these data from the two strategies were integrated to generate a nonredundant data set of repetitive elements in the final 2.4 | Correction, ordering, and orientation of the LMB genome assembly. temporary assembly with Hi-C data

To obtain a chromosome-level genome assembly of LMB, we em- 2.7 | Gene prediction and functional annotation ployed the Hi-C technology to process the temporary assembly (Belton et al., 2012). Blood samples from the same fish were fixed in Three routine methods were used to predict the final LMB gene set. formaldehyde. The restriction enzyme (Mbo I) was added to digest (i) ab initio annotation: First, we used “N” to replace the genomic the DNA, followed by repairing the 5′ overhangs with a biotinylated repetitive elements in our final assembly. augustus v2.5 (Stanke residue. A paired-end library with insert sizes of ~300 bp was con- et al., 2006) and genscan v1.0 (Burge & Karlin, 1997) were em - structed. The Hi-C library was sequenced on a BGISeq-500 platform ployed to annotate gene models. (ii) Homologous-gene-based an- (BGI). Finally, we generated a total of 70.93 Gb (~76×) raw reads notation: Protein sequences of zebrafish (Danio rerio), spotted gar (150 bp in length). (Lepisosteus oculatus), medaka (Oryzias latipes), Japanese puffer Detailed data processing procedures were described briefly as (Takifugu rubripes), green spotted puffer (Tetraodon nigroviridis), follows. (i) The paired-end Illumina reads were mapped onto the pol- Nile tilapia (Oreochromis niloticus), Southern platyfish (Xiphophorus ished temporary genome assembly using Hic-Pro V2.8.0 (Servant maculatus), and three-spined stickleback (Gasterosteus aculeatus) et al., 2015) with default parameters to filter the raw Hi-C reads. were downloaded from the Ensembl database (release version 90). 304 | SUN et al.

Meanwhile, protein sequences of European seabass (Dicentrarchus 10 -s 0.34” to identify the one-to-one orthologous proteins within labrax) and Asian seabass (Lates calcarifer) were downloaded from the 14 examined species. NCBI and the Asian Seabass Genome Project Database (http:// Subsequently, MrModeltest2 (https://github.com/nylander/ laszlo.tll.org.sg/asb_genome/), respectively. These sequences were MrModeltest2) was employed to obtain the best nucleotide substi- mapped onto our final LMB genome assembly to generate best- tution model. MrBayes v3.1.2 (Ronquist et al., 2012) with a gener - hit alignments using the traditional TBLASTN program (McGinnis & ation of 1,000,000 was applied to build the phylogenomic tree of Madden, 2004). Subsequently, GeneWise v2.2.0 (Birney et al., 2004) the 15 examined species. MCMCtree of the paml package (Yang & was used to predict the potential gene structure of each best-hit Rannala, 2006) was used to calculate the divergence times among alignment. (iii) Transcriptome-based annotation: Total RNA was ex- LMB and 14 other fish species. tracted from four tissues of the same LMB, including spleen, liver, muscle, and stomach tissues. Integrity of the RNAs was determined using an Agilent 2100 Bioanalyser (Agilent Technologies). Total RNA 2.9 | Gene family expansion and contraction samples with RIN values ≥8 were used to construct cDNA libraries for subsequent PacBio sequencing. We obtained 25,580,106 Regarding the phylogenomic tree described in the divergence time raw reads, which were aligned to the reference LMB genome using estimation, we used CAFE (Computational Analysis of Gene Family TopHat2.1.1. (Trapnell et al., 2009). Gene transcriptions were quan- Evolution, version 2.1; De Bie et al., 2006) to detect gene family ex- tified using cufflinks version 2.2.1 (http://cole-trapnell-lab.github. pansion and contraction in the assembled LMB genome with default io/cufflinks/) with the gene transfer format (GTF) annotation file. parameters.

Following the three above-mentioned procedures, we used GLEAN (Elsik et al., 2007) to integrate the results into a final gene set. This gene set was searched in five public functional databases, including 2.10 | Positively selected genes (PSGs) NCBI Nr (nonredundant protein sequences), Swiss-Prot (Boeckmann et al., 2003), TrEMBL (Boeckmann et al., 2003), Interpro (Hunter PSGs in the LMB genome were detected from one-to-one ortholo- et al., 2009), and Kyoto Encyclopedia of Genes and Genomes (KEGG; gous genes, in which the LMB was used as the foreground branch, Kanehisa & Goto, 2000), to identify the potential functional motifs and the European seabass, Asian seabass, spotted seabass, and and domains with BLASTP(McGinnis & Madden, 2004). three-spined stickleback were used as the background branches.

To detect positive selection genes, we used BLASTP based on the bi- directional best hit (BBH) method to screen out 3,454 one-to-one 2.8 | Phylogenomic analysis orthologous genes among largemouth bass, European seabass, Asian seabass, and spotted seabass. The multiple alignment was

We downloaded the protein sets of 14 teleost species (Table S2) performed by the Muscle software (http://www.drive5.com/muscl from public databases for phylogenomic analysis (Table 1). Then, we e/), and Gblocks (http://molevol.cmima.csic.es/castresana/Gbloc used BlASTP (McGinnis & Madden, 2004) to determine the best-hit of ks.html) was applied to polish the alignments. LMB was used as each protein, and Hcluster_sg (Li et al., 2006) with a parameter of “-w the foreground branch, and the European sea bass, Asian seabass,

TABLE 1 Comparison of the genome Largemouth Spotted European Asian assemblies between the present Assembly feature bass seabass1 seabass2 seabass3 largemouth bass and three published Size of assembly (Mb) 964 668 675 668 seabass species Contig N50 (kb) 1,227 31 53 1,066 Scaffolds N50 (Mb) 36.48 28.60 26.44 25.85 Anchored pseudo- 89.42 77.68 86 87.8 chromosomes (%) Repeat content (%) 33.79 20.73 21.47 18.6 Annotated protein-coding 23,701 19,215 26,719 22,184 genes Complete BUSCOs (%)* 97.2% 97.0% 96.0% 96.8% a Complete BUSCOs are those matches with the expected range of scores and length alignments in the BUSCO assessment profile. * b Shao et al. (2018). 1 c Tine et al. (2014). 2 d Vij et al. (2016). 3 SUN et al. | 305 spotted seabass, and three-spined stickleback were used as the To ensure the accuracy of subsequent analyses, we cleaned the background branches. The branch-site model incorporated in the raw reads to remove adaptor sequences and low-quality reads (>5% paml package was employed to detect PSGs. The null model used in unknown nucleotides and >50% bases with quality scores in Phred the branch-site test assumed that the comparison of the substitution scale <20). Subsequently, the clean reads were aligned onto the ref- rates at nonsynonymous and synonymous sites (Ka/Ks ratio) for all erence genome using hisat2 (version 2.0.5; Kim et al., 2015) with de- codons in all branches must be ≤1, whereas the alternative model fault parameters. SAM files were sorted using the sort procedure of assumed that the foreground branch included codons evolving at SAMtools (version 1.7; Li et al., 2009). With the program htseq-count Ka/Ks > 1. A maximum likelihood ratio test was used to compare the (version 0.11.2; Anders et al., 2015), we used the sorted SAM files two models. p-values were calculated through the chi-square dis- to generate read counts expressed per gene per library to calculate tribution with 1 degree of freedom (df = 1). The p-values were then the transcription level of each transcript (in fragments per kilobase adjusted for multiple testing using the false discovery rate (FDR) of exon per million mapped reads: FPKM; Roberts et al., 2011). Read method. Genes were identified as positively selected when the counts information was also used for analysing the differential gene FDR < 0.05. Furthermore, we required that at least one amino-acid expression by the r package DESeq2 (version 1.16.1; Love et al., 2014). site possessed a high probability of being positively selected (Bayes A gene was defined as being differentially expressed gene (DEG) probability >95%). If none of the amino acids passed this cutoff in when the Benjamini–Hochberg adjusted p-value was ≤0.05 and the PSGs, then these genes were identified as false positives and ex- fold changes ≥2. GO functional enrichment analysis was performed cluded. GO enrichment was derived with the Fisher's exact test and using the r package GOSeq (version 1.10.0; Young et al., 2010) and chi-square test and then adjusted using the Benjamini–Hochberg topGO (version 2.10.0; Alexa & Rahnenfuhrer, 2010), while KEGG procedure with a cutoff set at p < 0.05. pathway analysis was performed using kobas (version v2.0.12; Xie et al., 2011). DEGs were considered to be significantly enriched in GO terms and metabolic pathways when their Bonferroni-corrected 2.11 | Salinity challenge, sampling, and Illumina p-values were <0.05. transcriptome sequencing

