Whole Genome Sequencing of the Pirarucu (Arapaima gigas) Supports Independent Emergence of Major Teleost Clades

Abstract

The Pirarucu (Arapaima gigas) is one of the world’s largest freshwater fishes and member of the superorder Osteoglossomorpha (bonytongues), one of the oldest lineages of ray-finned fishes. This species is an obligate air-breather found in the basin of the Amazon River with an attractive potential for aquaculture. Its phylogenetic position among bony fishes makes the Pirarucu a relevant subject for evolutionary studies of early teleost diversification. Here, we present, for the first time, a draft genome version of the A. gigas genome, providing useful information for further functional and evolutionary studies. The A. gigas genome was assembled with 103-Gb raw reads sequenced in an Illumina platform. The final draft genome assembly was ∼661 Mb, with a contig N50 equal to 51.23 kb and scaffold N50 of 668 kb. Repeat sequences accounted for 21.69% of the whole genome, and a total of 24,655 protein-coding genes were predicted from the genome assembly, with an average of nine exons per gene. Phylogenomic analysis based on 24 fish species supported the postulation that Osteoglossomorpha and Elopomorpha (eels, tarpons, and bonefishes) are sister groups, both forming a sister lineage with respect to Clupeocephala (remaining teleosts). Divergence time estimations suggested that Osteoglossomorpha and Elopomorpha lineages emerged independently in a period of ∼30 Myr in the Jurassic. The draft genome of A. gigas provides a valuable genetic resource for further investigations of evolutionary studies and may also offer a valuable data for economic applications.

Introduction

Arapaima gigas, also known as Pirarucu or Paiche, is one of the world’s largest freshwater fishes (Wijnstekers 2011) whose body length and weight may attain 4.5 m (15 ft) and 200 Kg (440 lb), respectively (Nelson 1994; Froese and Pauly 2018). The genus Arapaima emerged in the Amazon floodplain basin and is presently distributed in Brazil, Colombia, Ecuador, and Peru (Hrbek et al. 2005, 2007; Froese and Pauly 2018), and also in Thailand and Malaysia where it has been introduced for commercial fishing (Froese and Pauly 2018). Arapaima gigas local name (Pirarucu) derives from the indigenous Tupi words “pira” and “urucum” for “fish” and “red,” respectively, presumably referring to its red tail scales flecks or to its reddish flesh (Marsden 1994; Godinho et al. 2005). The peculiarity of its breathing apparatus is characteristic of this Amazonian fish, comprising gills and a lung-like tissue devised for air-breathing derived from a modified and enlarged swim bladder (Burnie and Wilson 2001; Brauner et al. 2004). The Pirarucu has an attractive market value due to its low-fat and low bone content. Overfishing practices in the Amazonian region led to the banning of Pirarucu commercialization by the Brazilian government in 2001, although consumption by the native population is currently permitted under strict size and seasoning regulations (Bayley and Petrere 1989). Its main supply is provided by wild-caught fish and fish farming conducted by riverbank population of the Amazonas (Froese and Pauly 2018). Aquaculture production is attractive due to high carcass yields and rapid juvenile growth, with yearlings reaching up to 10 kg (22 lb) (Almeida et al. 2013).

Arapaima gigas belongs to the superorder Osteoglossomorpha of bony-tongued fishes whose tongue contains sharp bony teeth for disabling and shredding preys (Sanford and Lauder 1990; Burnie and Wilson 2001). Together with Elopomorpha (eels and tarpons) and Clupeocephala (most of extant fish species), the Osteoglossomorpha comprises one of the three main teleosts groups whose phylogenetic position has been controversial (Le et al. 1993; Inoue et al. 2003; Near et al. 2012; Betancur-R 2013; Faircloth et al. 2013; Chen et al. 2015; Hughes et al. 2018). Fossil records and some early molecular studies, including a recent comprehensive analysis of >300 Actinopterygii species (Hughes et al. 2018), placed Osteoglossomorpha as the oldest teleost group (Greenwood 1970; Inoue et al. 2003), while other studies placed Elopomorpha as the most ancestral one (Near et al. 2012; Betancur-R 2013; Faircloth et al. 2013). Recently, a phylogenetic study based on whole genome sequencing of the bony-tongued Asian arowana (Scleropages formosus) suggested that the branching of Elopomorpha and Osteoglossomorpha occurred almost simultaneously, placing them as sister lineages of Clupeocephala (Bian 2016). Within this context, the genome of the Pirarucu provides new insights to study the evolutionary history of teleosts as well as providing useful information for sustainable exploration of this giant Amazon fish. Here, we present the first whole genome assembly, gene annotation, and phylogenomic inference of the Pirarucu which should facilitate the molecular characterization and conservation of this economically important fish species.

Materials and Methods

Sample Collection and Sequencing

Genomic DNA was extracted from peripheral blood samples of four adult individuals (two males and two females) of Arapaima gigas: NCBI taxonomy ID 113544, FishBase ID: 2076. All samples were collected in accordance with the standards of the Federal University of Pará animal protocol. We applied a whole-genome shotgun sequencing strategy using two short-insert libraries (400 and 500 bp) in an Illumina HiSeq 2500 platform according to the manufacturer’s instructions (Illumina, San Diego, CA). HiSeq Rapid SBS Kits (FC-402-4021) and HiSeq Rapid Cluster Kits (PE-402-4002) were used to sequence paired-end read of 2 × 250 base pairs. Read quality was checked using FastQC, version 0.11.4 (Andrews 2010), and low-quality reads were trimmed with Sickle paired-end (pe), version 1.33 (Joshi and Fass 2011), under default parameters.

Genome Size Estimation and De Novo Assembly

Genome size was estimated based on the k-mer spectrum with the following formula: G= (N×(L−K + 1)−B)/D. Where N is the total read count, L is the read length, K is k-mer length (K = 31), B is the total low-frequency (frequency ≤1) k-mer count, D is the k-mer depth, and G is the genome size. Jellyfish 2.2.6 (Marçais and Kingsford 2011) was used to count k-mer frequencies of high-quality sequencing reads.

Genome assembly was performed using SOAPdenovo2 (version 2.04) (Luo et al. 2012) under default parameters (127mer version). Three assemblies were conducted: 1) using all reads; 2) with reads from male samples; and 3) with reads from female samples. Subsequently, gaps were filled using Redundants (Pryszcz and Gabaldón 2016) using three-run scaffolding steps: firstly with the default value of minimum read pairs to joining contigs (5 pairs), subsequently rerunning with previous data with a minimum value of four read pairs and, finally, using a minimum of three read pairs. Assembly quality and statistics were assessed with QUAST (version 4.4) (Gurevich et al. 2013).

Assessment of Genome Completeness

Assembly quality was measured by assessing gene completeness with Benchmarking Universal Single-Copy Orthologs (BUSCO) (Simão et al. 2015) based on 4,584 BUSCO groups derived from Actinopterygii orthologs.

Repeat Analysis

Transposable elements (TEs) and other repetitive elements of the Pirarucu genome were identified by a combined, homology-based method and a de novo annotation approach. Initially, tandem repeats were identified with Tandem Repeats Finder 4.09 (Benson 1999) with the following parameters: “Match=2, Mismatch=7, Delta=7, PM=80, PI=10, Minscore=50, and MaxPerid=2,000.” Additionally, a de novo repeat library was built with RepeatModeler 1.0.9 and LTR_FINDER (Xu and Wang 2007), and filtered with LTR_retriever (Ou and Jiang 2017) under default parameters. Subsequently, known and novel transposable elements were identified by mapping the assembled sequences to the Repbase TE 22.05 (Bao et al. 2015) and de novo repeat libraries using RepeatMasker 4.0 (Tarailo-Graovac and Chen 2009). In addition, we annotated TE-related proteins using RepeatProteinMask 4.0 (Tarailo-Graovac and Chen 2009).

Gene Structure and Function Annotation

Genome annotation was carried out with the MAKER2 pipeline (Holt and Yandell 2011) in a two-pass iteration. First, homology annotation was performed with protein data from Homo sapiens (human), Danio rerio (zebrafish), Takifugu rubripes (Japanese fugu), Tetraodon nigroviridis (spotted green pufferfish), Gasterosteus aculeatus (three-spined stickleback), Oryzias latipes (Japanese medaka), Latimeria chalumnae (coelacanth) (Ensembl release 88), together with Scleropages formosus (Asian arowana) protein sequences from NCBI RefSeq annotation data. Subsequently, de novo annotations were performed using the homology-based results achieved in the first step. We also used the RepeatModeller 1.0.9 (Smit and Hubley 2008) to build a de novo repeat library with default parameters. The GFF output from the first step was used to train the SNAP 20131129 (Korf 2004) and AUGUSTUS 3.2.3 (Stanke et al. 2008) predictors. GeneMark-ES 4.32 (Lomsadze et al. 2005) was trained using the genome assembly itself. InterProScan 5.24-63.0 (Jones et al. 2014) was run on the protein output of MAKER, providing gene ontologies and classifying protein domains and families. Protein output was compared using BLAST against the NCBI NR database (available on May 29, 2017) for identifying putative gene names. Blast2GO v5 (Conesa et al. 2005) was subsequently used to obtain Gene Ontology mapping and annotation (supplementary file S2, Supplementary Material online).

Phylogenomic Analysis

Phylogenomics was based on protein data from 24 fish species. Transcriptome data from ENA database were used for species whose genome had not been sequenced (supplementary table S5, Supplementary Material online). Transcripts were assembled with Trinity (Haas et al. 2013) and protein sequences were deduced with Transdecoder. All redundant sequences (>99.5% of identity) were later removed with CD-HIT (Fu et al. 2012), and those with <200 residues were discarded, resulting in a data set with a total number of 651,482 protein sequences.

