River drainage reorganization and reticulate evolution in the Two-Lined Salamander (Eurycea bislineata) species complex.

The origin and eventual loss of biogeographic barriers can create alternating periods of allopatry and secondary contact, facilitating gene flow among distinct metapopulations and generating reticulate evolutionary histories that are not adequately described by a bifurcating evolutionary tree. One such example may exist in the two-lined salamander (Eurycea bislineata) species complex, where discordance among morphological and molecular datasets has created a "vexing taxonomic challenge". Previous phylogeographic analyses of mitochondrial DNA (mtDNA) suggested that the reorganization of Miocene paleodrainages drove vicariance and dispersal, but the inherent limitations of a single-locus dataset precluded the evaluation of subsequent gene flow. Here, we generate triple-enzyme restriction site-associated DNA sequencing (3RAD) data for >100 individuals representing all major mtDNA lineages and use a suite of complementary methods to demonstrate that discordance among earlier datasets is best explained by a reticulate evolutionary history influenced by river drainage reorganization. Systematics of such groups should acknowledge these complex histories and relationships that are not strictly hierarchical.

Breeding behaviour predicts patterns of natural hybridization in North American minnows (Cyprinidae).

Premating barriers such as variation in reproductive behaviour can evolve quickly, but because gametic and postzygotic incompatibilities often evolve more slowly, circumstances that bring gametes into contact can breach the boundaries of premating isolation. In aquatic environments, the gametes of organisms with external fertilization are released into a constantly moving environment and may come into contact with heterospecific gametes. In fishes, nest association (spawning in another species' nest) is a behaviour that brings gametes from different species into close spatiotemporal proximity. These interactions might increase chances of hybridization, especially when multiple species associate with a single nest builder. This study addresses these interactions in the largest clade of North American freshwater fishes, the minnows (Cyprinidae). We compiled a list of over 17,000 hybrid specimens in conjunction with species distribution data, breeding behaviours, and an inferred phylogeny to test if breeding behaviour, in addition to evolutionary history, is an important predictor of hybridization. We find that breeding behaviour is a significant predictor of hybridization, even when phylogenetic relatedness and divergence time are accounted for. Specifically, nest associates are more likely to hybridize with other nest associates whereas non-nesting species had relatively low rates of hybridization.

Morphological Polymorphism Associated with Alternative Reproductive Tactics in a Plethodontid Salamander.

Understanding polymorphism is a central problem in evolution and ecology, and alternative reproductive tactics (ARTs) provide compelling examples for studying the origin and maintenance of behavioral and morphological variation. Much attention has been given to examples where "parasitic" individuals exploit the reproductive investment of "bourgeois" individuals, but some ARTs are instead maintained by environmental heterogeneity, with alternative tactics exhibiting differential fitness in discontinuous reproductive niches. We use genomic, behavioral, karyological, and field observational data to demonstrate one such example in plethodontid salamanders. These ARTs ("searching" and "guarding" males) are associated with different reproductive niches and, unlike most other examples in amphibians, demonstrate substantial morphological differences and inflexibility within a reproductive season. Evidence suggests the existence of these ARTs within three putative species in the two-lined salamander (Eurycea bislineata) species complex, with other members of this clade fixed for one of the two tactics. We highlight directions for future research in this system, including the relationship between these ARTs and parental care.

A hierarchical Bayesian model to incorporate uncertainty into methods for diversity partitioning.

Recently there have been major theoretical advances in the quantification and partitioning of diversity within and among communities, regions, and ecosystems. However, applying those advances to real data remains a challenge. Ecologists often end up describing their samples rather than estimating the diversity components of an underlying study system, and existing approaches do not easily provide statistical frameworks for testing ecological questions. Here we offer one avenue to do all of the above using a hierarchical Bayesian approach. We estimate posterior distributions of the underlying "true" relative abundances of each species within each unit sampled. These posterior estimates of relative abundance can then be used with existing formulae to estimate and partition diversity. The result is a posterior distribution of diversity metrics describing our knowledge (or beliefs) about the study system. This approach intuitively leads to statistical inferences addressing biologically motivated hypotheses via Bayesian model comparison. Using simulations, we demonstrate that our approach does as well or better at approximating the "true" diversity of a community relative to naïve or ad-hoc bias-corrected estimates. Moreover, model comparison correctly distinguishes between alternative hypotheses about the distribution of diversity within and among samples. Finally, we use an empirical ecological dataset to illustrate how the approach can be used to address questions about the makeup and diversities of assemblages at local and regional scales.

