Phylogenetic structure of body shape in a diverse inland ichthyofauna.

Body shape is a fundamental metric of animal diversity affecting critical behavioral and ecological dynamics and conservation status, yet previously available methods capture only a fraction of total body-shape variance. Here we use structure-from-motion (SFM) 3D photogrammetry to generate digital 3D models of adult fishes from the Lower Mississippi Basin, one of the most diverse temperate-zone freshwater faunas on Earth, and 3D geometric morphometrics to capture morphologically distinct shape variables, interpreting principal components as growth fields. The mean body shape in this fauna resembles plesiomorphic teleost fishes, and the major dimensions of body-shape disparity are similar to those of other fish faunas worldwide. Major patterns of body-shape disparity are structured by phylogeny, with nested clades occupying distinct portions of the morphospace, most of the morphospace occupied by multiple distinct clades, and one clade (Acanthomorpha) accounting for over half of the total body shape variance. In contrast to previous studies, variance in body depth (59.4%) structures overall body-shape disparity more than does length (31.1%), while width accounts for a non-trivial (9.5%) amount of the total body-shape disparity.

Biogeography of curimatid fishes reveals multiple lowland-upland river transitions and differential diversification in the Neotropics (Teleostei, Curimatidae).

The Neotropics harbors a megadiverse ichthyofauna comprising over 6300 species with approximately 80% in just three taxonomic orders within the clade Characiphysi. This highly diverse group has evolved in tropical South America over tens to hundreds of millions of years influenced mostly by re-arrangements of river drainages in lowland and upland systems. In this study, we investigate patterns of spatial diversification in Neotropical freshwater fishes in the family Curimatidae, a species-rich clade of the order Characiformes. Specifically, we examined ancestral areas, dispersal events, and shifts in species richness using spatially explicit biogeographic and macroevolutionary models to determine whether lowlands-uplands serve as museums or cradles of diversification for curimatids. We used fossil information to estimate divergence times in BEAST, multiple time-stratified models of geographic range evolution in BioGeoBEARS, and alternative models of geographic state-dependent speciation and extinction in GeoHiSSE. Our results suggest that the most recent common ancestor of curimatids originated in the Late Cretaceous likely in lowland paleodrainages of northwestern South America. Dispersals from lowland to upland river basins of the Brazilian and Guiana shields occurred repeatedly across independently evolving lineages in the Cenozoic. Colonization of upland drainages was often coupled with increased rates of net diversification in species-rich genera such as Cyphocharax and Steindachnerina. Our findings demonstrate that colonization of novel aquatic environments at higher elevations is associated with an increased rate of diversification, although this pattern is clade-dependent and driven mostly by allopatric speciation. Curimatids reinforce an emerging perspective that Amazonian lowlands act as a museum by accumulating species along time, whereas the transitions to uplands stimulate higher net diversification rates and lineage diversification.

Assessing extinction risk from geographic distribution data in Neotropical freshwater fishes

The IUCN Red List (RL) provides high-quality conservation assessments for individual species, yet the rate and scale of environmental deterioration globally challenges the conservation community to develop expedited methods for risk assessment. Here we compare threat assessments for 3,001 species of Neotropical freshwater fishes (NFF) in the IUCN-RL using readily accessible data types as proxies for extinction risk: geographic range, elevation, and species publication date. Furthermore, using geographic and taxonomic data alone, we generated preliminary conservation assessments for 2,334 NFF species currently awaiting IUCN assessment, identifying an additional 671 NFF species as potentially threatened. This number of potentially threatened species represents an increase of 59% over the number of species currently assigned to threat categories by the IUCN-RL. These results substantially expand the number of threatened NFF species from 422 currently on the IUCN RL to 1,093 species as threatened or potentially threatened, representing about 18% of all NFF species. Extinction risk is greater in species with smaller geographic ranges, which inhabit upland rivers, and which were described more recently. We propose the Central and Southern Andes, and Eastern Guiana Shield as priorities in the upcoming IUCN RL assessment of NFF species conservation risk.(AU)

A Lista Vermelha (IUCN) fornece avaliações precisas sobre status de conservação das espécies, porém a taxa e escala de deterioração ambiental desafia a comunidade conservacionista a desenvolver métodos rápidos para avaliações de riscos de extinção. Aqui, comparamos as avaliações da IUCN para 3.001 espécies de peixes dulcícolas neotropicais com dados facilmente acessíveis de risco de extinção: extensão de ocorrências, altitude e data de publicação das espécies. Além disso, usando apenas dados geográficos e taxonômicos, geramos avaliações preliminares de conservação para 2.334 espécies de peixes neotropicais aguardando avaliação da IUCN, e identificamos 671 espécies adicionais como potencialmente ameaçadas. Este número de espécies potencialmente ameaçadas representa um aumento de 59% em relação aquelas classificadas nas categorias de ameaça pela IUCN. Estes resultados expandem o número de espécies ameaçadas segundo a IUCN de 422 para 1.093 espécies ameaçadas ou potencialmente ameaçadas, representando cerca de 18% das espécies de peixes neotropicais. O risco de extinção é maior para espécies com distribuições geográficas restritas, que habitam rios de terras altas e que foram descritas mais recentemente. Sugerimos a região Central e Meridional do Andes e o Escudo das Guianas Orientais como prioridades para as próximas avaliações da IUCN sobre a conservação das espécies de peixes dulcícolas neotropicais.(AU)

A shocking discovery of threat risks on newly described species of weakly electric fishes.

This study aims to investigate relationships between species traits and publication date in the weakly electric osteoglossiform Mormyroidea (African knifefish and elephantfishes) and the ostariophysan Gymnotiformes (Neotropical knifefishes). It is investigated whether body size and geographic distribution area are correlated with publication date and whether extinction risk differs between both phylogenetically distant and geographically isolated clades. Statistical modelling indicates that the number of new species described annually is stable in mormyroids and clearly increasing in gymnotiforms. Best-fitting generalised linear models (GLM) indicate that the newly discovered species are more often of small-bodied, predominantly narrowly distributed and more likely to be threatened with extinction. These characteristics are more pronounced in mormyroids when compared with gymnotiforms, suggesting that some African electric fishes may live an ephemeral existence after formal description. Despite taxonomic work has been more intense in the Neotropics than in Africa in the recent decades, there is evidence that the African continent represents the next frontier of species descriptions. Taxonomic studies are fundamental for the understanding of richness and distribution and hence extinction risk assessment and conservation, of these remarkable convergent fish clades.

Using community phylogenetics to assess phylogenetic structure in the Fitzcarrald region of Western Amazonia

Here we explore the use of community phylogenetics as a tool to document patterns of biodiversity in the Fitzcarrald region, a remote area in Southwestern Amazonia. For these analyses, we subdivide the region into basin-wide assemblages encompassing the headwaters of four Amazonian tributaries (Urubamba, Yuruá, Purús and Las Piedras basins), and habitat types: river channels, terra firme (non-floodplain) streams, and floodplain lakes. We present a robust, well-documented collection of fishes from the region including 272 species collected from 132 field sites over 63 field days and four years, comprising the most extensive collection of fishes from this region to date. We conduct a preliminary community phylogenetic analysis based on this collection and recover results largely statistically indistinguishable from the random expectation, with only a few instances of phylogenetic structure. Based on these results, and of those published in other recent biogeographic studies, we conclude that the Fitzcarrald fish species pool accumulated over a period of several million years, plausibly as a result of dispersal from the larger species pool of Greater Amazonia.(AU)

Aquí exploramos el uso de la filogenética de comunidades como herramienta para documentar patrones de biodiversidad en la región de Fitzcarrald, un área remota en el suroeste de la Amazonía. Para estos análisis subdividimos la región en grupos de toda la cuenca que abarcan las cabeceras de cuatro tributarios del Amazonas (cuencas Urubamba, Yuruá, Purús y Las Piedras) y en los tipos de hábitat: canales fluviales, arroyos de tierra firme (sin planicie aluvial) y lagos de planicie aluvial. Presentamos una colección de peces robusta y bien documentada que incluye 272 especies, colectadas a lo largo de cuatro años y 63 días de campo, en 132 puntos de monitoreo. Convirtiéndose en la colección más extensa de peces de esta región hasta la fecha. Realizamos un análisis filogenético preliminar de la comunidad basado en esta recopilación y recuperamos resultados en gran medida estadísticamente indistinguibles de la expectativa aleatoria, con sólo unos pocos casos de estructura filogenética. Basándonos en estos resultados y los publicados en otros estudios biogeográficos recientes, concluimos que el grupo de especies de peces de Fitzcarrald acumulado durante un período de varios millones de años, se debe posiblemente al resultado de la dispersión del mayor grupo de especies de la Gran Amazonia.(AU)

Phylogenetic revision of Gymnotidae (Teleostei: Gymnotiformes), with descriptions of six subgenera.

