The complete mitochondrial genome of the threatened Neotropical catfish Lophiosilurus alexandri (Silurifomes: Pseudopimelodidae) and phylogenomic analysis indicate monophyly of Pimelodoidea

Abstract

Lophiosilurus alexandri is an endemic catfish from the São Francisco River Basin (Brazil) popularly known as pacamã, which has economic potential for aquaculture farming. The mitochondrial genome was sequenced for the threatened Neotropical catfish L. alexandri. Assembly into scaffolds using MIRA and MITObim software produced the whole, circularized mitochondrial genome, which comprises 16,445 bp and presents the typical gene arrangement of Teleostei mitochondria. A phylogenomic analysis was performed after the concatenation of all proteins obtained from whole mitogenomes of 20 Siluriformes and two outgroups. The results confirmed the monophyly of nine families of catfishes and also clustered L. alexandri as a sister group to the family Pimelodidae, thus confirming the monophyly of the superfamily Pimelodoidea. This is the first mitochondrial phylogenomics study for Pimelodoidea and the first mitogenome described for the Pseudopimelodidae family, representing an important resource for phylogeography, evolutionary biology, and conservation genetics studies in Neotropical fishes.

Keywords
Mitogenome; fish; next-generation-sequencing; Illumina; Pseudopimelodidae

Catfishes (Siluriformes) are a highly diverse order composed of 39 families and more than 3,700 living species (Eschmeyer and Fong, 2016). The fish Lophiosilurus alexandri (Steindachner, 1877), popularly known as pacamã, is an endemic catfish from the São Francisco River Basin (Brazil) and belongs to the Pseudopimelodidae family, a taxon broadly distributed in South America (Eschmeyer and Fong, 2016). L. alexandri is a carnivorous species that displays parental care and has economic potential for aquaculture farming (Sato et al., 2003; dos Santos and Luz, 2009).

Pacamã may be cultivated in captivity after adapting broodstock to aquaculture conditions (Costa et al., 2015), however its natural populations have declined over the last decades. Threats such as overfishing and environmental degradation led to the inclusion of L. alexandri in the Brazilian red list of threatened species (Brasil, 2014).

Here we present the complete, circularized version of the whole mitochondrial genome and the phylogenomic relationships of pacamã to 20 other related Siluriformes and two outgroups using a supermatrix approach.

Muscle fragments were obtained from a freshly captured L. alexandri. The voucher specimen was fixed in 10% formalin and later preserved in 70% ethanol. (voucher: LGC6088 at PUC Minas Natural History Museum). Genomic DNA was extracted using a modified salting-out method (Sunnucks and Hales, 1996) and nebulized for 6 min to obtain 200-600 bp fragments.

Partial genome sequencing was carried out using a Nextera kit in a sixth of an Illumina HiSeq 2000 lane. An initial mitogenome assembly generated by MIRA was used as an input for the MITObim algorithm (Hahn et al., 2013) using default parameters. Mitogenome coverage was obtained using Tablet software (Milne et al., 2013). Mitos WebServer (Bernt et al., 2013) and MitoFish (Iwasaki et al., 2013) were used for annotation. Blast searches (Altschul et al., 1997) against fish amino acid sequences confirmed gene boundaries. tRNA predictions were confirmed using tRNAscan-SE (Lowe and Eddy, 1997). Ribosomal RNA annotations were estimated through automatic analysis provided by Mitos Web Server and MitoFish followed by visual inspection of nucleotide sequence alignments against other Pimelodidae rRNAs.

The complete mitochondrial genome for L. alexandri was assembled using 0.06% (203,036 reads) of the total paired-end reads sequenced (33,839,478 reads of 100 bp each). The assembly provided a circular mitogenome with 134.1 x coverage comprised 16,445 bp, a size similar to the average mitogenome of catfishes. Its gene content also followed the typical pattern for teleost mitogenomes (Prosdocimi et al., 2012, Song et al., 2012, Zhang et al., 2013, Perini et al., 2014), being composed of 37 genes, including 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and 1 non-coding control region (Table S1, Supplementary Material). The mitogenome of Lophiosilurus alexandri is available in GenBank under the accession number KJ494387.

An in-house pipeline developed in Python (https://github.com/igorrcosta/phylomito) was used to: (i) concatenate individual alignments of mitochondrial proteins, (ii) retrotranslate these alignments into codons/nucleotides, and (iii) provide a supermatrix dataset that has been used as input into MEGA 7 for modeltest and phylogeny reconstruction (Kumar et al., 2016).