Pairwise beta diversity resolves an underappreciated source of confusion in calculating species turnover.

Beta diversity is an important metric in ecology quantifying differentiation or disparity in composition among communities, ecosystems, or phenotypes. To compare systems with different sizes (N, number of units within a system), beta diversity is often converted to related indices such as turnover or local/regional differentiation. Here we use simulations to demonstrate that these naive measures of dissimilarity depend on sample size and design. We show that when N is the number of sampled units (e.g., quadrats) rather than the "true" number of communities in the system (if such exists), these differentiation measures are biased estimators. We propose using average pairwise dissimilarity as an intuitive solution. That is, instead of attempting to estimate an N-community measure, we advocate estimating the expected dissimilarity between any random pair of communities (or sampling units)-especially when the "true" N is unknown or undefined. Fortunately, measures of pairwise dissimilarity or overlap have been used in ecology for decades, and their properties are well known. Using the same simulations, we show that average pairwise metrics give consistent and unbiased estimates regardless of the number of survey units sampled. We advocate pairwise dissimilarity as a general standardization to ensure commensurability of different study systems.

Extending the Concept of Diversity Partitioning to Characterize Phenotypic Complexity.

Most components of an organism's phenotype can be viewed as the expression of multiple traits. Many of these traits operate as complexes, where multiple subsidiary parts function and evolve together. As trait complexity increases, so does the challenge of describing complexity in intuitive, biologically meaningful ways. Traditional multivariate analyses ignore the phenomenon of individual complexity and provide relatively abstract representations of variation among individuals. We suggest adopting well-known diversity indices from community ecology to describe phenotypic complexity as the diversity of distinct subsidiary components of a trait. Using a hierarchical framework, we illustrate how total trait diversity can be partitioned into within-individual complexity (α diversity) and between-individual components (ß diversity). This approach complements traditional multivariate analyses. The key innovations are (i) addition of individual complexity within the same framework as between-individual variation and (ii) a group-wise partitioning approach that complements traditional level-wise partitioning of diversity. The complexity-as-diversity approach has potential application in many fields, including physiological ecology, ecological and community genomics, and transcriptomics. We demonstrate the utility of this complexity-as-diversity approach with examples from chemical and microbial ecology. The examples illustrate biologically significant differences in complexity and diversity that standard analyses would not reveal.

Tests of two methods for identifying founder effects in metapopulations reveal substantial type II error.

Genetic analysis has been promoted as a way to reconstruct recent historical dynamics ("historical demography") by screening for signatures of events, such as bottlenecks, that disrupt equilibrium patterns of variation. Such analyses might also identify "metapopulation" processes like extinction and recolonization or source-sink dynamics, but this potential remains largely unrealized. Here we use simulations to test the ability of two currently used strategies to distinguish between a set of interconnected subpopulations (demes) that have undergone bottlenecks or extinction and recolonization events (metapopulation dynamics) from a set of static demes. The first strategy, decomposed pairwise regression, provides a holistic test for heterogeneity among demes in their patterns of isolation-by-distance. This method suffered from a type II error rate of 59-100 %, depending on parameter conditions. The second strategy tests for deviations from mutation-drift equilibrium on a deme-by-deme basis to identify sites likely to have experienced recent bottlenecks or founder effects. Although bottleneck tests have good statistical power for single populations with recent population declines, their validity in structured populations has been called into question, and they have not been tested in a metapopulation context with immigration (or colonization) and population recovery. Our simulations of hypothetical metapopulations show that population recovery can rapidly eliminate the statistical signature of a bottleneck, and that moderate levels of gene flow can generate a false signal of recent population growth for demes in equilibrium. Although we did not cover all possible metapopulation scenarios, the performance of the tests was disappointing. Our results indicate that these methods might often fail to identify population bottlenecks and founder effects if population recovery and/or gene flow are influential demographic features of the study system.

Lethal effects of water quality on threatened California salamanders but not on co-occurring hybrid salamanders.

