Genetic relationships among freshwater mussel species from fifteen Amazonian rivers and inferences on the evolution of the Hyriidae (Mollusca: Bivalvia: Unionida) Guilherme da Cruz Santos-Neto a,b,⇑ , Colin Robert Beasley c , Horacio Schneider d , Daniel Mansur Pimpão e , Walter Randolph Hoeh f , Luiz Ricardo Lopes de Simone g , Claudia Helena Tagliaro a,⇑ a Laboratório de Conservação e Biologia Evolutiva, Instituto de Estudos Costeiros, Campus de Bragança, Universidade Federal do Pará, Alameda Leandro Ribeiro, s/n, Bairro Aldeia, Bragança, Pará CEP 68600-000, Brazil b Instituto Federal de Educação, Ciência e Tecnologia do Pará, Campus Abaetetuba, Rua Rio de Janeiro, 2233, Bairro Francilândia, Abaetetuba, Pará CEP 68440-000, Brazil c Laboratório de Moluscos, Instituto de Estudos Costeiros, Campus de Bragança, Universidade Federal do Pará, Alameda Leandro Ribeiro, s/n, Bairro Aldeia, Bragança, Pará CEP 68600-000, Brazil d Laboratório de Biologia Molecular, Instituto de Estudos Costeiros, Campus de Bragança, Universidade Federal do Pará, Alameda Leandro Ribeiro, s/n, Bairro Aldeia, Bragança, Pará CEP 68600-000, Brazil e Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis, Superintendência Regional no Goiás, Rua 229 n°95, Leste Universitário, Goiania, Goiás CEP 74605-090, Brazil f Department of Biological Sciences, Kent State University, Cunningham Hall, Summit Street, Kent, OH 44242, USA g Museu de Zoologia da Universidade de São Paulo, Caixa Postal 42494, CEP 04299-970 São Paulo, SP, Brazil article info Article history: Received 23 August 2015 Revised 19 March 2016 Accepted 9 April 2016 Available online 09 April 2016 Keywords: Freshwater bivalve Molecular phylogeny Neotropical mtDNA 18S-ITS1 abstract The current phylogenetic framework for the South American Hyriidae is solely based on morphological data. However, freshwater bivalve morphology is highly variable due to both genetic and environmental factors. The present study used both mitochondrial (COI and 16S) and nuclear (18S-ITS1) sequences in molecular phylogenetic analyses of nine Neotropical species of Hyriidae, collected from 15 South American rivers, and sequences of hyriids from Australia and New Zealand obtained from GenBank. The present molecular findings support traditional taxonomic proposals, based on morphology, for the South American subfamily Hyriinae, currently divided in three tribes: Hyriini, Castaliini and Rhipidodontini. Phylogenetic trees based on COI nucleotide sequences revealed at least four geographical groups of Castalia ambigua: northeast Amazon (Piriá, Tocantins and Caeté rivers), central Amazon, includ- ing C. quadrata (Amazon and Aripuanã rivers), north (Trombetas river), and C. ambigua from Peru. Genetic distances suggest that some specimens may be cryptic species. Among the Hyriini, a total evidence data set generated phylogenetic trees indicating that Paxyodon syrmatophorus and Prisodon obliquus are more closely related, followed by Triplodon corrugatus. The molecular clock, based on COI, agreed with the fossil record of Neotropical hyriids. The ancestor of both Australasian and Neotropical Hyriidae is estimated to have lived around 225 million years ago. Ó 2016 Elsevier Inc. All rights reserved. 1. Introduction Traditional taxonomic classifications of South American fresh- water mussels (for example, d’Orbigny, 1847; Graf, 2000; Lange- de-Morretes, 1949; Ortmann, 1921; Parodiz and Bonetto, 1963; von Ihering, 1890) have been based on morphological characteris- tics of the shell, soft parts and larval morphology. However, some authors (Alvarenga and Ricci, 1981; Graf, 2000; Hoeh et al., 1998) mention a number of difficulties arising from this approach. Intraspecific morphological variability due to environmental or genetic factors, or parallel evolution, can make it difficult to iden- tify the species (Nagel and Badino, 2001; Roper and Hickey, 1994). Morphologically similar cryptic species may go undetected in anal- yses based solely on shell characters, as happened, for example, with Velesunio wilsonii from Australia (Baker et al., 2003). The freshwater mussel family Hyriidae is traditionally placed in the subclass Palaeoheterodonta, order Unionida (Nevesskaja, http://dx.doi.org/10.1016/j.ympev.2016.04.013 1055-7903/Ó 2016 Elsevier Inc. All rights reserved. ⇑ Corresponding authors at: Laboratório de Conservação e Biologia Evolutiva, Instituto de Estudos Costeiros, Campus de Bragança, Universidade Federal do Pará, Alameda Leandro Ribeiro, s/n, Bairro Aldeia, Bragança, Pará CEP 68600-000, Brazil (G.C. Santos-Neto and C.H. Tagliaro). E-mail addresses: guilherneto@gmail.com (G.d.C. Santos-Neto), tagliaro@ufpa.br (C.H. Tagliaro). Molecular Phylogenetics and Evolution 100 (2016) 148–159 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev