Disentangling historical relationships within Poeciliidae (Teleostei: Cyprinodontiformes) using ultraconserved elements.

Poeciliids (Cyprinodontiformes: Poeciliidae), commonly known as livebearers, are popular fishes in the aquarium trade (e.g., guppies, mollies, swordtails) that are widely distributed in the Americas, with 274 valid species in 27 genera. This group has undergone various taxonomic changes recently, spurred by investigations using traditional genetic markers. Here we used over 1,000 ultraconserved loci to infer the relationships within Poeciliidae in the first attempt at understanding their diversification based on genome-scale data. We explore gene tree discordance and investigate potential incongruence between concatenation and coalescent inference methods. Our aim is to examine the influence of incomplete lineage sorting and reticulate evolution on the poeciliids' evolutionary history and how these factors contribute to the observed gene tree discordace. Our concatenated and coalescent phylogenomic inferences recovered four major clades within Poeciliidae. Most supra-generic level relationships we inferred were congruent with previous molecular studies, but we found some disagreements; the Middle American taxa Phallichthys and Poecilia (Mollienesia) were recovered as non-monophyletic, and unlike other recent molecular studies, we recovered Brachyrhaphis as monophyletic. Our study is the first to provide signatures of reticulate evolution in Poeciliidae at the family level; however, continued finer-scale investigations are needed to understand the complex evolutionary history of the family along with a much-needed taxonomic re-evaluation.

Investigating the utility of Anchored Hybrid Enrichment data to investigate the relationships among the Killifishes (Actinopterygii: Cyprinodontiformes), a globally distributed group of fishes.

The Killifishes (Cyprinodontiformes) are a diverse and well-known group of fishes that contains sixteen families inclusive of Anablepidae, Aphaniidae Aplocheilidae, Cubanichthyidae, Cyprinodontidae, Fluviphylacidae, Fundulidae, Goodeidae, Nothobranchiidae, Orestiidae, Pantanodontidae, Poeciliidae, Procatopodidae, Profundulidae, Rivulidae, and Valenciidae and more than 1,200 species that are globally distributed in tropical and temperate, freshwater and estuarine habitats. The evolutionary relationships among the families within the group, based on different molecular and morphological data sets, have remained uncertain. Therefore, the objective of this study was to use a targeted approach, anchored hybrid enrichment, to investigate the phylogenetic relationships among the families within the Cyprindontiformes. This study included more than 100 individuals, representing all sixteen families within the Cyprinodontiformes, including many recently diagnosed families. We recovered an average of 244 loci per individual. These data were submitted to phylogenetic analyses (RaxML and ASTRAL) and although we recovered many of the same relationships as in previous studies of the group, several novel sets of relationships for other families also were recovered. In addition, two well-established clades (Suborders Cyprinodontoidei and Aplocheilodei) were recovered as monophyletic and are in agreement with most previous studies. We also assessed the degree of gene tree discordance in our dataset to evaluate support for alternative topological hypotheses for interfamilial relationships within the Cyprinodontiformes using a variety of different analyses. The results from this study will provide a robust, historical framework needed to investigate a plethora of biogeographic, taxonomic, ecological, and physiological questions for this group of fishes.

Genetic differentiation among populations of the blackfin goodea Goodea atripinnis (Cyprinodontiformes: Goodeidae): implications for its evolutionary history.

Central Mexico is characterized by a complex topography that is the result of historic and contemporary tectonic and climatic factors. These events have influenced the evolutionary history of numerous freshwater fishes in the region. Nonetheless, recent studies have shown that life-history traits and ecological characteristics of species may influence dispersal capabilities and the degree of genetic connectivity. Goodea (Cyprinodontiformes: Goodeidae) is one of the most widely distributed and environmentally tolerant genera of goodeids. In this study, the authors analysed variation in the mitochondrial cytochrome b gene to evaluate the phylogeographic relationships, genetic structure, genetic diversity and demographic history of Goodea from across its distribution range. They found low genetic differentiation and identified shared haplotypes among several regions. Geographic segregation was found in samples southwest and northeast of the Lower Lerma region, with some internal isolated groups showing phylogeographic differentiation and unique haplotypes. The AMOVA best explained genetic structure when grouped by haplogroups rather than when grouped by recognized biogeographic regions. Several regions showed null genetic diversity, raising the possibility of dispersal mediated by humans. Finally, Bayesian Skyline Plot analysis showed a population expansion for the Southwest haplogroup, except for the Armería population and sub-group II of the Northeast haplogroup. All this suggests a recent colonization of Goodea atripinnis throughout some of the biogeographic regions currently inhabited by this species.