For the salinity challenge experiment, 60 LMB individuals with bod- 3 | RESULTS yweights of 6.43 ± 0.75 g and body lengths of 7.5 ± 0.42 cm were collected from our local breeding base in the Pearl River Fisheries 3.1 | Chromosome-level genome assembly of the Research Institute (Guangzhou, China). They were randomly divided largemouth bass into a control group and a salinity treated group. The salinity of the latter group was set at 12.5‰ (brackish water), and in the control We generated 82.01 gigabases (Gb) of PacBio sequencing reads group it was set at 0‰ (freshwater). Each group consisted of three from eight sequencing cells. The average N50 of these reads was replicates with 10 individuals in each replicate. Fishes were culti- 11,671 bp (Table S3). We also generated 183.2 Gb of Illumina short- vated in two 200 L aquaria under the same culturing conditions insert reads for polishing the primary PacBio assembly (Table S4). (water temperature maintained at 28°C; feeding twice per day with Following the Canu assembly and Pilon polishing, we produced 7,662 commercial feed). contigs for the LMB with contig N50 value of 1.23 Mb (Table S5), Three individuals from each replicate were randomly sampled which was longer than that of other bass species (more details of the on day 30 of the experiment. They were anaesthetized with a buff- comparison are shown in Table 1, Table S6). ered solution of tricaine methanesulphonate (MS-222 100 mg/ml; A total of 336 million raw reads (ca. 70.93 Gb) were generated Finquel). Gills and livers from each set of three fishes were pooled from the Hi-C sequencing. After removal of low-quality reads, we to reduce individual variation. In total, six pools composed of three obtained 121 million of clean reads for further analysis. We finally control samples and three challenged samples were eventually ob- applied the iterative clustering method in 3D-DNA (Dudchenko tained for RNA extraction. Extraction of total RNA was carried out et al., 2017) to cluster and order 2,591 contigs into super-scaffolds. with a TRIZOL Kit (Invitrogen) according to the manufacturer's in- The first 23 super-scaffolds are treated as the chromosomes of LMB structions, and then treated by DNase I to digest the DNA for ob- (Figure S1 and Table 1), which is consistent with a previous karyo- taining pure RNA products. Electrophoresis in 1.5% agarose gel type report (n = 23) for LMB (Hu et al., 1989). To our knowledge, was run to check the integrity of the RNA products. A NanoDrop this is the first high-quality chromosome-level genome assembly for 2000 Spectrophotometer (Thermo Scientific) set at an absor- LMB with 964 Mb genome size and scaffold N50 of 36.48 Mb. A bency of 260 nm was used to assess RNA quality and quantity. An total of 862 Mb (89.42%) were anchored to the 23 chromosomes Illumina Tru-Seq RNA Sample Preparation Kit (Illumina) was used to (Chr), although the remaining 102 Mb were unanchored and required generate the sequencing libraries according to the manufacturer's further investigation (Figure 1 and Table 1). protocol. After purification, these samples were sequenced on an Our BUSCO results showed that the genome module bench- Illumina Hiseq-2500 platform to generate 150 bp paired-end reads marking value was C: 97.2%, including S: 91.2%, D: 6.0%, F: 0.9%, at Novogene (Beijing, China). M: 1.9%, and n = 4,584 (C: complete, S: single-copy, D: duplicated, F: 306 | SUN et al.

FIGURE 1 The chromosome-level genome assembly and Circos atlas of the largemouth bass. (a) A photo of the sequenced largemouth bass. (b) A 17-mer distribution of the Illumina short reads for genome size estimation. The x-axis represents the sequencing depth of each unique 17-mer, and the y-axis represents the percentage of unique 17-mers. The peak depth was confirmed at 58. The yellow dot lines is a standard Poisson distribution. The total 17-mer number and the peak of 17-mer depth were 54,463,419,600 and 58, respectively. Therefore, we estimated that the genome size of largemouth bass was 939 Mb. (c) A Circos atlas of the chromosomal genome of the largemouth bass. From the outside to inside rings: (I) Chromosomes (Mb in length); (II) Distribution of gene density (in green colour) within 100 kb nonoverlapping windows; (III) Distribution of genomic GC content in 100 kb nonoverlapping windows; (IV) Distribution of repetitive elements (REs, in various colours) in 100 kb nonoverlapping windows. The most internal syntenic blocks are connected with lines, which were searched with i-Adhore 3.0 [Colour figure can be viewed at wileyonlinelibrary.com]

fragmental, M: missed, and n: total BUSCO groups of Actinopterygii_ element is normally identified at the end of a chromosome; however, odb9 data set for searching). The BUSCO assessment results vali- in the LMB Chr1 there is an additional segment with high-density of dated the high quality of our final genome assembly. Additionally, telomere elements, which is far away from either end of the special the variations in GC content between the genomes of LMB and other chromosome. The additional telomer region is located in the 53 Mb species revealed no sequencing-based GC preferences (Figure S2), of the Chr1, which is far away from both ends of this chromosome. indicating the high quality of our genome assembly (i.e., no contami- nation of prokaryote sequences). 3.3 | Annotation and comparative analyses of the largemouth bass genome 3.2 | Chromosomal fusion of the largemouth bass Repetitive elements accounted for 33.79% of the whole genome as- Using the Hi-C data, we constructed a haplotype map that spans 23 sembly. Detailed percentages of the predominant repetitive element chromosomes of LMB. Unlike other closely related Perciformes (with families are summarized in Table S7. We annotated a total of 23,701 the popular haploid chromosome number of 24), LMB has a haploid genes in the assembled genome with anchoring of 21,602 genes chromosome number of 23. Chr1 is the largest chromosome in LMB, onto the assembled chromosomal regions (Tables S8 and S9 and spanning more than 64 million bases (Mb). We confirmed that Chr1 Figure S3). BUSCO benchmarking value of this gene set was summa- results from an end-to-end fusion of two ancestral chromosomes. rized as C: 89.2%, including S: 82.9%, D: 6.3%, F: 7.1%, M: 3.7%, and Evidence for this conclusion includes: (i) correspondence of the Chr1 n = 4,584. Following a standard functional annotation, we observed to two chromosomes in closely related species, such as Chr20 and that 22,840 (96.37%) genes were annotated with at least one related Chr24 in the spotted seabass, and Chr18-21 and Chr24-25 in the functional assignment (Table S10 and Figure S4). Meanwhile, the European seabass (Figure 2a); and (ii) there is a high-density of tel- annotated transcripts from our transcriptomic data were realigned omere elements in the conjunction of two ancestral chromosomes onto the genome assembly, and thereby we predicted that 99.26% (within the 24th Mb region; Figure 2b). In general, the telomere of the gene regions were covered (Table S11), compared with the SUN et al. | 307

FIGURE 2 Chromosomal fusion in the largemouth bass (LMB). (a) Chromosomal syntenic relationships between the LMB and spotted sea bass or European sea bass. The syntenic blocks were calculated by MCscan. Macrosynteny connecting blocks >20 one-to-one gene pairs are shown. (b) Additional telomere element in the LMB Chr1. The upper section shows the genomic overview of the Chr1. Major DNA elements are clustered into exons, introns, repetitive elements, and other sequences. All clusters were determined for 100 kb windows. The lower section shows the detailed localization of the telomere element in the Chr1 with the same sliding windows. The identified telomere sequence is TTAGGG [Colour figure can be viewed at wileyonlinelibrary.com] 308 | SUN et al.