The diversity of gymnotid electric fishes has been intensely studied over the past 25 years, with 35 species named since 1994, compared to 11 species in the previous 236 years. Substantial effort has also been applied in recent years to documenting gymnotid interrelationships, with seven systematic studies published using morphological and molecular datasets. Nevertheless, until now, all gymnotids have been assigned to one of just two supraspecific taxa, the subfamily Electrophorinae with one genus Electrophorus and three valid species and the subfamily Gymnotine also with one genus Gymnotus and 43 valid species. This simple classification has obscured the substantial phenotypic and lineage diversity within the subfamily Gymnotine and hampered ecological and evolutionary studies of gymnotid biology. Here we present the most well-resolved and taxon-complete phylogeny of the Gymnotidae to date, including materials from all but one of the valid species. This phylogeny was constructed using a five-gene molecular dataset and a 115-character morphological dataset, enabling the inclusion of several species for which molecular data are still lacking. This phylogeny was time-calibrated using biogeographical priors in the absence of a fossil record. The tree topology is similar to those of previous studies, recovering all the major clades previously recognized with informal names. We propose a new gymnotid classification including two subfamilies (Electrophorinae and Gymnotinae) and six subgenera within the genus Gymnotus. Each subgenus exhibits a distinctive biogeographic distribution, within which most species have allopatric distributions and the subgenera are diverged from one another by an estimated 5-35 million years. We further provide robust taxonomic diagnoses, descriptions and identification keys to all gymnotid subgenera and all but four species. This new taxonomy more equitably partitions species diversity among supra-specific taxa, employing the previously vacant subgenus and subfamily ranks. This new taxonomy renders known gymnotid diversity more accessible to study by highlighting the deep divergences (chronological, geographical, genetic and morphological) among its several clades.

Bony Patchwork: Mosaic Patterns of Evolution in the Skull of Electric Fishes (Apteronotidae: Gymnotiformes).

Mosaic evolution refers to the pattern whereby different organismal traits exhibit differential rates of evolution typically due to reduced levels of trait covariation through deep time (i.e., modularity). These differences in rates can be attributed to variation in responses to selective pressures between individual traits. Differential responses to selective pressures also have the potential to facilitate functional specialization, allowing certain traits to track environmental stimuli more closely than others. The teleost skull is a multifunctional structure comprising a complex network of bones and thus an excellent system for which to study mosaic evolution. Here we construct an ultrametric phylogeny for a clade of Neotropical electric fishes (Apteronotidae: Gymnotiformes) and use three-dimensional geometric morphometrics to investigate patterns of mosaic evolution in the skull and jaws. We find strong support for a developmental, three-module hypothesis that consists of the face, braincase, and mandible, and we find that the mandible has evolved four times faster than its neighboring modules. We hypothesize that the functional specialization of the mandible in this group of fishes has allowed it to outpace the face and braincase and evolve in a more decoupled manner. We also hypothesize that this pattern of mosaicism may be widespread across other clades of teleost fishes.

Molecular phylogeny of the ghost knifefishes (Gymnotiformes: Apteronotidae).

Ghost knifefishes (Gymnotiformes: Apteronotidae) are weakly electric fishes that possess a high-frequency, neurogenic electric organ discharge. They are found throughout the humid Neotropics from Panama to Argentina and are most diverse and abundant in the channels of large lowland rivers. Apteronotidae is the most species-rich family of Neotropical electric knifefishes with 96 valid species in 15 genera. We present a phylogenetic hypothesis based on molecular sequence data from three mitochondrial genes (16S, coi, cytb) and four nuclear loci (glyt, rag2, ryr3, zic1). Our analysis includes sequence data for 203 samples in 54 species and 14 genera, making it the most densely-sampled and data-rich phylogeny of the Apteronotidae to date. Our results corroborate previous phylogenetic hypotheses with the placement of Orthosternarchus + Sternarchorhamphus sister to all other apteronotids, a non-monophyletic Apterontotus, and a sister relationship between Sternarchorhynchus and the Navajini. We also report several novel relationships, particularly within the Navajini and among several species of the nominal genus Apteronotus not previously included in phylogenetic analyses. We additionally provide a new classification for the family.

Resolving Deep Nodes in an Ancient Radiation of Neotropical Fishes in the Presence of Conflicting Signals from Incomplete Lineage Sorting.