The phylogenetic relationship between L. alexandri and the other 20 catfishes and two outgroups from the Gymnotiformes and Characiformes orders (Table 1) was recovered using a supermatrix approach of 11,468 nucleotides produced after the concatenation and reverse translation of protein sequence alignments for all the 13 mitochondrial proteins. A maximum likelihood tree was generated using all alignment sites with the best model found by MEGA (GTR+G+I). The phylogenomic analysis produced a consistent tree in accordance with phylogenetic evidence obtained using rag1 and rag2 nuclear genes recovered using 3,660 base pairs (Sullivan et al., 2006). Whole mitochondrial data, however, also evidenced new features, such as (i) a more ancestral split of Amblycipitidae species and also (ii) a highly supported clade (bootstrap=93) grouping families Ictaluridae, Cranogladidae and Pangasidae. L. alexandri, Pimelodus pictus and two species from the genus Pseudoplatystoma were clustered in the same clade with a very confidence support after 1000 resamplings (bootstrap=100) (Figure 1), corroborating the monophyletic relationship of the Pimelodoidea clade, as previously recovered elsewhere (Sullivan et al., 2006). The mitogenome described here is the first representative for the family Pseudopimelodidae.

Table 1
List of species, taxonomic information and accession numbers used in the phylogenetic analyses.

Order	Family	Species	GenBank ID	Size (bp)	Reference
Siluriformes	Doradidae	Amblydoras gonzalezi	NC_015745.1	16505	Nakatani et al., 2011
Siluriformes	Doradidae	Platydoras armatulus	NC_025585.1	16470	Liu et al., 2016
Siluriformes	Cranoglanididae	Cranoglanis bouderius	NC_008280.1	16539	Peng et al., 2006
Siluriformes	Clariidae	Clarias sp.	NC_015749.1	16508	Nakatani et al., 2011
Siluriformes	Clariidae	Clarias fuscus	NC_023924.1	16518	Zhou et al., 2015
Siluriformes	Ictaluridae	Ictalurus punctatus	NC_003489.1	16497	Waldbieser et al., 2003
Siluriformes	Ictaluridae	Ictalurus furcatus	NC_028151.1	16499	Liu et al. (Unpublished)
Siluriformes	Amblycipitidae	Liobagrus marginalis	NC_022923.1	16483	Li et al., 2014
Siluriformes	Amblycipitidae	Liobagrus nigricauda	NC_021407.1	16512	Jia et al., 2013b
Siluriformes	Amblycipitidae	Liobagrus obesus	NC_008232.1	16531	Kartavtsev et al., 2007
Siluriformes	Pangasiidae	Pangasianodon gigas	NC_006381.1	16533	Jondeung et al., 2007
Siluriformes	Pangasiidae	Pangasianodon hypophthalmus	NC_021752.1	16522	Zhao et al., 2014
Siluriformes	Pangasiidae	Pangasius larnaudii	NC_015839.1	16471	Nakatani et al., 2011
Siluriformes	Pangasiidae	Pangasius pangasius	NC_023924.1	16476	Mohindra et al., 2015
Siluriformes	Siluridae	Silurus glanis	NC_014261.1	16526	Vitta et al., 2011
Siluriformes	Siluridae	Silurus meridionalis	NC_014866.1	16527	Liang et al., unpublished
Siluriformes	Siluridae	Pterocryptis conchichinensis	NC_027107.1	16501	Xu et al., 2016
Siluriformes	Pseudopimelodidae	Lophiosilurus alexandri	KJ494387	16445	Present work
Siluriformes	Pimelodidae	Pimelodus pictus	NC_015797.1	16575	Nakatani et al., 2011
Siluriformes	Pimelodidae	Pseudoplatystoma corruscans	NC_026846.1	16123	Prosdocimi et al. (Unpublished)
Siluriformes	Pimelodidae	Pseudoplatystoma magdaleniatum	NC_026526.1	16568	Rangel-Medrano et al., 2015
Characiformes	Characidae	Paracheirodon axelrodi	NC_023270.1	17100	Zhang et al., 2014
Gymnotiformes	Hypopomidae	Brachyhypopomus occidentalis	NC_015078.1	16542	Lavoue et al., 2012

Figure 1
Molecular phylogenetic analysis of pacamã, 20 catfishes and two outgroups. A total of 11,468 positions were analyzed consisting in the concatenation of 13 mitochondrial genes. A tree was built using maximum likelihood approaches with GTR+G+I model. Outgroups were used to root the tree. All families and genera of catfishes analyzed were revealed as monophyletic (colored boxes). Bootstrap values (1000 replicates) are shown at the corresponding nodes.

Acknowledgments

The study received fincancial support from the Brazilian funding agencies FAPERJ (E-26/010.001625/2014 and E-26/202.810/2015) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico; fellowship grant 308537/2014-9).

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Internet Resources

• Eschmeyer WN and Fong JD (2016) Species by family/subfamily, http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamily.asp (May 2, 2016).
  » http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamily.asp.

Supplementary Material

The following online material is available for this article:

Table S1 - Mitochondrial genome characteristics of L. alexandri (KJ494387).

• Associate Editor: Houtan Noushmehr

Data availability

Data citations

Eschmeyer WN and Fong JD (2016) Species by family/subfamily, http://researcharchive.calacademy.org/research/ichthyology/catalog/SpeciesByFamily.asp (May 2, 2016).

Publication Dates

• Publication in this collection
  19 Sept 2016
• Date of issue
  Oct-Dec 2016

History

• Received
  19 Jan 2016
• Accepted
  20 June 2016