Subsequently, 17,031 orthogroups were built with OrthoFinder (Emms and Kelly 2015) using all-to-all BLASTP comparisons and MCL clustering (supplementary table S6, Supplementary Material online). Proteins in each group were aligned using MAFFT (Katoh and Standley 2013) and gene trees were estimated with FastTree (Price et al. 2010). Paralogous and spurious branches were removed by identifying clusters with monophyletic outgroups (“prune_ paralogs_MO.py”) as described by Yang and Smith (2014) and applying parameters suggested by Austin et al. (2015). Protein sequences in each cluster were realigned with MAFFT, trimmed with Gblocks (Talavera and Castresana 2007) and concatenated into a supermatrix of 282 loci and 188,505 aligned columns with an overall occupancy of 88.5%.

Phylogenomic analysis was conducted with ML and BI using the constructed supermatrix. ML analysis was conducted with RAxML (Stamatakis 2014) with 200 rapid bootstrap replicates considering each locus as a separate partition (278 loci, after merging partitions without occurrence of all amino acids). The final tree topology was selected as the tree with the best likelihood estimate. Bayesian inference was carried out using BEAST 2.4 (Bouckaert et al. 2014) under a LG substitution model. A Markov chain Monte Carlo (MCMC) was run for ten million generations and sampled every 5,000 generations. The consensus tree was determined after discarding (burn-in) 10% of initial trees.

Evolutionary rates were estimated by adding branch lengths from the tree tips to the teleost MRCA node (fig. 1, red star). Tajima’s relative rate tests (Tajima 1993) were performed with MEGA 7 (Kumar et al. 2016) with the same concatenated alignment used in phylogenomics analysis. Pirarucu rates were compared with rates of all other teleosts using the Spotted gar as outgroup; P < 0.05 was considered for rejecting the null hypothesis of equal rates between lineages.

Fig. 1.

—Phylogenomics inference. Phylogenomic tree inferred by maximum likelihood (ML) based on a supermatrix of 278 orthologs loci (188,505 amino acid sites) from 24 species using Elephant shark as outgroup. Dark gray circles indicate coincident nodes with Bayesian inference (BI) and maximum support values in both approaches (bootstrap=100% and Bayesian posterior probability=1). Branch lengths represent number of substitutions/site. Rates of molecular evolution (i.e., number of amino acids substitutions per site) estimated from the teleost split (red star) to the tips of the topology are indicated in red font close to the name of each taxon.

Divergence Times Estimation

Estimations of divergence times were carried out with MCMCTree of PAML package (Yang 2007). Calibration times were obtained from TimeTree (Hedges et al. 2015) (supplementary table S8, Supplementary Material online), a public knowledge-base providing information of the evolutionary time of the tree of life (TTOL) based on >3,000 studies and comprising >97,000 species (at the time of this work). Time estimations were calculated using the amino acid supermatrix as input and the ML topology.

Whole Genome Analysis

Distributions of synonymous substitutions per synonymous site (K_s) were estimated with the wgd Python package (https://github.com/arzwa/wgd). Briefly, for each species, CDS sequences were first translated to protein sequences, compared all-versus-all using BLASTP and clustered using the MCL algorithm (Enright et al. 2002). Then, for each cluster, sequences were aligned using MUSCLE (Edgar 2004) and protein sequences were subsequently reverse translated to nucleotide sequences according to the input CDS. Finally, to estimate the K_s distributions, a maximum likelihood phylogenetic analysis was performed using the CODEML program from the PAML package (Yang 2007), and K_s values were corrected based on a phylogenetic tree constructed for each family using FastTree (Price et al. 2010).

Gene Family Evolution Analysis

To investigate gene gain and loss dynamics among ancestral lineages the previously inferred phylogenetic tree and the 17,031 orthogroups identified using OrthoFinder (see Materials and Methods—Phylogenomic analysis section) were used. Orthogroups consisting of species-specific single-genes or with >50 genes in one species were considered to be artefacts and were removed from downstream analysis, resulting in 16,968 orthogroups (each one representing a gene family). A Wagner parsimony approach, using the Count software (Csurös 2010) was used for identifying gene family gain and loss events, as well as family expansions and contractions. Gain to loss penalty ratio was set to 1, assuming expansions and contractions to be equally likely.

Maximum Likelihood Estimation of Gene Family Rates

Gene family expansion and contraction rates were estimated for gene families (see Materials and Methods—Gene family evolution analysis) with at least one gene present in the MRCA of teleosts (16,402 families on total). Estimations were carried out with CAFE v4.1 (Han et al. 2013) and the time-calibrated tree with MCMCTree (fig. 2). To account for potential errors in the data set, we used the “caferror.py” script for estimating global errors without a priori information.

Fig. 2.

—Divergence time estimation between species. Numbers at nodes represent divergence time estimates in millions of years ago (Ma). Red squares indicate nodes calibrated by fossil records.

Rate estimations of gene family size evolution were assessed by equally considering rates of gains and losses (lambda) and different rates for each (lambdamu). We also performed estimations using a one-lambda(mu) model (i.e., average rate over all phylogenetic tree), and a two-lambda(mu) model (i.e., considering different rates for teleosts and nonteleosts). P threshold was set at 0.01.

Families Functional Annotation

Gene family functions were inferred by first selecting one representative sequence (the longest one) of each orthogroup. Subsequently, sequences were compared against the NR database with the DIAMOND tool (Buchfink et al. 2015) and against the InterProScan 5.24-63.0 (Jones et al. 2014) for domain annotation. Next, Gene Ontology terms were obtained with Blast2GO v5 (Conesa et al. 2005).