90.0% aligning rate of the transcriptomic data directly mapping to significantly (p < 0.05) changed (Table S13). Notably, LMB exhib- the genome assembly. These results also support the high accuracy ited the expansion of genes related to ion channel activity (GO: and completeness of our final genome assembly. 0005216), ion transport (GO: 0006811), transmembrane transport In the present study, we determined 872 one-to-one ortholo- (GO: 0055085), and voltage-gated potassium channel activity (GO: gous genes between LMB and 14 other teleost species (Figure 3, 0005249) (Table S14), among which potassium voltage-gated chan- Figures S5 and S6 and Table S2). LMB is phylogenetically closely re- nels showed the greatest extent of gene family expansion, followed lated to European seabass and spotted seabass, diverging 64.1 mya by voltage-dependent L-type calcium channels, and sodium volt- from the two seabass species, with a confidence interval of 49.4– age-gated channels (Table S15). In addition, there were three copies 80.6 mya (Figure 3). Our phylogenetic analysis placed European of the fatty acid desaturase 2 (Fads2) gene (Table S15), which were im- seabass as the sister to spotted seabass, which is consistent with portant for salinity adaptation (Ishikawa et al., 2019). They were de- previous research (Chen et al., 2019). termined to be localized on different chromosomes, and the locations The analysis of gene families determined that LMB has 167 unique of these three Fads2 in our LMB assembly are simplified as Ms_hic_ gene families (in comparison to these examined fishes in the present scaffold_3725:2857-8384, Ms_hic_scaffold_8:16761961-16770968, study) consisting of 566 genes, which are enriched for the following and Ms_hic_scaffold_20:10547844-10553382. However, there was four gene ontology (GO) terms: protein kinase activity, ATP binding, only one copy of Fads2 in the published spotted seabass, European protein phosphorylation, and ubiquitin-protein transferase activity. seabass and Asian seabass. Meanwhile, these included multiple genes associated with ion trans- Using LMB as the foreground branch, European seabass, Asian port, such as sodium-channel proteins, sodium-driven chloride bicar- seabass, spotted seabass and three-spined stickleback as the back- bonate exchanger (NDCBE)-like proteins, voltage-dependent T-type ground branches, we incorporated the branch-site model in the calcium channels, voltage-dependent P/Q-type calcium channels, cin- paml package to detect positively selected genes (PSGs). A total of gulin, and ATP-binding cassette sub-family members (Table S12). 387 PSGs were identified in the LMB (p < .05), which were espe- Compared with the common ancestor of bass species, LMB cially enriched in GO terms related to “transmembrane receptor presented 120 gene family expansion events and 109 gene fam- protein tyrosine kinase signalling pathway”, “ephrin receptor activ- ily contraction events (p <0 .05, Figure S7). Among these families, ity”, “protein tyrosine kinase activity”, and “ATP binding”. PSGs en- 120 expanded gene families and 109 contracted gene families were riched in GO terms related to “ATP binding” included sodium- and

FIGURE 3 Phylogenomic tree and comparative BUSCO assessments of the largemouth bass (LMB) and 14 other fish species. The left section shows the evolutionary position of LMB. The red dot nodes have been validated by the records of the TimeTree (http://www.timet ree.org/). Each blue number set represents the estimated divergence time with 95% confidence intervals. The right section summarizes the results of the BUSCO assessment of each genome assembly [Colour figure can be viewed at wileyonlinelibrary.com] SUN et al. | 309 chloride-dependent GABA transporter, sodium/potassium-trans- sequencing of LMB, yielding the first chromosome-level genome porting ATPase, and potassium/sodium hyperpolarization-activated and high-quality genome annotations for this economically im- cyclic nucleotide-gated channel 2. It is worth noting that these PSGs portant species. Using the Hi-C sequencing, we obtained 121 mil- included claudins, which regulate the extent of tight junctions among lion raw reads and finally clustered 2,591 contigs into 23 groups, cells (Tables S16-S18). which is consistent with a previous karyotype analysis of LMB (Hu et al., 1989). The chromosomal-level genome assembly for LMB may serve as an important resource for future ecological and evo- 3.4 | Gene duplication and evolution for genetic lutionary analysis, germplasm conservation, mapping of traits with adaptation to both fresh and brackish water economic importance, and genetic improvements for LMB, as well as provide important reference information for studying other In the LMB genome, we predicted some expanded gene families with economically important fish species. essential roles in adaptation to salinity, and found eight different It was hypothesized that the ray-finned fishes have a fresh- gene families comprising 294 genes associated with ionic regulation. water or brackish origin, which is supported by certain molecular These included claudin, aquaporins, fatty acid desaturase 2 (Fads2), phylogenetic analysis (Carrete Vega & Wiens, 2012). Likelihood re- sodium channel protein type, potassium voltage-gated channel, cal- constructions of the time-calibrated phylogeny of 22 major clades cium voltage-gated channel, sodium- and chloride-transporter, so- in actinopterygians suggested that the common ancestor of living dium/calcium exchanger, and sodium/potassium-transporting. Our actinopterygians occurred in freshwater (either partially or exclu- gene mapping analysis showed that these genes are distributed on sively). In addition, the phylogeny clearly shows that there were re- the 23 chromosomes in a scattered way (Figure S8). peated invasions of freshwater habitats by marine ancestors within LMB has 68 claudin copies (Figures S9, S10a and S11), as the larg- the Percomorpha (accounting for ~33% of all freshwater species) est copy number among the 15 examined fishes, including euryha- ~100 mya (Carrete Vega & Wiens, 2012). These findings indicate a line and freshwater species. The other 14 fish species have 56–67 complicated history of transitions between sea and fresh water in claudin copies. Chromosomal blocks contain claudin duplications in the evolutionary history of ray-finned fishes. Chr3 (18 claudin genes) and Chr9 (14 claudins; Figure S9b,c). We also Comparative genomics analysis of 14 species of Osteichthyes detected the syntenic blocks of claudins among spotted seabass, (including spotted seabass, Europeansea bass, Asian seabass, European seabass and three-spined stickleback, but some copies Gasterosteiformes, Tetraodontiformes, Pleuronectiformes, and were lost in these fishes. Lepisosteiformes) found that LMB had the most recent divergence from European seabass and spotted seabass (64.1 mya), followed by three-spined stickleback (Gasterosteiformes) (69.6 mya) and 3.5 | Transcriptome changes in the largemouth bass Asian seabass (Perciformes) (91.8 mya). We previously (Hughes after exposure to salinity treatments et al., 2018) performed a similar phylogenetic analysis on the transcriptome and genome sequencing data of 303 ray-finned fishes The transcriptome sequencing of LMB reared in fresh (0‰) and (Actinopterygii), designing to trace the effect of whole-genome brackish water (12.5‰) for 30 days yielded 99.61 Gb of clean data, duplication events on gene trees and find paralogy-free loci using a of which 91.67% can be mapped to the assembled genome. In ad- bioinformatics approach. Our results in the present study showed dition, the transcriptions of 17,530 (73.96%) genes were detected that LMB is relatively closely related to European seabass and in at least one of the samples (FPKM ≥ 1). There were 121 and 34 spotted seabass. Interestingly, these more closely related species DEGs in the gill and liver tissues of LMB in the brackish water group (i.e., European seabass, spotted seabass, three-spined stickleback, compared with the freshwater group, respectively (Table S19). There and Asian seabass) are euryhaline fishes that can live in fresh, were 74 upregulated genes and 47 downregulated genes in the gill brackish, and seawater; whereas LMB is primarily distributed in tissues of LMB in the brackish water group, among them the tran- freshwater and can also be found in brackish water affected by scription levels of potassium voltage-gated channels, sodium-driven the tide (1‰–12‰ salinity) along the North American coast and chloride bicarbonate exchangers, claudins, and aquaporins increased the Gulf of Mexico, but LMB is unable to tolerate high levels of sa- significantly (Padj ≤ 0.05). linity. Phylogenetically informed studies have demonstrated that freshwater species diversified from euryhaline ancestors through processes such as landlocking (Schultz & McCormick, 2012). Some 4 | DISCUSSION euryhaline taxa are particularly susceptible to changes in halohab- itat and subsequent diversification, and some geographic regions 4.1 | Relationship of largemouth bass to other represent hotspots for the transition to freshwater (Schultz & Perciform fishes McCormick, 2012). Based on the genome-wide evolutionary analysis in the present study, we speculate that LMB shares a common In the present study, the third-generation PacBio sequenc- ancestor with European seabass and spotted sea bass and gradu- ing combined with Hi-C approach was used for whole-genome ally evolved into a freshwater fish. 310 | SUN et al.