Molecular systematics of the Awaous banana complex (River gobies; Teleostei: Oxudercidae).

Diadromous fishes can exhibit interesting evolutionary and population-level patterns given their use of freshwater and marine environments as part of their life histories. The River goby genus Awaous are prominent members of riverine ichthyofaunas and occur throughout Atlantic and Pacific slopes of the Americas from the southern United States to Ecuador and Brazil. Here we study the widespread and polymorphic Awaous banana complex to assess phylogeographic patterns and test previous hypotheses that all populations of this species in the Americas belong to the same species. Analysis of sequence data based on the mitochondrial cytochrome oxidase I gene shows multiple clades within the Atlantic and Pacific basins, which correspond to previously described species. Additionally, haplotype analysis demonstrates unique and unconnected networks between these species. Within these clades we document biogeographic patterns that are congruent with results of other co-occurring diadromous species, as well as a novel biogeographic pattern for the region. Our results support the recognition of distinct species of Awaous in the Atlantic (A. banana and A. tajasica) and Pacific (A. transandeanus) basins. These results are concordant with previously established morphological characters permitting the separation of these species.

Assessing phylogenetic information to reveal uncertainty in historical data: An example using Goodeinae (Teleostei: Cyprinodontiformes: Goodeidae).

A major emerging challenge to resolution of a stable phylogenetic Tree of Life has been incongruent inference among studies. Given the increasing ubiquity of incongruent studies, analyzing the predicted phylogenetic utility and quantitative evidence regarding contributions toward resolution of commonly-used markers in historical studies over the last decade represents an important, yet neglected, component of phylogenetics. Here we examine the phylogenetic utility of two sets of commonly-used legacy markers for understanding the evolutionary relationships among goodeines, a group of viviparous freshwater fishes endemic to central Mexico. Our analyses reveal that the validity of existing inferences is compromised by both lack of information and substantially biased patterns of nucleotide substitution. Our analyses demonstrate that many of the evolutionary relationships of goodeines remain uncertain - despite over a century of work. Our results provide an updated baseline of critically needed areas of investigation for the group and underscore the importance of quantifying phylogenetic information content as a fundamental step towards eroding false confidence in results based on weak and biased evidence.

Disentangling the drivers of diversification in an imperiled group of freshwater fishes (Cyprinodontiformes: Goodeidae).

BACKGROUND: One of the most perplexing questions in evolutionary biology is why some lineages diversify into many species, and others do not. In many cases, ecological opportunity has played an important role, leading to diversification along trophic or habitat-based axes. The Goodeidae (Teleostomi: Cyprinodontiformes) are a family of freshwater fishes with two subfamilies: Goodeinae (42 species, viviparous, heterogeneous habitats, Mesa Central of Mexico) and Empetrichthyinae (4 species, oviparous, homogeneous habitats, Great Basin of the United States). These discrepant sets of characteristics and their sister-group relationship make the goodeids amenable to a comparative study of diversification. We gathered lateral body images from more than 1600 specimens of all extant species in the family. Geometric morphometric, and phylogenetic comparative analyses were used to address whether higher species diversity correlates with higher rates of morphological shape evolution and whether there are differences in functional/habitat modules between the two subfamilies. RESULTS: This study recovered a higher rate of overall body shape evolution in the Goodeinae that is nearly double in magnitude compared to the Empetrichthyinae. A modularity test indicated that the Goodeinae displayed elevated rates of morphological evolution in comparison to the Empetrichthyinae when only trunk (locomotor) regions were compared between subfamilies. No significant differences in evolutionary shape rates were recovered when the trophic (head) regions were compared between subfamilies. DISCUSSION: These results support the hypothesis that Mexican goodeids radiated via an ecological opportunity scenario into a wide-array of novel habitats in the island-like Mesa Central as evidenced by their high rate of shape evolution, relative to the Empetrichthyinae. This study quantitatively unraveled the drivers of evolution and eliminated trophic specialization as a driving force within the Goodeidae. CONCLUSIONS: A combination of phylogenetic and morphometric data, and phylogenetic comparative analyses were used to examine body shape rate evolution within the Goodeidae. Results support the hypothesis that species in the subfamily Goodeinae on the central Mexican plateau had a higher rate of body shape evolution relative to its sister subfamily Empetrichthyinae in the Great Basin suggesting that the Goodeinae diversified via an ecological opportunity scenario along habitat, rather than trophic axes.