Sex Comparisons

To identify sex-specific genomic regions, we performed three different approaches:

1. Sex-specific trimmed reads were aligned against the main genome assembly (sex-mixed) using BWA (Li and Durbin 2009) and read mapping statistics was evaluated using SAMStat with default parameters (Lassmann et al. 2011). SAMtools package (Li et al. 2009) was used for creating and sorting binary (“.bam”) alignment files and, subsequently, for extracting only sequences mapped against the reference assembly, generating a file with genome length data. Statistics for assessing genome coverage were retrieved with BEDTools suit (Quinlan and Hall 2010) and shell command lines were used to calculate proportions of genomes/contigs coverage. Additionally, sex-specific coverages were summarized with Mosdepth (Pedersen and Quinlan 2018) considering read depths in window frames of 50 kb, and R for figure plotting.

2. In order to evaluate sex-specific genome assemblies, we used the Quality Assessment Tool for Genome Assemblies (QUAST) software (Gurevich et al. 2013). Comparisons were performed using the main genome assembly as reference and gene annotation data (“.gff”).

3. Lastly, a cross-read-assembly comparison was carried out for identifying sex-specific regions in each assembly. Firstly, reads of a given sex were aligned against the genome assembly of the opposite sex using BWA (Li and Durbin 2009). Unaligned reads considered to be sex-specific and were subsequently realigned against the genome assembly of the same sex for identifying regions mapped on the assembly. Mapped regions without mapped sex-specific reads were masked, and the remaining sequences were compared against the NR database using BLASTx to identify likely protein-coding genes associated with the sex-specific regions.

Results and Discussion

Genome Sequencing, Assembly, and Annotation

Whole genome sequencing of four adults (two males and two females) was performed with two paired-end short insert libraries using an Illumina HiSeq 2500 platform (2×250 bp), producing a total of 103.01-Gb raw sequences. After removing low-quality and redundant reads, we obtained ∼76.91 Gb of high-quality data for de novo assembling. Using a k-mer-based approach, genome size was estimated as 761 Mb (with ∼135× coverage) (supplementary table S1 and fig. S1, Supplementary Material online). Subsequently, de novo assembling generated a draft genome comprising 661,278,939 bp, 5,301 scaffolds, with scaffold N50 = 668 kb, and contig N50 = 51.23 kb. Assembly quality was measured with BUSCO (Simão et al. 2015) showing high-level completeness with 94.61% of complete BUSCOs groups (supplementary table S2, Supplementary Material online).

Repeat analysis showed a total of 143 Mb repetitive sequences, with DNA transposons representing the most predominant repeat, accounting for 46% of all transposable elements (TE) and 8.51% of the genome. Long repeat elements, like LTRs and LINEs, accounted for 3.07% and 4%, respectively (supplementary table S3, Supplementary Material online). In view that only short-insert libraries (400 and 500 bp long) were sequenced, complete LTR and LINE transposons were not expected to be fully identified.

Genome annotation predicted 24,655 protein-coding genes, covering 33.9% of the genome. Comparisons with the nonredundant (NR) NCBI database identified putative identities for 99% of the genes and Gene Ontology (GO) terms assigned to 12,460 proteins (50.5% of total) (supplementary table S4, Supplementary Material online). A summary of the sequencing data, genome assembly, and annotation is shown in table 1.

Table 1.

Summary Statistics of the Pirarucu Genome

Sequencing Information
Library insert size (bp)	400–500
Read length (bp)	2×250
Total raw bases sequenced (Gb)	103.01
Total filtered bases sequenced (Gb)	76.91
Genome Features
Assembled genome size (Mb)	661.28
# scaffolds	5,301
Scaffold N50 (kb)	668
Contig N50 (kb)	51.23
Largest scaffold (bp)	5,332,704
GC (%)	43.18
Repeat content (% of genome)	21.69
Genome Annotation
Protein-coding gene number	24,655
% of genome covered by genes	33.9
Mean transcript length (bp)	9,150
Mean exons per gene	9
Mean CDS length (bp)	1,603
Mean exon length (bp)	174
Mean intron length (bp)	920

Compared with the Asian arowana genome, the genome of the Pirarucu is considerably smaller, with ∼60 Mb of difference in estimated genome size and 120 Mb in assembled genome size. However, the Pirarucu had more protein-coding genes identified (>2,000) and lower repeat content (21% against 27% in the Arowana) (Bian 2016).

Phylogenomic Analysis

Orthology inference was carried out with OrthoFinder (Emms and Kelly 2015) based on amino acid sequence data from 24 fish species (table 2). Due to the scarcity of genome data from Elopomorpha and Osteoglossomorpha species, available transcriptome data were also used for enriching these lineages and providing a better understanding of divergence between these taxa (supplementary table S5, Supplementary Material online). We identified 17,031 orthogroups, comprising a total of 630,993 genes, with 651 species-specific orthogroups and 1,436 orthogroups shared by all 24 fish species (supplementary table S6, Supplementary Material online). Following stringent procedures for identifying orthologs and excluding likely paralogs, 278 orthologous loci were found to be shared across species. Ortholog concatenation resulted in a supermatrix with 188,505 amino acid sites and overall occupancy of 88.5%. Tree topologies were inferred by maximum-likelihood (ML) and Bayesian inference (BI) with maximum values of bootstrap support and posterior probability for all nodes. Discordant arrangements were observed for the Cladistia and Chondrostei clades with ML supporting reedfishes as the sister lineage of all other Actinopterygii while BI placed reedfishes as the sister lineage of American paddlefishes (fig. 1). Evolutionary rates, based on number of amino acid substitutions per site, were found to be highly heterogeneous among teleost fishes (fig. 1; red numbers in brackets), indicating a rapid and divergent teleost evolution. The evolutionary rates of the Pirarucu were significantly different from all other teleost species, including the Asian arowana (P < 0.05; Tajima’s relative rate test, supplementary table S7, Supplementary Material online).

Table 2.

List of Species Included in Phylogenomic Analysis

Organismsource	Scientific Name	Order	Reference
Ray-finned fish (Teleostei–Osteoglossomorpha)
Pirarucu*	Arapaima gigas	Osteoglossiformes	This study
Asian arowanaU	Scleropages formosus	Osteoglossiformes	Austin et al. (2015)
aFreshwater butterflyfishENA	Pantodon buchholzi	Osteoglossiformes	Pasquier et al. (2016)
aPeters’ elephantnose fishENA	Gnathonemus petersii	Osteoglossiformes	Pasquier et al. (2016)
Ray-finned fish (Teleostei–Elopomorpha)
European EelZ	Anguilla anguilla	Anguilliformes	Henkel et al. (2012)
aFalse morayENA	Kaupichthys hyoproroides	Anguilliformes	Gruber et al. (2015)
aIndo-Pacific tarponENA	Megalops cyprinoides	Elopiformes	Sun et al. (2016)
Ray-finned fish (Teleostei–Clupeocephala)
MedakaQFO	Oryzias latipes	Beloniformes	Kasahara et al. (2007)
Blind cave fishU	Astyanax mexicanus	Characiformes	McGaugh et al. (2014)
Nile tilapiaU	Oreochromis niloticus	Cichliformes	Brawand et al. (2014)
Common carpR	Cyprinus carpio	Cypriniformes	Xu et al. (2014)
ZebrafishQFO	Danio rerio	Cypriniformes	Howe et al. (2013)
Amazon mollyU	Poecilia formosa	Cyprinodontiformes	Unpublished
Southern platyfishU	Xiphophorus maculatus	Cyprinodontiformes	Schartl et al. (2013)
Atlantic codE	Gadus morhua	Gadiformes	Star et al. (2011)
Electric EelF	Electrophorus electricus	Gymnotiformes	Gallant et al. (2014)
Three-spined sticklebackU	Gasterosteus aculeatus	Perciformes	Jones et al. (2012)
Spotted green pufferfishU	Tetraodon nigroviridis	Tetraodontiformes	Jaillon et al. (2004)
FuguU	Takifugu rubripes	Tetraodontiformes	Kai et al. (2011)
Ray-finned fish (Holostei)
Spotted garQFO	Lepisosteus oculatus	Semionotiformes	Unpublished
Ray-finned fish (Chondrostei)
aAmerican paddlefishENA	Polyodon spathula	Acipenseriformes	Sun et al. (2016)
Ray-finned fish (Cladistia)
aReedfishENA	Erpetoichthys calabaricus	Polypteriformes	Sun et al. (2016)
Lobe-finned fish
CoelacanthU	Latimeria chalumnae	Coelacanthiformes	Unpublished
Cartilaginous fish
Elephant sharkR	Callorhinchus milii	Chimaeriformes	Venkatesh et al. (2014)

Note.—Codes for source: Ensembl (E), efish genomics (F), Quest of Orthologs (QFO), RefSeq (R), EBI ENA (ENA), UniProt (U), ZF Genomics (Z), and this study (*).

^aRaw transcriptomics reads.

The branching order of the teleost superorders Osteoglossomorpha, Elopomorpha, and Clupeocephala has been controversial (Patterson and Rosen 1977; Le et al. 1993; Inoue et al. 2003; Near et al. 2012; Betancur-R 2013; Faircloth et al. 2013; Chen et al. 2015; Hughes et al. 2018). Our findings placed Osteoglossomorpha as a sister branch of Elopomorpha, both forming a monophyletic sister lineage with respect to Clupeocephala in a topology consistent with recent studies, suggesting a rapid, near-simultaneous emergence of teleost lineages (Bian 2016). This contradicts the current morphological view of basal teleost lineages, in which Osteoglossomorpha and Elopomorpha do not present identified synapomorphies, and the Elopomorpha is placed alone as the sister lineage to all other teleosts (Arratia 1997). Therefore, this might suggest a revaluation of morphological characters used to define these major teleost clades (Bian 2016), or a revaluation of phylogenetic assumptions by considering independent data sets or different hypothesis-testing procedures (Hughes et al. 2018).