FIGURE 4 Schematic diagram of proposed ion regulation gene network in the largemouth bass. These genes are involved in two mechanisms of ion transport: (1) facilitated diffusion driven by the concentration gradient of ions, including claudin, aquaporins, sodium channel protein, potassium voltage-gated channel, and calcium voltage-gated channel; (2) active transport of ions against the concentration gradient, including sodium- and chloride- transporter, sodium/calcium exchanger, and sodium/potassium-transporting ATPase [Colour figure can be viewed at wileyonlinelibrary.com]

4.2 | Molecular mechanisms for LMB to adapt to without the need for energy consumption, including claudins, aqua- brackish water porins, sodium channel proteins, potassium voltage-gated channels, and calcium voltage-gated channels. Facilitated diffusion can be fur- A previous genome analysis revealed that European seabass adapt ther divided into paracellular and transcellular based on the route to high salinity fluctuations by expanding gene families associated of ion transport. Paracellular transport of ions and water is mainly with ion and water regulation (Tine et al., 2014). To adapt to brackish controlled by claudins that regulate the extent of tight junctions be- water with a certain level of salinity, LMB requires a comprehen- tween cells. Transcellular transport involves the transportation of sive ionic regulatory network to maintain its osmotic homeostasis ions and water via various ion channels and aquaporins. (ii) Other by controlling the uptake or excretion of ions and water under dif- genes are involved in the active transport of ions against the con- ferent levels of osmotic pressure. Analysis of contraction and expan- centration gradient with assistance of energy consumption, includ- sion, positive selection, and transcriptomes in the genome of LMB ing sodium- and chloride- transporters, sodium/calcium exchangers, identified eight gene families comprising 294 genes associated with and sodium/potassium-transporting ATPase. Comparative genome ionic regulation, such as claudins, aquaporins, and sodium channel analysis of the published genomes of 14 Osteichthyes species fo- proteins. These genes can be divided into two categories based on cusing on these eight mentioned gene families found that claudins, the mode of ion transport (i) Certain genes involved facilitated diffu- sodium/calcium exchangers, and potassium voltage-gated channels sion, i.e., passive transport driven by a concentration gradient of ions have the highest copy numbers in LMB. The remaining five gene SUN et al. | 311 families also have relatively high numbers of gene copies, which may in looser tight junctions in the gills and facilitated the expulsion of provide molecular evidence for the adaptation of LMB to brackish water molecules from the body. This is a key aspect of osmoregu- water (Figure 4) (Madsen et al., 2015; Sundell & Sundh, 2012). lation in freshwater. The expression of claudin-10 homologues was European seabass has previously been shown, via gene family significantly upregulated in the euryhaline mummichog (Fundulus expansion and contraction analysis, to have undergone signifi- heteroclitus) transferred from freshwater to seawater or from seawa- cant expansion of gene families involved in intracellular, olfactory ter to hypersaline seawater (Marshall et al., 2018). receptor activity, G-protein coupled receptor signalling pathway, Our study on LMB identified two PSGs encoding claudins, i.e., and G-protein coupled receptor activity (Tine et al., 2014). Spotted Claudin tight junction (Ms.hic_GLEAN_10009003) and Claudin-7-A seabass has also undergone significant expansion of gene fami- (Ms.hic_GLEAN_10015140). There were three significantly upreg- lies involved in DNA integration, transposition, DNA-mediated, ulated claudin genes in the gill tissues of LMB after exposure to olfactory receptor activity, and NAD(P)+-protein-arginine ADP- 12.5‰ salinity for 30 days (p < 0.01), indicating that LMB tolerates ribosyltransferase activity (Shao et al., 2018). Three-spined stickle- certain levels of salinity by up-regulating the expression of claudin back has undergone significant expansion of gene families involved genes in gill tissues to increase the extent of tightness of junctions in nucleosome, intracellular, DNA binding, and olfactory receptor and reduce the loss of water. The high expression in LMB in response activity (Jones et al., 2012). Our study found that LMB had under- to salinity stresses may play an important role in adaptation to sa- gone significant expansion of gene families involved in ion trans- linity variations. port, such as ion channel activity, ion transport, transmembrane Taken together, the gene family contraction and expansion transport, and voltage-gated potassium channel activity, especially analysis and positive selection analysis of the LMB genome and the in channels used for sodium, potassium, and chloride ion transport. transcriptome profiling of LMB exposed to salinity treatment, sug- Ions move from an area of higher concentration to an area of lower gest that LMB can regulate the extent of tight junctions between concentration via facilitated diffusion with the aid of ion channels cell membranes via multiple members of the claudin gene family to in cell membranes (Armstrong & Hollingworth, 2018). Subsequent control the flow of water and solutes across the intercellular space. positive selection analysis that compared LMB to four species of eu- In the presence of a concentration gradient across cell membranes, ryhaline fishes (European seabass, Asian seabass, Spotted seabass, ions may be transported via facilitating diffusion using the ion chan- and three-spine stickleback) yielded 387 PSGs, which were mainly nels in cell membranes (passive transport). However, ions may also enriched for ATP binding and transmembrane receptor protein tyro- be actively transported by sodium and potassium pumps in cell sine kinase signalling pathways. Genes in the ATP binding pathway membranes at the expense of ATP for salinity adaptation. Therefore, provide necessary energy for the active transport of ions across cell despite being considered as a freshwater fish, LMB can also inhabit membranes (Melkikh & Seleznev, 2012). in brackish water at certain levels of salinity. Claudins are a family of transmembrane proteins and important components of tight junctions between cells. They tightly join the membranes of adjacent cells to prevent the flow of molecules across 4.3 | Chr1 of the largemouth bass is a putatively the intercellular space into cell, thereby regulating the extent of tight fused chromosome junctions between cells and the permeability to water and solutes (Baltzegar et al., 2013; Marshall et al., 2018; Mineta et al., 2011). Using Hi-C data we constructed 23 chromosome sequences of LMB, Tight junctions also close or seal the space between cells and thus corresponding to the karyotype of the species (Hu et al., 1989). This set up a semipermeable barrier to prevent or reduce paracellular dif- number of known Perciformes usually ranges from 21 to 24, most of fusion (“barrier function”; Findley & Koval, 2009). Changes in tight which are telocentric chromosomes. European seabass and spotted junction morphology may be important for minimizing diffusive seabass, which are the most closely related with LMB, have a haploid fluxes of water and ions in freshwater, while allowing for Na+ and chromosome number of 24 (Shao et al., 2018; Tine et al., 2014). The other ion counterion diffusion in seawater or hypersaline conditions comparative genomics analysis revealed that the Chr1 of LMB cor- (Laverty & Skadhauge, 2012). Claudins are four-transmembrane pro- responds to Chr20 and Chr24 of spotted seabass and Chr18–21 and teins acting to collectively regulate the paracellular movement of Chr24–25 of European seabass. Meanwhile, there is a special region water and ions across cellular tight junctions in vertebrate tissues with a high density of telomeres (usually found at the ends of chro- (Baltzegar et al., 2013). Some claudins form tight junction-associated mosomes) in the upper and middle segments of the Chr1 of LMB, pores allow paracellular ion transport (Anderson & Van Itallie, 2009). indicating that this chromosome may have resulted from a fusion Claudins play an important regulatory role in the adaptation of of two telocentric chromosomes, and one of the centromeres may fishes to salinity via tight junctions (Tipsmark et al., 2016). Tilapia lost or degraded. These results indicate that the fusion is a relatively transferred from freshwater to seawater showed increased tran- recent evolutionary event, but the exact time of this fusion event scription levels of claudins, Na+-K+-ATPase, and Na+-K+-2Cl- cotrans- still needs more in-depth investigations. A similar observation was porters with increased plasma osmotic pressure (Tipsmark et al., reported in human. In fact, the human chromosome 2 is a product 2016). On the contrary, tilapia transferred from seawater to fresh- of a telomere fusion of two ancestral chromosomes and the loss/ water showed decreased transcription levels of claudins, resulting degeneration of one of the two original centromeres (Miga, 2017). 312 | SUN et al.