Resolving patterns of ancient and rapid diversifications is one of the most challenging tasks in evolutionary biology. These difficulties arise from confusing phylogenetic signals that are associated with the interplay of incomplete lineage sorting (ILS) and homoplasy. Phylogenomic analyses of hundreds, or even thousands, of loci offer the potential to resolve such contentious relationships. Yet, how much useful phylogenetic information these large data sets contain remains uncertain and often goes untested. Here, we assess the utility of different data filtering approaches to maximize phylogenetic information and minimize noise when reconstructing an ancient radiation of Neotropical electric knifefishes (Order Gymnotiformes) using ultraconserved elements. We found two contrasting hypotheses of gymnotiform evolutionary relationships depending on whether phylogenetic inferences were based on concatenation or coalescent methods. In the first case, all analyses inferred a previously-and commonly-proposed hypothesis, where the family Apteronotidae was found as the sister group to all other gymnotiform families. In contrast, coalescent-based analyses suggested a novel hypothesis where families producing pulse-type (viz., Gymnotidae, Hypopomidae, and Rhamphichthyidae) and wave-type electric signals (viz., Apteronotidae, Sternopygidae) were reciprocally monophyletic. Nodal support for this second hypothesis increased when analyzing loci with the highest phylogenetic information content and further increased when data were pruned using targeted filtering methods that maximized phylogenetic informativeness at the deepest nodes of the Gymnotiformes. Bayesian concordance analyses and topology tests of individual gene genealogies demonstrated that the difficulty of resolving this radiation was likely due to high gene-tree incongruences that resulted from ILS. We show that data filtering reduces gene-tree heterogeneity and increases nodal support and consistency of species trees using coalescent methods; however, we failed to observe the same effect when using concatenation methods. Furthermore, the targeted filtering strategies applied here support the use of "gene data interrogation" rather than "gene genealogy interrogation" approaches in phylogenomic analyses, to extract phylogenetic signal from intractable portions of the Tree of Life.

Estimating Improved Partitioning Schemes for Ultraconserved Elements.

Ultraconserved (UCEs) are popular markers for phylogenomic studies. They are relatively simple to collect from distantly-related organisms, and contain sufficient information to infer relationships at almost all taxonomic levels. Most studies of UCEs use partitioning to account for variation in rates and patterns of molecular evolution among sites, for example by estimating an independent model of molecular evolution for each UCE. However, rates and patterns of molecular evolution vary substantially within as well as between UCEs, suggesting that there may be opportunities to improve how UCEs are partitioned for phylogenetic inference. We propose and evaluate new partitioning methods for phylogenomic studies of UCEs: Sliding-Window Site Characteristics (SWSC), and UCE Site Position (UCESP). The first method uses site characteristics such as entropy, multinomial likelihood, and GC content to generate partitions that account for heterogeneity in rates and patterns of molecular evolution within each UCE. The second method groups together nucleotides that are found in similar physical locations within the UCEs. We examined the new methods with seven published data sets from a variety of taxa. We demonstrate the UCESP method generates partitions that are worse than other strategies used to partition UCE data sets (e.g., one partition per UCE). The SWSC method, particularly when based on site entropies, generates partitions that account for within-UCE heterogeneity and leads to large increases in the model fit. All of the methods, code, and data used in this study, are available from https://github.com/Tagliacollo/PartitionUCE. Simplified code for implementing the best method, the SWSC-EN, is available from https://github.com/Tagliacollo/PFinderUCE-SWSC-EN.

Shift from slow- to fast-water habitats accelerates lineage and phenotype evolution in a clade of Neotropical suckermouth catfishes (Loricariidae: Hypoptopomatinae).

Identifying habitat characteristics that accelerate organismal evolution is essential to understanding both the origins of life on Earth and the ecosystem properties that are most critical to maintaining life into the future. Searching for these characteristics on a large scale has only recently become possible via advances in phylogenetic reconstruction, time-calibration, and comparative analyses. In this study, we combine these tools with habitat and phenotype data for 105 species in a clade of Neotropical suckermouth catfishes commonly known as cascudinhos. Our goal was to determine whether riverine mesohabitats defined by different flow rates (i.e., pools vs. rapids) and substrates (plants vs. rocks) have affected rates of cascudinho cladogenesis and morphological diversification. In contrast to predictions based on general theory related to life in fast-flowing, rocky riverine habitats, Neoplecostomini lineages associated with these habitats exhibited increased body size, head shape diversity, and lineage and phenotype diversification rates. These findings are consistent with a growing understanding of river rapids as incubators of biological diversification and specialization. They also highlight the urgent need to conserve rapids habitats throughout the major rivers of the world.

