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Occurrence; Specimen

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Subfamily Trichomycteridae is endemic to the Neotropics. The biological samples collected and preserved by my team come exclusively from Brazil.

The fish are known as catfish and are delimited at the level of genus and species, contemplating a large representation of the subfamily Trichomycterinae, mainly of the genus Trichomycterus.

(1) Background: Trichomycterinae represent 60% of taxa in the family and, while seven genera comprise 1–3 species each, Trichomycterus and Cambeva have over 180 known species between them. Although integrative studies aimed to clarify the relationships within the subfamily, the diversity of Trichomycterus species remains an open question. Herein, we explored an unprecedented sample to investigate the divergence in the lineages of Trichomycterus; (2) Methods: we recovered the phylogenetic relationships of the subfamily using 566 sequences (999 bp) of the mitochondrial gene cytochrome b, calculated intra- and intergroup distance percentages, and estimate divergence times; (3) Results: we recovered 13 highly supported and geographically structured lineages; inter-genus divergence was 11–20%, while inter-species divergence was 3–11%; Trichomycterus, Cambeva, Scleronema, Hatcheria, Eremophilus and Ituglanis were recovered as monophyletic, with three other highly divergent clades: Guiana Shield, Magdalena basin and Tapajós basin; (4) Conclusions: we propose that the Trans-Andean austral clades be allocated into Hatcheria, and the Guiana clade supports a new genus. We also observed that the headwaters nearest the Magdalena and Orinoco basins showed a high diversity and endemism of Trichomycterinae lineages. We discussed the role of geomorphological events and the climatic features which may explain cladogenesis events in Trichomycterinae.

The personnel involved in the project:

Our analysis involved geo-referenced 566 sequences of 33 species of Trichomycterinae (Table S1), 364 of which belong to Trichomycterus sensu stricto (including sequences of the type species of the genus T. nigricans Valenciennes 1832). The samples from the Brazilian Atlantic coast comprised 13 sequences of Cambeva spp., four of Scleronema spp., and 14 of Ituglanis spp. The Andean samples consisted of 125 sequences of Trichomycterus areolatus Valenciennes 1846, 10 of Hatcheria macraei Girard 1855, and 12 of Bullockia maldonadoi Eigenmann 1928. The sequences from the northern Amazon basin consisted of 14 sequences of the T. guianense group Eigenmann 1909 from the Guiana Shield, eight from the Magdalena basin, and one Trichomycterus sp. from the Tapajós basin. We also used four sequences as an external group, two from Listrura spp. (Glanapteryginae) and two from Trichogenes longipinnis Britski & Ortega 1983 (Trichogeninae). Of the 324 Brazilian sequenced catfish (323 from Trichomycterus and one from Cambeva), 182 were collected by our team, and 142 were obtained from fish collections. We sequenced these 324 samples and obtained another 246 sequences from GenBank® (ncbi.nlm.nih.gov). Fieldwork was carried out between June 2013 and October 2014 on expeditions that revisited the type-localities of the eastern Brazilian species between the Jequitinhonha River basin in the north and the Itabapoana River basin in the south. Specimen sampling procedures followed the standard protocol, mainly using trawl nets. Captured specimens were anesthetized by submersion in eugenol solution and the guidelines for fish euthanasia. Tissue samples of the right flank muscle were obtained from specimens fixed in 90% ethanol and preserved in absolute ethanol for molecular analyses. For morphological comparisons, specimens were fixed in formalin for 15 days and then transferred to 70% ethanol. All specimens were registered in the fish collection at the Instituto Nacional da Mata Atlântica (INMA), formerly the Museu de Biologia Professor Mello Leitão, in Santa Teresa, Espírito Santo, Brazil. We also used sequences from Genbank® of cis- and trans-Andean species of Trichomycterinae, the locations of which ranged from the Isthmus of Panama in the north to Patagonia at the extreme southern limit of the subfamily’s range.

Method step description:

1. DNA samples were extracted following Bruford et al. (1992) and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific™). For the in vitro amplification of the mitochondrial gene cytochrome b (cytb), we used the primers CytbSiluF and CytbSiluR (Villa-Verde et al., 2012). The amplicons were purified by adding 1 μL Exosap to each 10 μL of PCR product. Sequencing reactions were performed using the Big Dye Terminator Cycle Sequencing Kit, following the manufacturer’s protocol. The 332 samples were sequenced in an ABI310 automatic sequencer (Applied Biosystems) at the Núcleo de Genética Aplicada à Conservação da Biodiversidade (NGACB), Universidade Federal do Espírito Santo (UFES).