Variations in chromosomal arrangement within and among These data were also deposited in the NCBI under accession number species are ubiquitous in nature and play important roles in adap- PRJNA640112. tation and reproductive isolation (Dobigny et al., 2017; Guerrero & Kirkpatrick, 2014). Molecular genomic studies have suggested ORCID that chromosomal rearrangement plays a key role in speciation and Chengfei Sun https://orcid.org/0000-0002-7390-4241 has been instrumental in the evolution of genomes (Guerrero & Qiong Shi https://orcid.org/0000-0002-6358-976X Kirkpatrick, 2014; Heslop-Harrison, 2012; Sankoff & Nadeau, 2003). Xing Ye https://orcid.org/0000-0003-3456-9839 A significant overrepresentation of a large (~5 Mb) chromosomal rearrangement in a population of Atlantic cod, which was previously REFERENCES considered to comprise genes critical for survival at low salinity, Alexa, A., & Rahnenfuhrer, J. (2010). topGO: Enrichment analysis for gene suggests local adaptation to the environment may be enabled by ontology. R package version.2, (45–46). Anders, S., Pyl, P. T., & Huber, W. (2015). HTSeq—A Python framework the suppression of recombination in the rearranged region (Barth to work with high-throughput sequencing data. Bioinformatics, 31(2), et al., 2017). A similar phenomenon was also observed in Atlantic 166–169. https://doi.org/10.1093/bioinformatics/btu638 salmon populations in the Miramichi River, North America. Fusion Anderson, J. M., & Van Itallie, C. M. (2009). Physiology and function of of chromosomes 8 and 29 and life-history-associated genomic vari- the tight junction. Cold Spring Harbor Perspectives in Biology, 1(2), a002584. https://doi.org/10.1101/cshperspect.a002584 ations contribute to the within-river local adaptation of Atlantic Armstrong, C. M., & Hollingworth, S. (2018). A perspective on Na and salmon (Lehnert et al., 2018; Wellband et al., 2019). K channel inactivation. Journal of General Physiology, 150(1), 7–18. In this study, genes associated with LMB adaptation to salinity https://doi.org/10.1085/jgp.201711835 were investigated. We identified nine families comprising 277 as- Bailey, R. M., Winn, H. E., & Smith, C. L. (1954). Fishes from the Escambia sociated genes on the assembled 23 chromosomes, among which River, Alabama and Florida, with ecologic and taxonomic notes. Proceedings of the Academy of Natural Sciences of Philadelphia, 106, there were six chromosomes containing at least 20 associated genes. 109–164. There were 20 genes associated with salinity adaptation on the Chr1, Bain, M. B., & Boltz, S. E. (1992). Effect of aquatic plant control on the which seems to be resulted from a chromosomal fusion event and microdistribution and population characteristics of largemouth bass. thus carries an additional telomere and centromere compared with Transactions of the American Fisheries Society, 121(1), 94–103. https:// doi.org/10.1577/1548-8659(1992)1212.3.CO;2 the nonfused chromosomes. Telomeric and centromeric interphase Baltzegar, D. A., Reading, B. J., Brune, E. S., & Borski, R. J. (2013). chromatins could easily become heterochromatins, which are poly- Phylogenetic revision of the claudin gene family. Marine Genomics, condensed chromatin fibers with higher levels of folding and com- 11, 17–26. https://doi.org/10.1016/j.margen.2013.05.001 pression that can inhibit the expression of nearby genes (Schoeftner & Barth, J. M. I., Berg, P. R., Jonsson, P. R., Bonanomi, S., Corell, H., Hemmer- Hansen, J., Jakobsen, K. S., Johannesson, K., Jorde, P. E., Knutsen, Blasco, 2009). The role of chromosome fusion on adaptation to fresh H., Moksnes, P.-O., Star, B., Stenseth, N. C., Svedäng, H., Jentoft, S., and brackish water in LMB remains to be further explored. & André, C. (2017). Genome architecture enables local adaptation of Atlantic cod despite high connectivity. Molecular Ecology, 26(17), ACKNOWLEDGEMENTS 4452–4466. https://doi.org/10.1111/mec.14207 Barthel, B. L., Lutz-Carrillo, D. J., Norberg, K. E., Porak, W. F., Tringali, The project was supported by China Agriculture Research System M. D., Kassler, T. W., Johnson, W. E., Readel, A. M., Krause, R. A., (No. CARS-46), Central Public-interest Scientific Institution Basal & Philipp, D. P. (2010). Genetic relationships among populations of Research Fund, CAFS (2020TD23), Youth Program of National Florida Bass. Transactions of the American Fisheries Society, 139(6), Natural Science Foundation of China (No. 31902354), Central 1615–1641. https://doi.org/10.1577/T09-185.1 Belton, J. M., McCord, R. P., Gibcus, J. H., Naumova, N., Zhan, Y., & Public-interest Scientific Institution Basal Research Fund of the Dekker, J. (2012). Hi-C: A comprehensive technique to capture the Chines Academy of Fishery Sciences (No. 2017HY-XKQ0208), and conformation of genomes. Methods, 58(3), 268–276. https://doi. Special Fund for Scientific Research in Public Welfare and Capacity org/10.1016/j.ymeth.2012.05.001 Building of Guangdong Province (No. 2017A030303002). Bian, C., Huang, Y. U., Li, J., You, X., Yi, Y., Ge, W., & Shi, Q. (2019). Divergence, evolution and adaptation in ray-finned fish genomes. Science China Life Sciences, 62(8), 1003–1018. https://doi. AUTHOR CONTRIBUTIONS org/10.1007/s11427-018-9499-5 X.Y., C.S., and Q.S. designed the research. C.S., J.D., J.H., W.L., and Birney, E., Clamp, M., & Durbin, R. (2004). GeneWise and genomew- Y.T. collected the sample and performed the experiment. Jia L., Y.N., ise. Genome Research, 14(5), 988–995. https://doi.org/10.1101/ C.S., Jinmin L., Jiang L. and J.D. analysed the data. C.S., X.Y., J.L., gr.1865504 Boeckmann, B., Bairoch, A., Apweiler, R., Blatter, M. C., Estreicher, A., Y.N., J.D., and Q.S. wrote the paper. All the authors participated in Gasteiger, E., & Pilbout, S. (2003). The SWISS-PROT protein knowl- the discussion. All authors read and approved the final manuscript. edgebase and its supplement TrEMBL in 2003. Nucleic Acids Research, 31(1), 365–370. https://doi.org/10.1093/nar/gkg095 DATA AVAILABILITY STATEMENT Boutet, I., Ky, C. L., & Bonhomme, F. (2006). A transcriptomic approach of salinity response in the euryhaline teleost, Dicentrarchus labrax. The largemouth bass genome assembly and the sequencing data Gene, 379, 40–50. https://doi.org/10.1016/j.gene.2006.04.011 used for de novo whole-genome assembly are available from the Burge, C., & Karlin, S. (1997). Prediction of complete gene structures China National GenBank (CNGB; https://db.cngb.org/) Nucleotide in human genomic DNA. Journal of Molecular Biology, 268(1), 7894. Sequence Archive (CNSA) under accession number CNP0000894. https://doi.org/10.1006/jmbi.1997.0951 SUN et al. | 313

