Neotropical tetras of the family Characidae form the largest and most taxonomically complex clade within the order Characiformes. Previous phylogenetic relationships concur on the recognition of four major subclades, whereas knowledge on intergeneric and interspecific relationships remains largely incomplete or nonexistent. We sampled 575 specimens of 494 species and 123 genera classified in Characidae, generated new molecular data of 1.75 ultraconserved elements (UCEs), and used likelihood and Bayesian analyses. The phylogeny (1348 UCE loci: 538 472 bp) yielded clades with unprecedented resolution at species- and genus-levels, allowing us to propose a new classification of former Characidae into four families: Spintherobolidae, Stevardiidae, Characidae, and Acestrorhamphidae. The family Stevardiidae includes nine subfamilies: Landoninae, Xenurobryconinae, Glandulocaudinae, Argopleurinae, Hemibryconinae, Stevardiinae, Planaltininae, Creagrutinae, and Diapominae. The family Characidae includes five subfamilies: Aphyocharacinae, Cheirodontinae, Exodontinae, Tetragonopterinae, and Characinae. The family Acestrorhamphidae congregates 15 subfamilies: Oxybryconinae, Trochilocharacinae, Stygichthyinae, Megalamphodinae, Stichonodontinae, unnamed subfamily, Stethaprioninae, Pristellinae, Jupiabinae, Tyttobryconinae, Hyphessobryconinae, Thayeriinae, Rhoadsiinae, Grundulinae, and Acestrorhamphinae. The phylogeny resolves intergeneric relationships and supports revalidation of Myxiops, Megalamphodus, Ramirezella, Holopristis, and Astyanacinus, synonymy of Aphyodite, Genycharax, and Psellogrammus, and expansion of Cyanogaster, Makunaima, Deuterodon, Hasemania, Hemigrammus, Bario, Ctenobrycon, and Psalidodon. The phylogeny opens avenues for new systematic reviews and redefinitions of included genera.

We sampled 575 specimens of 494 species and 123 genera of Characidae s.l. and outgroups representing 39.4% of the 1,255 species and 86.7% of the genera currently classified in Characidae. Staff from Arbor Biosciences quantified and enriched genomic libraries utilizing the MYbaits Target Enrichment system (MYcroarray) with the Ostariophysans-UCE-2.7Kv1 probe-set containing 6,737 baits to capture 2,708 nuclear ultraconserved element (UCE) loci. Sequencing was performed on the Illumina platform at Arbor Biosciences with additional details about laboratory procedures available in previous publications for Characiformes. We used the PHYLUCE v1.5.0 pipeline for analyses of UCE data that included the assembly of raw read sequences to contigs, the identification and separation of UCI loci from contigs, and the building of trimmed alignments of the individual UCE loci. We removed adapter contamination and low-quality bases using Illumiprocessor and Trimmomatic, and used Velvet v1.5.0 to assemble fastq reads into fasta contigs. We then searched for the UCE loci using the Ostariophysans-UCE-2.7Kv1 probe-set and the “phyluce_assembly_match_contigs_to_probes” to remove duplicated or paralog regions. After extracting the UCE loci, we aligned them using the edge trimming method implemented in MUSCLE. We used the 75% complete matrix with loci present in at least 75% of taxa (i.e., loci present in at least 432 terminals), and the 90% complete matrix with loci present in at least 90% of taxa (i.e., loci present in at least 518 terminals).