The relationships within Osteoglossomorpha are also subject of controversy (Kumazawa and Nishida 2000; Hilton 2003; Lavoué and Sullivan 2004; Wilson and Murray 2008), and our findings showed the pantodontid freshwater butterflyfish (Pantodon buchholzi) as a sister lineage to all other Osteoglossiformes, while Mormyridae (represented by the Peters’ elephantnose fish) was placed as a sister branch of Osteoglossidae (Pirarucu and Asian arowana), in agreement with previous molecular studies (Lavoué and Sullivan 2004).

Estimation of Divergence Times

The divergence times of the ML topology were estimated with alignment data from 278 orthologous loci and calibration points (supplementary table S8, Supplementary Material online) obtained from the TimeTree database (Hedges et al. 2015) (fig. 2). Our findings were consistent with previous studies, including: 1) cartilaginous (chondrichthyes) and bony (Osteichthyes) fishes diverging at 450 Ma (Inoue et al. 2010; Dos Reis et al. 2015); 2) Actinopterygii and Sarcopterygii splitting ∼446 Ma (Patterson and Rosen 1977; Blair and Hedges 2005; Inoue et al. 2005; Azuma et al. 2008; Nakatani et al. 2011; Wei et al. 2014), and 3) teleosts emerging ∼245 Ma (Chen et al. 2013; Dornburg et al. 2014). Interestingly, in agreement with the proposed emergence of a monophyletic clade comprising Osteoglossomorpha and its sister Elopomorpha lineage, the divergence between these two superorders was estimated to have taken place 223 Ma, in the Late Triassic, with Osteoglossomorpha originating ∼187 Ma, in the Early Jurassic, and Elopomorpha almost 30 Myr after, in the Late Jurassic. These rapid cladogenic events occurring during this period (including the diversification of the Clupeocephala subgroups, Otomorpha and Euteleosteomorpha) might be attributed to the amelioration of restrictive environmental conditions ensuing periods of mass extinction (Broughton et al. 2013).

Whole Genome and Lineage-Specific Duplications among Teleosts

Events of whole genome duplication (WGD) are characterized by the occurrence of nondisjunction during meiosis that results in the duplication of the entire genome, including coding and noncoding regions like intronic and regulatory sequences. Along the history of vertebrates, at least two known WGD events occurred ∼500–600 Ma (Moriyama and Koshiba-Takeuchi 2018) while another event took place in the teleost lineage following divergence from land vertebrates, which is usually designated teleost-specific (TS) WGD or third round (3R) WGD (Glasauer and Neuhauss 2014). WGD events can be detected by estimating the number of synonymous substitutions per synonymous site (denoted as K_s, or d_s). Since K_s is assumed to have remained constant throughout time, relict of WGDs are expected to be visualized by peaks in K_s distributions when comparing paralogous gene pairs (Lynch and Conery 2000; Zwaenepoel and Van de Peer 2017).

Analyses of K_s distributions of paralogous genes (paranome) of Pirarucu and other species in key branches of the phylogeny were analyzed before and after TS-WGD. Inferred paralogous families showed highly variable estimates across species, with fractions of outlying pairs (K_s > 5) ranging from 52% (Zebrafish) to 79% (Fugu) (supplementary table S9, Supplementary Material online). Peaks representing past duplication events were identified in teleost species, with evident differences in range between major teleost lineages (fig. 3). Osteoglossomorpha and Elopomorpha showed peaks with wide ranges around K_s=1.50, while Clupeocephala species showed higher peaks around K_s=2.30. Low K_s values indicate low mutational distances between duplicated genes, pointing to a recent evolutionary event (Lu et al. 2012). Previous studies suggested that peaks around K_s=2.30 and K_s=1.50 could be identified as remnants of the three major WGD events in teleost lineages (Vanneste et al. 2013). Differences in peak range across teleost groups may suggest a higher conservation of TS-WGD duplications in Osteoglossomorpha and Elopomorpha lineages than in Clupeocephala. Interestingly, an even more recent peak (K_s=0.85), with a wide range, was specifically observed in the European eel, which could support a recent hypothesis of a likely fourth WGD event in this species (Rozenfeld et al. 2017), however, we do not discard that such peak could be an artefact resulted from wrong fitting assumptions or due to sequencing or annotation faults. More research efforts should be directed at this topic to provide more reliable evidence.

Fig. 3.

—Empirical age distributions. Age distributions based on number of synonymous substitutions per synonymous site (K_s) estimated for paralogous gene families of each species. Distributions were modelled using a four component Gaussian mixture model (GMM). Solid black lines show mixture distributions, and dashed lines represent individual components. Vertical dashed lines correspond to the geometric mean of each component. K_s estimates (X axis) can be interpreted as age divergence between paralogous genes of a given species. The initial peak represents newly duplicated genes (usually derived from small-scale duplication events). Over time, duplications are eventually lost, and a decreasing slope is observed following the initial peak, outlining the steady decrease of retained duplicates. WGD events create distinct peaks to the distribution and can usually be observed as different components in a mixture distribution.