Carpenter, S. R., & Kitchell, J. F. (Eds.) (1996). The trophic cascade Carolina Rivers. Proceedings of the Southeastern Association of Fish in lakes. Cambridge University Press. https://doi.org/10.104 and Wildlife Agencies, 32, 391–400. 6/j.1365-2400.1998.5403492.x Heslop-Harrison, J. P. (2012). Genome evolution: Extinction, continua- Carrete Vega, G., & Wiens, J. J. (2012). Why are there so few fish in the tion or explosion? Current Opinion in Plant Biology, 15(2), 115–121. sea? Proceedings of the Royal Society B: Biological Sciences, 279(1737), https://doi.org/10.1016/j.pbi.2012.03.006 2323–2329. https://doi.org/10.2307/41549542 Hirai, N., Tagawa, M., Kaneko, T., Seikai, T., & Tanaka, M. (1999). Chen, B., Li, Y., Peng, W., Zhou, Z., Shi, Y., Pu, F., Luo, X., Chen, L., & Distributional changes in branchial chloride cells during freshwater Xu, P. (2019). Chromosome-level assembly of the Chinese seabass adaptation in Japanese sea bass Lateolabrax japonicus. Zoological (Lateolabrax maculatus) genome. Frontiers in Genetics, 10, 275. https:// Science, 16(1), 43–49. https://doi.org/10.2108/zsj.16.43 doi.org/10.3389/fgene.2019.00275 Hu, H., Wu, G., Zhu, X., & Chen, K. (1989). Karyotype of Micropterus sal- Chen, N. (2004). Using Repeat Masker to identify repetitive elements moides. Freshwater Fisheries, 4, 21–23. in genomic sequences. Current Protocols in Bioinformatics, 5(1), 4–10. Hughes, L. C., Ortí, G., Huang, Y. U., Sun, Y., Baldwin, C. C., Thompson, https://doi.org/10.1093/geront/4 A. W., Arcila, D., Betancur-R., R., Li, C., Becker, L., Bellora, N., Copeland, J. R., & Noble, R. L. (1994). Movements by young-of-year and Zhao, X., Li, X., Wang, M., Fang, C., Xie, B., Zhou, Z., Huang, H., yearling largemouth bass and their implications for supplemental Chen, S., … Shi, Q. (2018). Comprehensive phylogeny of ray-finned stocking. North American Journal of Fisheries Management, 14(1), 119– fishes (Actinopterygii) based on transcriptomic and genomic data. 124. https://doi.org/10.1577/1548-8675(1994)014<0119:MBYOY Proceedings of the National Academy of Sciences, 115(24), 6249–6254. A>2.3.CO;2 https://doi.org/10.1073/pnas.1719358115 De Bie, T., Cristianini, N., Demuth, J. P., & Hahn, M. W. (2006). CAFE: Hunter, S., Apweiler, R., Attwood, T. K., Bairoch, A., Bateman, A., Binns, A computational tool for the study of gene family evolution. D., Bork, P., Das, U., Daugherty, L., Duquenne, L., Finn, R. D., Gough, Bioinformatics, 22(10), 1269–1271. https://doi.org/10.1093/bioin J., Haft, D., Hulo, N., Kahn, D., Kelly, E., Laugraud, A., Letunic, I., formatics/btl097 Lonsdale, D., … Yeats, C. (2009). InterPro: The integrative protein Dekayne, R., Zoller, S., & Feulner, P. G. (2020). A de novo chro- signature database. Nucleic Acids Research, 37(suppl_1), D211–D215. mosome-level genome assembly of Coregonus sp. “Balchen”: https://doi.org/10.1093/nar/gkn785 One representative of the Swiss Alpine whitefish radiation. Ishikawa, A., Kabeya, N., Ikeya, K., Kakioka, R., Cech, J. N., Osada, N., Molecular Ecology Resources, 20(4), 1093–1109. https://doi. Leal, M. C., Inoue, J., Kume, M., Toyoda, A., Tezuka, A., Nagano, org/10.1111/1755-0998.13187 A. J., Yamasaki, Y. Y., Suzuki, Y., Kokita, T., Takahashi, H., Lucek, DeVries, D. R., Wright, R. A., Glover, D. C., Farmer, T. M., Lowe, M. R., K., Marques, D., Takehana, Y., … Kitano, J. (2019). A key metabolic Norris, A. J., & Peer, A. C. (2015). Largemouth bass in coastal estu- gene for recurrent freshwater colonization and radiation in fishes. aries: A comprehensive study from the Mobile-Tensaw River Delta, Science, 364(6443), 886–889. https://doi.org/10.1126/scien Alabama. In Black bass diversity, multidisciplinary science for conser- ce.aau5656 vation. American Fisheries Society symposium (Vol. 82, pp. 297–310). Jensen, M. K., Madsen, S. S., & Kristiansen, K. (1998). Osmoregulation Dobigny, G., Britton-Davidian, J., & Robinson, T. J. (2017). Chromosomal and salinity effects on the expression and activity of Na+, K+-ATPase polymorphism in mammals: An evolutionary perspective. Biological in the gills of European sea bass, Dicentrarchus labrax (L.). Journal of Reviews, 92(1), 1–21. https://doi.org/10.1111/brv.12213 Experimental Zoology, 282(3), 290–300. https://doi.org/10.1002/ Dudchenko, O., Batra, S. S., Omer, A. D., Nyquist, S. K., Hoeger, M., (SICI)1097-010X(19981015)282:3<290:AID-JEZ2>3.0.CO;2-H Durand, N. C., & Aiden, E. L. (2017). De novo assembly of the Aedes Jones, F. C., Grabherr, M. G., Chan, Y. F., Russell, P., Mauceli, E., Johnson, aegypti genome using Hi-C yields chromosome-length scaffolds. J., Swofford, R., Pirun, M., Zody, M. C., White, S., Birney, E., Searle, Science, 356(6333), 92–95. https://doi.org/10.1126/science.aal3327 S., Schmutz, J., Grimwood, J., Dickson, M. C., Myers, R. M., Miller, C. Durand, N. C., Shamim, M. S., Machol, I., Rao, S. S., Huntley, M. H., T., Summers, B. R., Knecht, A. K., … Kingsley, D. M. (2012). The ge- Lander, E. S., & Aiden, E. L. (2016). Juicer provides a one-click system nomic basis of adaptive evolution in threespine sticklebacks. Nature, for analyzing loop-resolution Hi-C experiments. Cell Systems, 3(1), 484(7392), 55–61. https://doi.org/10.1038/nature10944 95–98. https://doi.org/10.1016/j.cels.2016.07.002 Jurka, J., Kapitonov, V. V., Pavlicek, A., Klonowski, P., Kohany, O., & Elsik, C. G., Mackey, A. J., Reese, J. T., Milshina, N. V., Roos, D. S., & Walichiewicz, J. (2005). Repbase Update, a database of eukaryotic Weinstock, G. M. (2007). Creating a honey bee consensus gene set. repetitive elements. Cytogenetic and Genome Research, 110(1–4), Genome Biology, 8(1), R13. https://doi.org/10.1186/gb-2007-8-1-r13 462–467. https://doi.org/10.1186/s13100-015-0041-9 FAO (2017). The state of world fisheries and aquaculture 2017. Food Kanehisa, M., & Goto, S. (2000). KEGG: Kyoto encyclopedia of genes and Agriculture Organisation of the United Nations. https://doi. and genomes. Nucleic Acids Research, 28(1), 27–30. https://doi. org/10.18356/8c28d3e2-en org/10.1093/nar/28.1.27 Findley, M. K., & Koval, M. (2009). Regulation and roles for claudin-fam- Kim, D., Langmead, B., & Salzberg, S. L. (2015). HISAT: A fast spliced ily tight junction proteins. IUBMB Life, 61(4), 431–437. https://doi. aligner with low memory requirements. Nature Methods, 12(4), 357– org/10.1002/iub.175 360. https://doi.org/10.1038/nmeth.3317 Forsyth, I. A., & Wallis, M. (2002). Growth hormone and prolactin-mo- Koren, S., Walenz, B. P., Berlin, K., Miller, J. R., Bergman, N. H., & Phillippy, lecular and functional evolution. Journal of Mammary Gland Biology A. M. (2017). Canu: Scalable and accurate long-read assembly via and Neoplasia, 7(3), 291–312. https://doi.org/10.1023/A:10228 adaptive k-mer weighting and repeat separation. Genome Research, 04817104 27(5), 722–736. https://doi.org/10.1101/gr.215087.116 Glover, D. C., DeVries, D. R., & Wright, R. A. (2012). Effects of tem- Laverty, G., & Skadhauge, E. (2012). Adaptation of teleosts to very high perature, salinity and body size on routine metabolism of coastal salinity. Comparative Biochemistry and Physiology Part A: Molecular largemouth bass Micropterus salmoides. Journal of Fish Biology, 81(5), & Integrative Physiology, 163(1), 1–6. https://doi.org/10.1016/j. 1463–1478. https://doi.org/10.1111/j.1095-8649.2012.03385.x cbpa.2012.05.203 Guerrero, R. F., & Kirkpatrick, M. (2014). Local adaptation and the evo- Lehmann, R., Lightfoot, D. J., Schunter, C., Michell, C. T., Ohyanagi, H., lution of chromosome fusions. Evolution, 68(10), 2747–2756. https:// Mineta, K., & Gojobori, T. (2019). Finding Nemo's Genes: A chromo- doi.org/10.1111/evo.12481 some-scale reference assembly of the genome of the orange clown- Guier, C. R., Miller, W. G., Mullis, A. W., & Nichols, L. E. (1978). Comparison fish Amphiprion percula. Molecular Ecology Resources, 19(3), 570–585. of growth rates and abundance of largemouth bass in selected North https://doi.org/10.1111/1755-0998.12939 314 | SUN et al.