Fluctuations in Evolutionary Integration Allow for Big Brains and Disparate Faces.

In theory, evolutionary modularity allows anatomical structures to respond differently to selective regimes, thus promoting morphological diversification. These differences can then influence the rate and direction of phenotypic evolution among structures. Here we use geometric morphometrics and phenotypic matrix statistics to compare rates of craniofacial evolution and estimate evolvability in the face and braincase modules of a clade of teleost fishes (Gymnotiformes) and a clade of mammals (Carnivora), both of which exhibit substantial craniofacial diversity. We find that the face and braincase regions of both clades display different degrees of integration. We find that the face and braincase evolve at similar rates in Gymnotiformes and the reverse in Carnivora with the braincase evolving twice as fast as the face. Estimates of evolvability and constraints in these modules suggest differential responses to selection arising from fluctuations in phylogenetic integration, thus influencing differential rates of skull-shape evolution in these two clades.

Coordinated Dispersal and Pre-Isthmian Assembly of the Central American Ichthyofauna.

We document patterns of coordinated dispersal over evolutionary time frames in heroine cichlids and poeciliine live-bearers, the two most species-rich clades of freshwater fishes in the Caribbean basin. Observed dispersal rate (DO) values were estimated from time-calibrated molecular phylogenies in Lagrange+, a modified version of the ML-based parametric biogeographic program Lagrange. DO is measured in units of "wallaces" (wa) as the number of biogeographic range-expansion events per million years. DO estimates were generated on a dynamic paleogeographic landscape of five areas over three time intervals from Upper Cretaceous to Recent. Expected dispersal rate (DE) values were generated from alternative paleogeographic models, with dispersal rates proportional to target area and source-river discharge volume, and inversely proportional to paleogeographic distance. Correlations between DO and DE were used to assess the relative contributions of these three biogeographic parameters. DO estimates imply a persistent dispersal corridor across the Eastern (Antillean) margin of the Caribbean plate, under the influence of prevailing and perennial riverine discharge vectors such as the Proto-Orinoco-Amazon river. Ancestral area estimation places the earliest colonizations of the Greater Antilles and Central America during the Paleocene-Eocene (ca. 58-45 Ma), potentially during the existence of an incomplete Paleogene Arc (∼59 Ma) or Lesser Antilles Arc (∼45 Ma), but predating the GAARlandia land bridge (∼34-33 Ma). Paleogeographic distance is the single best predictor of DO. The Western (Central American) plate margin did not serve as a dispersal corridor until the Late Neogene (12-0 Ma), and contributed relatively little to the formation of modern distributions.

Data supporting phylogenetic reconstructions of the Neotropical clade Gymnotiformes.

Data is presented in support of model-based total evidence (MBTE) phylogenetic reconstructions of the Neotropical clade of Gymnotiformes "Model-based total evidence phylogeny of Neotropical electric knifefishes (Teleostei, Gymnotiformes)" (Tagliacollo et al., 2016) [1]). The MBTE phylogenies were inferred using a comprehensive dataset comprised of six genes (5277 bp) and 223 morphological characters for an ingroup taxon sample of 120 of 218 valid species and 33 of the 34 extant genera. The data in this article include primer sequences for gene amplification and sequencing, voucher information and GenBank accession numbers, descriptions of morphological characters, morphological synapomorphies for the recognized clades of Gymnotiformes, a supermatrix comprised of concatenated molecular and morphological data, and computer scripts to replicate MBTE inferences. We also included here Maximum-likelihood and Bayesian topologies, which support two main gymnotiform clades: Gymnotidae and Sternopygoidei, the latter comprised of Rhamphichthyoidea (Rhamphichthyidae+Hypopomidae) and Sinusoidea (Sternopygidae+Apteronotidae).

Transcontinental dispersal, ecological opportunity and origins of an adaptive radiation in the Neotropical catfish genus Hypostomus (Siluriformes: Loricariidae).