Evolution of Gene Families

Gene gains and losses have been considered major sources of genomic variation and main drivers of phenotypic diversity (Ohno 1970; Zhang 2003). Based on homology data inferred with OrthoFinder, family gains and losses were mapped as discrete character-state changes. A parsimony approach was applied for inferring ancestral states and determining family gains, losses, expansions, and contraction events (fig. 4). In our inferred topology, 9,684 gene families were identified at the root of all sampled species, and 10,692 families were found in the Most Recent Common Ancestor (MRCA)—that is, the lowest common ancestor of two phylogenetic clades in an evolutionary tree—of Teleostei and Holostei. With respect to the MRCA of the three major teleost clades, Clupeocephala, Osteoglossomorpha, and Elopomorpha, 10,640, 10,925, and 9,790 gene families were respectively identified. Inference of ancestral gene duplication events revealed 485 gene family expansions in the MRCA of teleosts, followed by 656 expansions in the MRCA of Elopomorpha and Osteoglossomorpha, and 166 expansions in the MRCA of Clupeocephala. Osteoglossomorpha showed even further expansions (884 gene families) after diverging from Elopomorpha. Considerably losses were found in Elopomorpha, mainly in the Anguilliformes order (represented by the European eel and false moray) with 877 contractions, contrary to the Indo-Pacific tarpon (a representative of the Elopiformes order) with only 11.

Fig. 4.

—Reconstruction of gene family evolution. Events of gene family gains, losses, expansions, and contractions were inferred with Wagner parsimony. Number of families are indicated in black fonts near nodes. Gains (green numbers) indicate the number of families acquired along lineages leading to their respective MRCA node. Losses (red numbers) indicate lost families along lineages leading to their respective MRCA node. Expansions are indicated by numbers (in blue font) of expanded families (from size 1) and contractions by the number (in yellow font) of contracted families (to size 1) to their respective MRCA node. Gene Ontology (GO) terms associated with changes observed in key points of the phylogeny are shown near each node. GO enrichment was estimated based on Fisher’s exact test (FDR < 0.05) using, as background, population families present in each respective MRCA node. Arrows indicate terms associated to gains and/or expansions (upward) and losses and/or contractions (downward).

Analyses of the biological role of gene families that have gone through evolutionary change were carried out by assigning Gene Ontology (GO) terms at each major teleost group (fig. 4). Significant, enriched (FDR adjusted Fisher’s exact test; P < 0.05) GO terms of gained and expanded families in the MRCA of Teleostei and Holostei pointed to the G-protein couple receptor signalling pathway (GO: 0007186), phototransduction (GO: 0007602), sensory perception (GO: 0007600), and detection stimulus (GO: 0051606). At the teleost’s MRCA, enriched terms associated to gained or expanded families were not found, although terms related to metabolic processes (GO: 0008152) were enriched for lost families respective to the MRCA node. In Elopomorpha, considerably losses of functions related to G-protein couple receptor signalling pathway (GO: 0007186), reproduction (GO: 0000003), and organelle fission (GO: 0048285) were observed. In Clupeocephala, terms associated to sensory perception (GO: 0007600) and visual perception (GO: 0007601) were found to be enriched in gained and expanded families, while terms of cell differentiation in spinal cord (GO: 0021515), mammillary body development (GO: 0021767), and central nervous system neuron differentiation (GO: 0021953) were significant. With respect to Osteoglossomorpha, no significantly enriched terms were found in association with gained/expanded or lost/contracted families.

Likelihood Estimation of Gene Family Evolutionary Rates

The birth-and-death evolutionary model has been observed in several gene families, including sensory receptor and immune systems genes (Demuth and Hahn 2009; Innan and Kondrashov 2010). Using a ML framework of birth-and-death models, we carried out estimations of gain and loss rates across the tree. To account for potential erroneous gene number estimations, usually derived from low-quality genome assemblies or transcriptome-only data, we estimated the global error in the data set. We found that ∼14% (ɛ = 0.1417) of size groups estimates at the tree tips were prone to errors. The average rate of gene gain (λ) and loss (µ) was estimated for the 16,402 orthogroups with at least one representative teleost-species (supplementary table S10, Supplementary Material online). This was carried out for each group, namely teleosts and nonteleosts. The estimated rates of gene turnover of nonteleosts showed gains (λ₀=0.0031), accounting for duplications/gene/Ma and losses (μ₀=0.0016) for losses/gene/Ma. In teleosts, a more balanced gain/loss rates were observed, with λ₁=0.0029 and µ₁=0.0028 (supplementary table S10, Supplementary Material online; two-lambdamu model with global error correction).

Evidence of accelerated evolution was also inferred for some gene families based on the probability of any gene family of evolving under a birth-and-death process. Among the 16,402 gene families, 758 were found to be unlikely evolving under a random gain and loss process (P < 0.01; supplementary tables S11 and S12, Supplementary Material online). Of these, 714 were found at the tips of the topology, four shared among all teleosts, eight in at least two Osteoglossomorpha species, 13 shared by Anguilliformes, and a total of 19 rapidly evolving families in different branches of Clupeocephala (supplementary fig. S2, Supplementary Material online).