Lehnert, S. J., Bentzen, P., Kess, T., Lien, S., Horne, J. B., Clement, M., & genome-detection of a supergene and other signals of selection. Bradbury, I. R. (2018). Chromosome polymorphisms track trans-At- Genome Research, 29(11), 1919–1928. https://doi.org/10.1101/ lantic divergence, admixture and adaptive evolution in salmon. gr.253435.119 BioRxiv, 351338, https://doi.org/10.1101/351338 Ravi, V., & Venkatesh, B. (2018). The divergent genomes of tele- Li, H., Coghlan, A., Ruan, J., Coin, L. J., Heriche, J. K., Osmotherly, L., & osts. Annual Review of Animal Biosciences, 6(1), 47–68. https://doi. Wong, G. K. S. (2006). TreeFam: A curated database of phylogenetic org/10.1146/annurev-animal-030117-014821 trees of animal gene families. Nucleic Acids Research, 34(suppl_1), Renfro, W. C. (1959). Survival and migration of fresh-water fishes in salt D572–D580. https://doi.org/10.1093/nar/gkj118 water. Texas Journal of Science, 11, 172–180. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, Roberts, A., Trapnell, C., Donaghey, J., Rinn, J. L., & Pachter, L. (2011). G., Abecasis, G., & Durbin, R. (2009). The sequence alignment/map Improving RNA-Seq expression estimates by correcting for frag- format and SAMtools. Bioinformatics, 25(16), 2078–2079. https://doi. ment bias. Genome Biology, 12(3), R22. https://doi.org/10.1186/ org/10.1093/bioinformatics/btp352 gb-2011-12-3-r22 Liu, B., Shi, Y., Yuan, J., Hu, X., Zhang, H., Li, N., & Fan, W. (2013). Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Estimation of genomic characteristics by analyzing k-mer frequency Höhna, S., Larget, B., Liu, L., Suchard, M. A., & Huelsenbeck, J. P. in de novo genome projects. Quantitative Biology, 35, 62–67. (2012). MrBayes 3.2: Efficient Bayesian phylogenetic inference and Love, M. I., Huber, W., & Anders, S. (2014). Moderated estimation of model choice across a large model space. Systematic Biology, 61(3), fold change and dispersion for RNA-seq data with DESeq2. Genome 539–542. https://doi.org/10.1093/sysbio/sys029 Biology, 15(12), 550. https://doi.org/10.1186/s13059-014-0550-8 Sankoff, D., & Nadeau, J. H. (2003). Chromosome rearrangements in evo- Madsen, S. S., Engelund, M. B., & Cutler, C. P. (2015). Water transport lution: From gene order to genome sequence and back. Proceedings and functional dynamics of aquaporins in osmoregulatory organs of of the National Academy of Sciences, 100(20), 11188–11189. https:// fishes. The Biological Bulletin, 229(1), 70–92. https://doi.org/10.1086/ doi.org/10.1073/pnas.2035002100 BBLv229n1p70 Schoeftner, S., & Blasco, M. A. (2009). A ‘higher order’ of telomere Marshall, W. S., Breves, J. P., Doohan, E. M., Tipsmark, C. K., Kelly, S. P., regulation: Telomere heterochromatin and telomeric RNAs. The Robertson, G. N., & Schulte, P. M. (2018). claudin-10 isoform expres- EMBO Journal, 28(16), 2323–2336. https://doi.org/10.1038/ sion and cation selectivity change with salinity in salt-secreting epi- emboj.2009.197 thelia of Fundulus heteroclitus. Journal of Experimental Biology, 221(1), Schultz, E. T., & McCormick, S. D. (2012). Euryhalinity in an evolution- 1–11. https://doi.org/10.1242/jeb.168906 ary context. Fish Physiology, 32, 477–533. https://doi.org/10.1016/ McGinnis, S., & Madden, T. L. (2004). BLAST: At the core of a powerful B978-0-12-396951-4.00010-4 and diverse set of sequence analysis tools. Nucleic Acids Research, Servant, N., Varoquaux, N., Lajoie, B. R., Viara, E., Chen, C.-J., Vert, J.-P., 32(suppl_2), W20–W25. https://doi.org/10.1093/nar/gkh435 Heard, E., Dekker, J., & Barillot, E. (2015). HiC-Pro: An optimized and Meador, M. R., & Kelso, W. E. (1990). Growth of largemouth bass flexible pipeline for Hi-C data processing. Genome Biology, 16(1), 259. in low-salinity environments. Transactions of the American https://doi.org/10.1186/s13059-015-0831-x Fisheries Society, 119(3), 545–552. https://doi.org/10.1577/1548- Shao, C., Li, C., Wang, N. A., Qin, Y., Xu, W., Liu, Q., Zhou, Q., Zhao, 8659(1990)119<0 5 4 5 : G O L B I L >2.3.CO;2 Y., Li, X., Liu, S., Chen, X., Mahboob, S., Liu, X., & Chen, S. (2018). Melkikh, A. V., & Seleznev, V. D. (2012). Mechanisms and models of the Chromosome-level genome assembly of the spotted sea bass, active transport of ions and the transformation of energy in intra- Lateolabrax maculatus. GigaScience, 7(11), giy114. https://doi. cellular compartments. Progress in Biophysics and Molecular Biology, org/10.1093/gigascience/giy114 109(1–2), 33–57. https://doi.org/10.1016/j.pbiomolbio.2012.04.008 Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V., & Miga, K. H. (2017). Chromosome-specific centromere sequences provide Zdobnov, E. M. (2015). BUSCO: Assessing genome assembly and an estimate of the ancestral chromosome 2 fusion event in hominin annotation completeness with single-copy orthologs. Bioinformatics, genomes. Journal of Heredity, 108(1), 45–52. https://doi.org/10.1093/ 31(19), 3210–3212. https://doi.org/10.1093/bioinformatics/btv351 jhered/esw039 Stanke, M., Keller, O., Gunduz, I., Hayes, A., Waack, S., & Morgenstern, Mineta, K., Yamamoto, Y., Yamazaki, Y., Tanaka, H., Tada, Y., Saito, K., B. (2006). AUGUSTUS: Ab initio prediction of alternative transcripts. Tamura, A., Igarashi, M., Endo, T., Takeuchi, K., & Tsukita, S. (2011). Nucleic Acids Research, 34(suppl_2), W435–W439. https://doi. Predicted expansion of the claudin multigene family. FEBS Letters, org/10.1093/nar/gkl200 585(4), 606–612. https://doi.org/10.1016/j.febslet.2011.01.028 Sundell, K., & Sundh, H. (2012). Intestinal fluid absorption in anadromous Nack, S. B., Bunnell, D., Green, D. M., & Forney, J. L. (1993). Spawning salmonids: Importance of tight junctions and aquaporins. Frontiers in and nursery habitats of largemouth bass in the tidal Hudson River. Physiology, 3, 388. https://doi.org/10.3389/fphys.2012.00388 Transactions of the American Fisheries Society, 122(2), 208–216. Susanto, G. N., & Peterson, M. S. (1996). Survival, osmoregulation and https://doi.org/10.1577/1548-8659(1993)122<0208:SANHO oxygen consumption of YOY coastal largemouth bass, Micropterus L>2.3.CO;2 salmoides (Lacepede) exposed to saline media. Hydrobiologia, 323(2), Page, L. M. (2008). Handbook of European freshwater fishes. Copeia, 3, 119–127. https://doi.org/10.1007/BF00017589 725–727. https://doi.org/10.1643/OT-08-098a.1 Tarailo-Graovac, M., & Chen, N. (2009). Using RepeatMasker to iden- Peterson, M. S., & Meador, M. R. (1994). Effects of salinity on freshwater tify repetitive elements in genomic sequences. Current Protocols in fishes in coastal plain drainages in the southeastern US. Reviews in Bioinformatics, 25(1), 4–10. https://doi.org/10.1002/0471250953. Fisheries Science, 2(2), 95–121. https://doi.org/10.1080/1064126940 b i 0 4 1 0 s 2 5 9388554 Tine, M., Kuhl, H., Gagnaire, P.-A., Louro, B., Desmarais, E., Martins, R. S. Peterson, M. S., & Ross, S. T. (1991). Dynamics of littoral fishes and T., Hecht, J., Knaust, F., Belkhir, K., Klages, S., Dieterich, R., Stueber, decapods along a coastal river-estuarine gradient. Estuarine, Coastal K., Piferrer, F., Guinand, B., Bierne, N., Volckaert, F. A. M., Bargelloni, and Shelf Science, 33(5), 467–483. https://doi.org/10.1016/0272- L., Power, D. M., Bonhomme, F., … Reinhardt, R. (2014). European sea 7714(91)90085-P bass genome and its variation provide insights into adaptation to eu- Pettersson, M. E., Rochus, C. M., Han, F., Chen, J., Hill, J., Wallerman, O., ryhalinity and speciation. Nature Communications, 5(1), 1–10. https:// Fan, G., Hong, X., Xu, Q., Zhang, H. E., Liu, S., Liu, X., Haggerty, L., doi.org/10.1038/ncomms6770 Hunt, T., Martin, F. J., Flicek, P., Bunikis, I., Folkvord, A., & Andersson, Tipsmark, C. K., Breves, J. P., Rabeneck, D. B., Trubitt, R. T., Lerner, D. T., L. (2019). A chromosome-level assembly of the Atlantic herring & Grau, E. G. (2016). Regulation of gill claudin paralogs by salinity, SUN et al. | 315