Ecological opportunity is often proposed as a driver of accelerated diversification, but evidence has been largely derived from either contemporary island radiations or the fossil record. Here, we investigate the potential influence of ecological opportunity on a transcontinental radiation of South American freshwater fishes. We generate a species-dense, time-calibrated molecular phylogeny for the suckermouth armored catfish subfamily Hypostominae, with a focus on the species-rich and geographically widespread genus Hypostomus. We use the resulting chronogram to estimate ancestral geographical ranges, infer historical rates of cladogenesis and diversification in habitat and body size and shape, and test the hypothesis that invasions of previously unoccupied river drainages accelerated evolution and contributed to adaptive radiation. Both the subfamily Hypostominae and the included genus Hypostomus originated in the Amazon/Orinoco ecoregion. Hypostomus subsequently dispersed throughout tropical South America east of the Andes Mountains. Consequent to invasion of the peripheral, low-diversity Paraná River basin in southeastern Brazil approximately 12.5 Mya, Paraná lineages of Hypostomus, experienced increased rates of cladogenesis and ecological and morphological diversification. Contemporary lineages of Paraná Hypostomus are less species rich but more phenotypically diverse than their congeners elsewhere. Accelerated speciation and morphological diversification rates within Paraná basin Hypostomus are consistent with adaptive radiation. The geographical remoteness of the Paraná River basin, its recent history of marine incursion, and its continuing exclusion of many species that are widespread in other tropical South American rivers suggest that ecological opportunity played an important role in facilitating the observed accelerations in diversification.

Model-based total evidence phylogeny of Neotropical electric knifefishes (Teleostei, Gymnotiformes).

This study provides the most comprehensive Model-Based Total Evidence (MBTE) phylogenetic analyses of the clade Gymnotiformes to date, reappraising relationships using a dataset comprised of six genes (5277bp) and 223 morphological characters, and an ingroup taxon sample including 120 of 212 valid species representing 34 of the 35 extant genera. Our MBTE analyses indicate the two main gymnotiform clades are Gymnotidae and Sternopygoidei, the latter comprised of Rhamphichthyoidea (Rhamphichthyidae+Hypopomidae) and Sinusoidea (Sternopygidae+Apteronotidae). Within Gymnotidae, Electrophorus and Gymnotus are sister taxa, and Gymnotus includes the following six clades: (i) G. pantherinus clade, (ii) G. coatesi clade, (iii) G. anguillaris clade, (iv) G. tigre clade, (v) G. cylindricus clade, and (vi) G. carapo clade. Within Rhamphichthyoidea, Steatogenae (Steatogenys+Hypopygus) is a member of Rhamphichthyidae, and Hypopomidae includes the following clades: (i) Akawaio, (ii) Hypopomus, (iii) Microsternarchini, and (iv) Brachyhypopomus. Within Sternopygidae, Sternopygus and Eigenmanninae are sister groups, Rhabdolichops is the sister to other Eigenmanninae, Archolaemus+Distocyclus is the sister to Eigenmannia, and Japigny is nested within Eigenmannia. Within Apteronotidae, Sternarchorhamphinae (Sternarchorhamphus+Orthosternarchus) is the sister to Apteronotinae, Adontosternarchus is the sister group to other Apteronotinae, Sternarchorhynchini (Sternarchorhynchus+Platyurosternarchus) is the sister to Navajini, and species assigned to Apteronotus are members of two separate clades: (i) A. sensu stricto in the Apteronotini, and (ii) the "A." bonapartii clade in the Navajini.

Molecular phylogeny of Aphyocharacinae (Characiformes, Characidae) with morphological diagnoses for the subfamily and recognized genera.

The subfamily Aphyocharacinae was recently redefined to comprise eight genera: Aphyocharax, Prionobrama, Paragoniates, Phenagoniates, Leptagoniates, Xenagoniates, Rachoviscus and Inpaichthys. This new composition, however, is partially incongruent with published results of molecular studies especially concerning the positions of Rachoviscus and Inpaichthys. Our goal was to investigate the monophyly of Aphyocharacinae and its interrelationships using three distinct phylogenetic methodologies: Maximum-likelihood and Bayesian analyses of molecular data, and also Parsimony analysis of a concatenated molecular and morphological dataset. All tree topologies recovered herein suggest that Rachoviscus, Inpaichthys and Leptagoniates pi do not belong to the Aphyocharacinae. The remaining aphyocharacin taxa analyzed do form a monophyletic group, which is itself composed of two subgroups being one comprised of Paragoniates, Phenagoniates, Leptagoniates and Xenagoniates, and the other comprised of Aphyocharax and Prionobrama. Internal relationships among these genera are statistically well supported and morphological synapomorphies are presented at the generic level. All tree topologies also indicate that Aphyocharacidium is closely related to Aphyocharacinae suggesting that it should be included in this subfamily. As recognized in the present study, the Aphyocharacinae is diagnosed by a single morphological synapomorphy: two dorsal-fin rays articulating with the first dorsal pterygiophore.