A New Cryptic Species of South American Freshwater Pufferfish of the Genus Colomesus (Tetraodontidae), Based on Both Morphology and DNA Data

Based on Both Morphology and DNA Data. PLoS ONE 8(9): e74397. doi:10.1371/journal.pone.0074397 A New Cryptic Species of South American Freshwater Pufferfish of the Genus Colomesus (Tetraodontidae), Based on Both Morphology and DNA Data Cesar R. L. Amaral 0 Paulo M. Brito 0 Dayse A. Silva 0 Elizeu F. Carvalho 0 Dorothee Huchon, Tel-Aviv University, Israel 0 1 Department of Zoology, Universidade Estadual do Rio de Janeiro , Rio de Janeiro , Brazil , 2 Department of Ecology, Universidade Estadual do Rio de Janeiro , Rio de Janeiro , Brazil The Tetraodontidae are an Acantomorpha fish family with circumglobal distribution composed of 189 species grouped in 19 genera, occurring in seas, estuaries, and rivers between the tropical and temperate regions. Of these, the genus Colomesus is confined to South America, with what have been up to now considered only two species. C. asellus is spread over the entire Amazon, Tocantins-Araguaia drainages, and coastal environments from the Amazon mouth to Venezuela, and is the only freshwater puffers on that continent. C. psittacus is found in coastal marine and brackish water environments from Cuba to the northern coast of South America as far south as to Sergipe in Brazil. In the present contribution we used morphological data along with molecular systematics techniques to investigate the phylogeny and phylogeography of the freshwater pufferfishes of the genus Colomesus. The molecular part is based on a cytochrome C oxidase subunit I dataset constructed from both previously published and newly determined sequences, obtained from specimens collected from three distinct localities in South America. Our results from both molecular and morphological approaches enable us to identify and describe a new Colomesus species from the Tocantins River. We also discuss aspects of the historical biogeography and phylogeography of the South American freshwater pufferfishes, suggesting that it could be more recent than previously expected. - Funding: This study was supported by the Brazilian National Counsel of Technological and Scientific Development and Fundacao Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The Tetraodontidae is an Acantomorpha fish family with circumglobal distribution composed of 189 species in 19 genera, occurring in seas, estuaries, and rivers between the tropical and temperate regions [1]. They are mainly characterized by their typical four large dental plates; the ability to inflate their body in stressful situations; the presence of the neurotoxin Tetrodotoxin/ Saxitoxin in its tissues, being responsible for numerous cases of fatal poisoning in many countries, including Brazil; and by having the smallest genome among vertebrates, therefore being considered as a model for the genome evolution of the group. Among the Amazonian taxa exploited by the ornamental fish industry in South America are those of Colomesus [2], a genus confined to South America, with what is presently considered two species, C. asellus and C. psittacus. C. asellus [3] is spread in the entire Amazon, Tocantins-Araguaia drainages, and coastal environments from the Amazon mouth to Venezuela, being the only freshwater puffers on that continent. C. psittacus [4] is found in coastal marine and brackish water environments from Cuba and the northern coast of South America to Sergipe in Brazil. Mainly located in tropical and subtropical regions all around the world, including the Amazon region, the ornamental fish industry is one of the largest transporters of live animals and plants with an annual trade volume estimated at U$1525 billion [57], in a scenario where species identification problems, mainly related to border biosecurity are not rare. The DNA barcode is a widely accepted tool for species determination mainly due to its enhanced attention on standardization and data validation [8], being a rapid and low cost method of identification [9]. The use of DNA barcoding techniques has been utilized in many taxa, including bacteria, birds, bivalves, butterflies, fishes, flies, macroalgae, mammals, spiders, sprigtails, and also for plants [1027]. The DNA barcode technique for Metazoans uses a short (,650 bp) and standardized gene region from the mitochondrial 59 region of the cytochrome C oxidase subunit I (COI) for a rapid and cost-effective animal identification. This has been demonstrated to be an effective fish identification tool in numerous situations, including consumer protection [2830], fisheries management/conservation [31], border biosecurity in the ornamental fish trade [5], and in the identification of overlooked or cryptic species [32]. Here we used both morphological and molecular methodologies in an integrative taxonomical approach to investigate the diversity of the Amazonian freshwater pufferfishes of the genus Colomesus based on specimens collected from three distinct populations from both Brazil and Peru. Additionally, we describe a new Colomesus species from the Upper Tocantins drainage based on both morphological and molecular data. Specimens of Colomesus asellus were collected from three distinct populations with about 2200 km of mean distance separating them. The collection localities were Ilha do Mosqueiro, Belem, Brazil; Upper Tocantins River - Porto Nacional, Tocantins, Brazil; and Nanay River - Iquitos, Peru (Figure 1). Ethics Statement No statement from an ethics committee was necessary, and the manuscript did not involve any endangered or protect species. All samples were extracted from dead specimens collected with appropriate permissions under authorization number 22512 issued by SISBIO/Instituto Chico Mendes de Conservac ao da Biodiversidade. We used the ice-slurry method for killing following [33] as they are tropical warm water species and the collected specimens are all smaller than 5 cm SL. All specimens were preserved in alcohol. The reported localities do not include protected areas. Nomenclatural Acts The electronic version of this document does not represent a published work according to the International Code of Zoological Nomenclature (ICZN), and hence the nomenclatural acts contained in the electronic version are not available under that Code from the electronic edition. Therefore, a separate edition of Sphoeroides annulatus Sphoeroides testudineus (*)Sequences newly determined in this study. doi:10.1371/journal.pone.0074397.t001 this document was produced by a method that assures numerous identical and durable copies, and those copies were simultaneously obtainable (from the publication date noted on the first page of this article) for the purpose of providing a public and permanent scientific record, in accordance with Article 8.1 of the Code. The separate print-only editi (...truncated)