Biological invasions and habitat alteration are often detrimental to native species, but their interactions are difficult to predict. Interbreeding between native and introduced species generates novel genotypes and phenotypes, and human land use alters habitat structure and chemistry. Both invasions and habitat alteration create new biological challenges and opportunities. In the intensively farmed Salinas Valley, California (U.S.A.), threatened California tiger salamanders (Ambystoma californiense) have been replaced by hybrids between California tiger salamander and introduced barred tiger salamanders (Ambystoma tigrinum mavortium). We conducted an enclosure experiment to examine the effects habitat modification and relative frequency of hybrid and native California tiger salamanders have on recruitment of salamanders and their prey, Pacific chorus frogs (Pseudacris regilla). We tested whether recruitment differed among genetic classes of tiger salamanders (hybrid or native) and pond hydroperiod (seasonal or perennial). Roughly 6 weeks into the experiment, 70% (of 378 total) of salamander larvae died in 4 out of 6 ponds. Native salamanders survived (n = 12) in these ponds only if they had metamorphosed prior to the die-offs. During die-offs, all larvae of native salamanders died, whereas 56% of hybrid larvae died. We necropsied native and hybrid salamanders, tested water quality, and queried the California Department of Pesticide Regulation database to investigate possible causes of the die-offs. Salamander die-offs, changes in the abundance of other community members (invertebrates, algae, and cyanobacteria), shifts in salamander sex ratio, and patterns of pesticide application in adjacent fields suggest that pesticide use may have contributed to die-offs. That all survivors were hybrids suggests that environmental stress may promote rapid displacement of native genotypes.

Rapid spread of invasive genes into a threatened native species.

When introduced or cultivated plants or animals hybridize with their native relatives, the spread of invasive genes into native populations might have biological, aesthetic, and legal implications. Models suggest that the rate of displacement of native by invasive alleles can be rapid and inevitable if they are favored by natural selection. We document the spread of a few introduced genes 90 km into a threatened native species (the California Tiger Salamander) in 60 years. Meanwhile, a majority of genetic markers (65 of 68) show little evidence of spread beyond the region where introductions occurred. Using computer simulations, we found that such a pattern is unlikely to emerge by chance among selectively neutral markers. Therefore, our results imply that natural selection has favored both the movement and fixation of these exceptional invasive alleles. The legal status of introgressed populations (native populations that are slightly genetically modified) is unresolved by the US Endangered Species Act. Our results illustrate that genetic and ecological factors need to be carefully weighed when considering different criteria for protection, because different rules could result in dramatically different geographic areas and numbers of individuals being protected.

Biofluorescent sexual dimorphism revealed in a southern Appalachian endemic salamander, Plethodon metcalfi.

Biofluorescence occurs when a living organism absorbs high energy light and reemits it at longer wavelengths. Many species within clades of vertebrates are known to fluoresce including mammals, reptiles, birds, and fish. Most, if not all, amphibians exhibit biofluorescence when exposed to either blue (440-460 nm) or ultra-violet (360-380 nm) wavelengths of light. Salamanders (Lissamphibia: Caudata) appear to consistently fluoresce in green wavelengths (520-560 nm) when excited by blue light. Biofluorescence is theorized to have many ecological functions including mate signaling, camouflage, and mimicry. Despite the discovery of their biofluorescence, its role in salamander ecology and behavior remains unresolved. In this study we present the first case of biofluorescent sexual dimorphism within Amphibia and the first documentation of the biofluorescent pattern of a salamander within the Plethodon jordani species complex. This sexually dimorphic trait was discovered in the southern Appalachian endemic species, Southern Gray-Cheeked Salamander (Plethodon metcalfi, Brimley in Proc Biol Soc Wash 25:135-140, 1912), and may extend into other species within the Plethodon jordani and Plethodon glutinosus species complexes. We propose that this sexually dimorphic trait could be related to fluorescence of ventral modified granular glands used in plethodontid chemosensory communication.

Estimating ancestry and heterozygosity of hybrids using molecular markers.

BACKGROUND: Hybridization, genetic mixture of distinct populations, gives rise to myriad recombinant genotypes. Characterizing the genomic composition of hybrids is critical for studies of hybrid zone dynamics, inheritance of traits, and consequences of hybridization for evolution and conservation. Hybrid genomes are often summarized either by an estimate of the proportion of alleles coming from each ancestral population or classification into discrete categories like F1, F2, backcross, or merely "hybrid" vs. "pure". In most cases, it is not realistic to classify individuals into the restricted set of classes produced in the first two generations of admixture. However, the continuous ancestry index misses an important dimension of the genotype. Joint consideration of ancestry together with interclass heterozygosity (proportion of loci with alleles from both ancestral populations) captures all of the information in the discrete classification without the unrealistic assumption that only two generations of admixture have transpired. METHODS: I describe a maximum likelihood method for joint estimation of ancestry and interclass heterozygosity. I present two worked examples illustrating the value of the approach for describing variation among hybrid populations and evaluating the validity of the assumption underlying discrete classification. RESULTS: Naively classifying natural hybrids into the standard six line cross categories can be misleading, and false classification can be a serious problem for datasets with few molecular markers. My analysis underscores previous work showing that many (50 or more) ancestry informative markers are needed to avoid erroneous classification. CONCLUSION: Although classification of hybrids might often be misleading, valuable inferences can be obtained by focusing directly on distributions of ancestry and heterozygosity. Estimating and visualizing the joint distribution of ancestry and interclass heterozygosity is an effective way to compare the genetic structure of hybrid populations and these estimates can be used in classic quantitative genetic methods for assessing additive, dominant, and epistatic genetic effects on hybrid phenotypes and fitness. The methods are implemented in a freely available package "HIest" for the R statistical software ( http://cran.r-project.org/web/packages/HIest/index.html).

Invasive hybrid tiger salamander genotypes impact native amphibians.

Although the ecological consequences of species invasions are well studied, the ecological impacts of genetic introgression through hybridization are less understood. This is particularly true of the impacts of hybridization on "third party" community members not genetically involved in hybridization. We also know little about how direct interactions between hybrid and parental individuals influence fitness. Here, we examined the ecological effects of hybridization between the native, threatened California Tiger Salamander (Ambystoma californiense) and the introduced Barred Tiger Salamander (Ambystoma tigrinum mavortium). Native x introduced hybrids are widespread in California, where they are top predators in seasonal ponds. We examined the impacts of early generation hybrids (first 2 generations of parental crosses) and contemporary hybrids derived from ponds where hybrids have been under selection in the wild for 20 generations. We found that most classes of hybrid tiger salamander larvae dramatically reduced survival of 2 native community members, the Pacific Chorus Frog (Pseudacris regilla) and the California Newt (Taricha torosa). We also found that native A. californiense larvae were negatively impacted by the presence of hybrid larvae: Native survival and size at metamorphosis were reduced and time to metamorphosis was extended. We also observed a large influence of Mendelian dominance on size, metamorphic timing and predation rate of hybrid tiger salamanders. These results suggest that both genetic and ecological factors are likely to influence the dynamics of admixture, and that tiger salamander hybridization might constitute a threat to additional pond-breeding species of concern in the region.

Retention of low-fitness genotypes over six decades of admixture between native and introduced tiger salamanders.

BACKGROUND: Introductions of non-native tiger salamanders into the range of California tiger salamanders have provided a rare opportunity to study the early stages of secondary contact and hybridization. We produced first- and second-generation hybrid salamanders in the lab and measured viability among these early-generation hybrid crosses to determine the strength of the initial barrier to gene exchange. We also created contemporary-generation hybrids in the lab and evaluated the extent to which selection has affected fitness over approximately 20 generations of admixture. Additionally, we examined the inheritance of quantitative phenotypic variation to better understand how evolution has progressed since secondary contact. RESULTS: We found significant variation in the fitness of hybrids, with non-native backcrosses experiencing the highest survival and F2 hybrids the lowest. Contemporary-generation hybrids had similar survival to that of F2 families, contrary to our expectation that 20 generations of selection in the wild would eliminate unfit genotypes and increase survival. Hybrid survival clearly exhibited effects of epistasis, whereas size and growth showed mostly additive genetic variance, and time to metamorphosis showed substantial dominance. CONCLUSIONS: Based on first- and second- generation cross types, our results suggest that the initial barrier to gene flow between these two species was relatively weak, and subsequent evolution has been generally slow. The persistence of low-viability recombinant hybrid genotypes in some contemporary populations illustrates that while hybridization can provide a potent source of genetic variation upon which natural selection can act, the sorting of fit from unfit gene combinations might be inefficient in highly admixed populations. Spatio-temporal fluctuation in selection or complex genetics has perhaps stalled adaptive evolution in this system despite selection for admixed genotypes within generations.

Relatedness and genetic structure in a socially polymorphic population of the spider Anelosimus studiosus.