cortisol and prolactin in Mozambique tilapia (Oreochromis mossam- Plateau. Molecular Ecology Resources, 19(4), 1027–1036. https://doi. bicus). Comparative Biochemistry and Physiology Part A: Molecular org/10.1111/1755-0998.13021 & Integrative Physiology, 199, 78–86. https://doi.org/10.1016/j. Yang, Z., & Rannala, B. (2006). Bayesian estimation of species divergence cbpa.2016.05.014 times under a molecular clock using multiple fossil calibrations with Trapnell, C., Pachter, L., & Salzberg, S. L. (2009). TopHat: Discovering soft bounds. Molecular Biology and Evolution, 23(1), 212–226. https:// splice junctions with RNA-Seq. Bioinformatics, 25(9), 1105–1111. doi.org/10.1093/molbev/msj024 https://doi.org/10.1093/bioinformatics/btp120 Young, M. D., Wakefield, M. J., Smyth, G. K., & Oshlack, A. (2010). Gene Varsamos, S., Diaz, J. P., Charmantier, G. U. Y., Flik, G., Blasco, C., & ontology analysis for RNA-seq: Accounting for selection bias. Genome Connes, R. (2002). Branchial chloride cells in sea bass (Dicentrarchus Biology, 11(2), R14. https://doi.org/10.1186/gb-2010-11-2-r14 labrax) adapted to fresh water, seawater, and doubly concentrated Zdobnov, E. M., Tegenfeldt, F., Kuznetsov, D., Waterhouse, R. M., Simão, seawater. Journal of Experimental Zoology, 293(1), 12–26. https://doi. F. A., Ioannidis, P., Seppey, M., Loetscher, A., & Kriventseva, E. V. org/10.1002/jez.10099 (2017). OrthoDB v9. 1: Cataloging evolutionary and functional an- Vij, S., Kuhl, H., Kuznetsova, I. S., Komissarov, A., Yurchenko, A. A., Van notations for animal, fungal, plant, archaeal, bacterial and viral or- Heusden, P., & Saju, J. M. (2016). Chromosomal-level assembly of thologs. Nucleic Acids Research, 45(D1), D744–D749. https://doi. the Asian seabass genome using long sequence reads and multi-lay- org/10.1093/nar/gkw1119 ered scaffolding. PLOS Genetics, 12(4), e1005954. https://doi. Zhang, Z., Zhang, K., Chen, S., Zhang, Z., Zhang, J., You, X., Bian, C., Xu, J., org/10.1371/journal.pgen.1006500 Jia, C., Qiang, J., Zhu, F., Li, H., Liu, H., Shen, D., Ren, Z., Chen, J., Li, Walker, B. J., Abeel, T., Shea, T., Priest, M., Abouelliel, A., Sakthikumar, S., J., Gao, T., Gu, R., … Xu, P. (2018). Draft genome of the protandrous & Earl, A. M. (2014). Pilon: An integrated tool for comprehensive mi- Chinese black porgy, Acanthopagrus schlegelii. GigaScience, 7(4), crobial variant detection and genome assembly improvement. PLoS giy012. https://doi.org/10.1093/gigascience/giy012 One, 9(11), e112963. https://doi.org/10.1510/icvts.2008.0000S1 Zhou, Q., Gao, H., Zhang, Y., Fan, G., Xu, H., Zhai, J., & Niu, Y. (2019). Wellband, K., Mérot, C., Linnansaari, T., Elliott, J. A. K., Curry, R. A., & A chromosome-level genome assembly of the giant grouper Bernatchez, L. (2019). Chromosomal fusion and life history-associ- (Epinephelus lanceolatus) provides insights into its innate immunity ated genomic variation contribute to within-river local adaptation and rapid growth. Molecular Ecology Resources, 19(5), 1322–1332. of Atlantic salmon. Molecular Ecology, 28(6), 1439–1459. https://doi. https://doi.org/10.1111/1755-0998.13048 org/10.1111/mec.14965 Xiao, Y., Xiao, Z., Ma, D., Liu, J., & Li, J. (2019). Genome sequence of the barred knifejaw Oplegnathus fasciatus (Temminck & Schlegel, SUPPORTING INFORMATION 1844): The first chromosome-level draft genome in the family Additional supporting information may be found online in the Oplegnathidae. GigaScience, 8(3), giz013. https://doi.org/10.1093/ gigascience/giz013 Supporting Information section. Xie, C., Mao, X., Huang, J., Ding, Y., Wu, J., Dong, S., Kong, L., Gao, G. E., Li, C.-Y., & Wei, L. (2011). KOBAS 2.0: A web server for annotation and identification of enriched pathways and diseases. Nucleic Acids How to cite this article: Sun C, Li J, Dong J, et al. Research, 39(suppl_2), W316–W322. https://doi.org/10.1093/nar/ Chromosome-level genome assembly for the largemouth gkr483 Xu, Z., & Wang, H. (2007). LTR_FINDER: An efficient tool for the pre- bass Micropterus salmoides provides insights into adaptation diction of full-length LTR retrotransposons. Nucleic Acids Research, to fresh and brackish water. Mol Ecol Resour. 2021;21:301– 35(suppl_2), W265–W268. https://doi.org/10.1093/nar/gkm286 315. https://doi.org/10.1111/1755-0998.13256 Yang, X., Liu, H., Ma, Z., Zou, Y., Zou, M., Mao, Y., & Yang, R. (2019). Chromosome-level genome assembly of Triplophysa tibetana, a fish adapted to the harsh high-altitude environment of the Tibetan