Sex-Specific Genomic Regions

Studies on the mechanism of sex determination in fishes are important for preservation and commercial purposes. The Pirarucu does not show evident sexual dimorphism, and adults can only be reliably sexed during the reproductive phase (Chu-Koo et al. 2009). Previous karyotypic studies failed to identify chromosome differences between sexes, suggesting a nonchromosomal system of sex determination or recent loss of the sex-determining locus (SDL) in a carrier chromosome (Almeida et al. 2013). We herewith carried out computational analyses of genomic data from both sexes to identify potential genetic differences. Comparisons of sex-specific sequencing reads against the main genome assembly (built with data from both sexes) allowed for an initial evaluation of read depths across the genome. Female and male reads covered, with at least one read depth per base, 99.88% and 99.87% of the 661-Mb assembly, respectively. The average read depth per base over the whole genome equalled 55 female reads and 59 male reads without evident large regions of different depth coverage (fig. 5A).

Fig. 5.

—Comparison of sex-specific sequences and assemblies. Samples from each sex were compared against the main genome assembly containing data from both sexes. (A) Depth of coverage (i.e., average number of reads mapped to a specific region) of female (in red) and male (in blue) reads compared with the main assembly. Coverage was estimated for windows of 50 kb using Mosdepth (Pedersen and Quinlan 2018) and regions were ordered by female coverage estimates (ascending, left to right). Genome scaffolds with <50 kb were not included in the plot. Y axis was restricted to 200 for better visualization. (B) Number of complete and partial genes identified in each sex-specific assembly.

Comparative analysis of male and female genomes, carried out between sex-specific genome assemblies, showed minimal differences. Despite having higher contig N50 values and a slightly higher genome size, the female assembly was more fragmented, with 8,324 contigs and scaffold N50 of 295 kb than male reads, with 6,058 contigs and scaffold N50 of 471 kb (table 3). Comparisons of sex-specific assemblies against the main (mixed) assembly showed similar results, with both assemblies presenting 99.7% of aligned bases to the reference genome (genome fraction). The female assembly showed fewer misassemblies and longer continuous alignment with the reference genome than the male assembly despite being more fragmented. Considering the predicted gene regions using the main assembly as a reference, 19,737 complete genes and 5,076 partial ones were found in the female assembly, while 19,540 complete and 5,274 partial genes were found in the male assembly (fig. 5B). This suggested ∼2,000 specific genes for each sex, if only complete genes were considered. However, as a complete gene in one sex may be a partial gene in the opposite sex, these differences might be actually due to misassemblies. Functional analysis did not find significantly enriched GO terms in either set of sex-specific genes.

Table 3.

Comparison of Sex-Specific Genome Assemblies

Genome Features	Arapaima gigas (female)	Arapaima gigas (male)
Assembled genome size (Mb)	660.71	660.43
Genome fraction (%)	99.74	99.73
# scaffolds	8,324	6,058
Scaffold N50 (kb)	295	471
Contig N50 (kb)	40.75	35.19
Largest scaffold (bp)	2,179,931	2,199,363
Largest alignment to the reference (bp)	2,178,748	1,935,564
GC (%)	43.18	43.18
# misassemblies	2,041	2,342
# complete genes	19,737	19,540
# partial genes	5,076	5,274
Number of sex-specific bases in assembly (bp)	103,749 (0.0157%)	64,323 (0.0097%)
Longest sex-specific sequence length (bp)	2,881	1,658

In order to map sex-specific regions in each assembly, we mapped sex-specific reads to the opposite sex-specific assembly. We found that sex-specific regions were minimal, accounting for ∼0.01% of total base pairs and with continuous sequences as long as 2,881 and 1,658 bp in the female and the male, respectively (table 3). Comparison of sex-specific regions against the NR protein database showed inconclusive results, with few regions indicating coding potential, many of which with similarities to hypothetical or partial proteins (supplementary file S1, Supplementary Material online).

Conclusions

We report the first draft of the genome of Arapaima gigas, a fascinating Osteoglossomorpha species of bony-tongued fishes. The final draft assembly comprised ∼661.3 Mb, accounting for 86.9% of the estimated genome size (761.1 Mb). We also predicted 24,655 protein-coding genes from the generated assembly. Our phylogenomic analysis supported the postulation that Osteoglossomorpha and Elopomorpha are sister groups that diverged in a short period of time during the Jurassic. The data and resources produced in this study will be valuable to future analysis to understand the species evolutionary history, its breeding mechanism, and its large size. Additionally, these findings might contribute to the environmental protection of this ancient teleost species.

Supplementary Material

Supplementary data are available at Genome Biology and Evolution online.

Author Contributions

S.S., S.J.S., A.R.S., P.P.A., E.H.C.O., and I.G.H. designed the study. J.E.S.S. and P.A.A.S. performed sequencing quality control and assembled the genome. R.A.V. performed the genome annotation. R.A.V., K.P.L., A.M.R.S., and F.A.O.S. analyzed the data. R.A.V., T.S., D.G.T., and J.P.M.S.L. performed the evolutionary analysis. C.F. performed the DNA sequencing. R.A.V., H.N.S., K.P.L., and S.J.S. wrote the manuscript. All authors participated in discussions and provided advice. All authors read and approved the final manuscript.