The evolution of sociality remains a challenge in evolutionary biology and a central question is whether association between kin is a critical factor favouring the evolution of cooperation. This study examines genetic structure of Anelosimus studiosus, a spider exhibiting polymorphic social behaviour. Two phenotypes have been identified: an 'asocial' phenotype with solitary female nests and a 'social' phenotype with multi-female/communal nests. To address the questions of whether these phenotypes are differentiated populations and whether cooperative individuals are closely related, we used microsatellites to analyse individuals from both communal and solitary nests. We found no evidence of differentiation between social and solitary samples, implying high rates of interbreeding. This is consistent with the hypothesis that these phenotypes coexist as a behavioural polymorphism within populations. Pairwise relatedness coefficients were used to test whether cooperating individuals are more closely related than expected by chance. Pairwise relatedness of females sharing communal webs averaged 0.25, the level expected for half-siblings and significantly more closely related than random pairs from the population. Solitary females collected at similar distances to the communal spider pairs were also more closely related than expected by chance (mean relatedness = 0.18), but less related than social pairs. These results imply that low dispersal contributes to increase likelihood of interaction between kin, but relatedness between social pairs is not explained by spatial structure alone. We propose that these phenotypes represent stages in the evolution of sociality, where viscous population structure creates opportunities for kin selection and cooperation is favoured under certain environmental conditions.

Geography disentangles introgression from ancestral polymorphism in Lake Malawi cichlids.

Phenotypically diverse Lake Malawi cichlids exhibit similar genomes. The extensive sharing of genetic polymorphism among forms has both intrigued and frustrated biologists trying to understand the nature of diversity in this and other rapidly evolving systems. Shared polymorphism might result from hybridization and/or the retention of ancestrally polymorphic alleles. To examine these alternatives, we used new genomic tools to characterize genetic differentiation in widespread, geographically structured populations of Labeotropheus fuelleborni and Metriaclima zebra. These phenotypically distinct species share mitochondrial DNA (mtDNA) haplotypes and show greater mtDNA differentiation among localities than between species. However, Bayesian analysis of nuclear single nucleotide polymorphism (SNP) data revealed two distinct genetic clusters corresponding perfectly to morphologically diagnosed L. fuelleborni and M. zebra. This result is a function of the resolving power of the multi-locus dataset, not a conflict between nuclear and mitochondrial partitions. Locus-by-locus analysis showed that mtDNA differentiation between species (F(CT)) was nearly identical to the median single-locus SNP F(CT). Finally, we asked whether there is evidence for gene flow at sites of co-occurrence. We used simulations to generate a null distribution for the level of differentiation between co-occurring populations of L. fuelleborni and M. zebra expected if there was no hybridization. The null hypothesis was rejected for the SNP data; populations that co-occur at rock reef sites were slightly more similar than expected by chance, suggesting recent gene flow. The coupling of numerous independent markers with extensive geographic sampling and simulations utilized here provides a framework for assessing the prevalence of gene flow in recently diverged species.

Population differences in behaviour are explained by shared within-population trait correlations.

Correlations in behavioural traits across time, situation and ecological context (i.e. 'behavioural syndromes' or 'personality') have been documented for a variety of behaviours, and in diverse taxa. Perhaps the most controversial inference from the behavioural syndromes literature is that correlated behaviour may act as an evolutionary constraint and evolutionary change in one's behaviour may necessarily involve shifts in others. We test the two predictions of this hypothesis using comparative data from eighteen populations of the socially polymorphic spider, Anelosimus studiosus (Araneae, Theriidae). First, we ask whether geographically distant populations share a common syndrome. Second, we test whether population differences in behaviour are correlated similarly to within-population trait correlations. Our results reveal that populations separated by as much as 36 degrees latitude shared similar syndromes. Furthermore, population differences in behaviour were correlated in the same manner as within-population trait correlations. That is, population divergence tended to be along the same axes as within-population covariance. Together, these results suggest a lack of evolutionary independence in the syndrome's constituent traits.

Rapid fixation of non-native alleles revealed by genome-wide SNP analysis of hybrid tiger salamanders.

BACKGROUND: Hybrid zones represent valuable opportunities to observe evolution in systems that are unusually dynamic and where the potential for the origin of novelty and rapid adaptation co-occur with the potential for dysfunction. Recently initiated hybrid zones are particularly exciting evolutionary experiments because ongoing natural selection on novel genetic combinations can be studied in ecological time. Moreover, when hybrid zones involve native and introduced species, complex genetic patterns present important challenges for conservation policy. To assess variation of admixture dynamics, we scored a large panel of markers in five wild hybrid populations formed when Barred Tiger Salamanders were introduced into the range of California Tiger Salamanders. RESULTS: At three of 64 markers, introduced alleles have largely displaced native alleles within the hybrid populations. Another marker (GNAT1) showed consistent heterozygote deficits in the wild, and this marker was associated with embryonic mortality in laboratory F2's. Other deviations from equilibrium expectations were idiosyncratic among breeding ponds, consistent with highly stochastic demographic effects. CONCLUSION: While most markers retain native and introduced alleles in expected proportions, strong selection appears to be eliminating native alleles at a smaller set of loci. Such rapid fixation of alleles is detectable only in recently formed hybrid zones, though it might be representative of dynamics that frequently occur in nature. These results underscore the variable and mosaic nature of hybrid genomes and illustrate the potency of recombination and selection in promoting variable, and often unpredictable genetic outcomes. Introgression of a few, strongly selected introduced alleles should not necessarily affect the conservation status of California Tiger Salamanders, but suggests that genetically pure populations of this endangered species will be difficult to maintain.

Power and sample size for nested analysis of molecular variance.

Analysis of molecular variance (AMOVA) is a widely used tool for quantifying the contribution of various levels of population structure to patterns of genetic variation. Implementations of AMOVA use permutation tests to evaluate null hypotheses of no population structure within groups and between groups. With few populations per group, between-group structure might be impossible to detect because only a few permutations of the sampled populations are possible. In fact, with fewer than six total populations, permutation tests will never result in P-values <0.05 for higher-level population structure. I present minimum numbers of replicates calculated from multinomial coefficients and an r script that can be used to evaluate the minimum P-value for any sampling scheme. While it might seem counterintuitive that a large sample of individuals is uninformative about hierarchical structure, the power to detect between-group differences depends on the number of populations per group and investigators should sample appropriately.

Frequency-dependent selection by wild birds promotes polymorphism in model salamanders.

BACKGROUND: Co-occurrence of distinct colour forms is a classic paradox in evolutionary ecology because both selection and drift tend to remove variation from populations. Apostatic selection, the primary hypothesis for maintenance of colour polymorphism in cryptic animals, proposes that visual predators focus on common forms of prey, resulting in higher survival of rare forms. Empirical tests of this frequency-dependent foraging hypothesis are rare, and the link between predator behaviour and maintenance of variation in prey has been difficult to confirm. Here, we show that predatory birds can act as agents of frequency-dependent selection on terrestrial salamanders. Polymorphism for presence/absence of a dorsal stripe is widespread in many salamander species and its maintenance is a long-standing mystery. RESULTS: We used realistic food-bearing model salamanders to test whether selection by wild birds maintains a stripe/no-stripe polymorphism. In experimental manipulations, whichever form was most common was most likely to be attacked by ground-foraging birds, resulting in a survival advantage for the rare form. CONCLUSION: This experiment demonstrates that frequency-dependent foraging by wild birds can maintain colour polymorphism in cryptic prey.

Extensive Cryptic Diversity Within the Physalaemus cuvieri-Physalaemus ephippifer Species Complex (Amphibia, Anura) Revealed by Cytogenetic, Mitochondrial, and Genomic Markers.

Previous cytogenetic and phylogenetic analyses showed a high variability in the frog taxa Physalaemus cuvieri and Physalaemus ephippifer and suggested the presence of undescribed diversity in this species complex. Here, by 1) adding specimens from the Brazilian Amazon region, 2) employing sequence-based species delimitation approaches, and 3) including RADseq-style markers, we demonstrate that the diversity in the P. cuvieri-P. ephippifer species complex is even greater than previously suspected. Specimens from Viruá and Western Pará, located at the Guiana Amazonian area of endemism, were recovered as distinct from all previously identified lineages by the phylogenetic analyses based on mitochondrial DNA and RAD markers, a PCA from RAD data, and cytogenetic analysis. The sequence-based species delimitation analyses supported the recognition of one or two undescribed species among these Amazonian specimens and also supported the recognition of at least three other species in the P. cuvieri-P. ephippifer species complex. These new results reinforce the need for a comprehensive taxonomic revision.