Multi-locus fossil-calibrated phylogeny of Atheriniformes (Teleostei, Ovalentaria).

Phylogenetic relationships among families within the order Atheriniformes have been difficult to resolve on the basis of morphological evidence. Molecular studies so far have been fragmentary and based on a small number taxa and loci. In this study, we provide a new phylogenetic hypothesis based on sequence data collected for eight molecular markers for a representative sample of 103 atheriniform species, covering 2/3 of the genera in this order. The phylogeny is calibrated with six carefully chosen fossil taxa to provide an explicit timeframe for the diversification of this group. Our results support the subdivision of Atheriniformes into two suborders (Atherinopsoidei and Atherinoidei), the nesting of Notocheirinae within Atherinopsidae, and the monophyly of tribe Menidiini, among others. We propose taxonomic changes for Atherinopsoidei, but a few weakly supported nodes in our phylogeny suggests that further study is necessary to support a revised taxonomy of Atherinoidei. The time-calibrated phylogeny was used to infer ancestral habitat reconstructions to explain the current distribution of marine and freshwater taxa. Based on these results, the current distribution of Atheriniformes is likely due to widespread marine dispersal along the margins of continents, infrequent trans-oceanic dispersal, and repeated invasion of freshwater habitats. This conclusion is supported by post-Gondwanan divergence times among families within the order, and a high probability of a marine ancestral habitat.

Life in the fastlane? A comparative analysis of gene expression profiles across annual, semi-annual, and non-annual killifishes (Cyprinodontiformes: Nothobranchiidae).

The Turquoise Killifish is an important vertebrate for the study of aging and age-related diseases due to its short lifespan. Within Nothobranchiidae, species possess annual, semi-annual, or non-annual life-histories. We took a comparative approach and examined gene expression profiles (QuantSeq) from 62 individuals from eleven nothobranchid species that span three life-histories. Our results show significant differences in differentially expressed genes (DEGs) across life-histories with non-annuals and semi-annuals being most similar, and annuals being the most distinct. At finer scales, we recovered significant differences in DEGs for DNA repair genes and show that non-annual and semi-annuals share similar gene expression profiles, while annuals are distinct. Most of the GO terms enriched in annuals are related to metabolic processes. However, GO terms, including translation, protein transport, and DNA replication initiation also are enriched in annuals. Non-annuals are enriched in Notch signaling pathway genes and downregulated in the canonical Wnt signaling pathway compared to annual species, which suggests that non-annuals have stronger regulation in cellular processes. This study provides support for congruency in DEGs involved in these life-histories and provides strong evidence that a particular set of candidate genes may be worthy of study to investigate their role in the aging process.

It's a family matter: molecular phylogenetics of Atheriniformes and the polyphyly of the surf silversides (family: Notocheiridae).

Phylogenetic relationships among families of Atheriniformes have long been problematic. The affinities of one of the most enigmatic lineages, surf silversides (Notocheiridae), have proven particularly elusive due to this taxon's unique morphology and rarity in museum collections. In this study, we use mitochondrial and nuclear sequence data to generate a phylogeny for seven of the eight families of Atheriniformes. Our results reveal that four families within Atheriniformes (Atherinopsidae, Notocheiridae, Atherinidae, Melanotaeniidae) are not monophyletic. Most notably, Notocheiridae is polyphyletic, with Notocheirus hubbsi nested within New World silversides (Atherinopsidae), while members of Iso are sister to all other Old World Atheriniforms. These data suggest that the unique morphology of Notocheirus and Iso is a result of adaptive convergent evolution to the high-energy surf habitat where these species live.

Genomic data support the taxonomic validity of Middle American livebearers Poeciliopsis gracilis and Poeciliopsis pleurospilus (Cyprinodontiformes: Poeciliidae).

Poeciliopsis (Cyprinodontiformes: Poeciliidae) is a genus comprised of 25 species of freshwater fishes. Several well-known taxonomic uncertainties exist within the genus, especially in relation to the taxonomic status of Poeciliopsis pleurospilus and P. gracilis. However, to date, no studies have been conducted to specifically address the taxonomic status of these two species. The goal of this study was to examine the taxonomic validity of P. pleurospilus and P. gracilis using genomic data (ddRADseq) in phylogenetic, population genetic, and species delimitation frameworks. Multiple analyses support the recognition of both taxa as distinct species and also permits us to revise their respective distributions. A species delimitation analysis indicates that P. pleurospilus and P. gracilis are distinct species, each of which consists of two distinct lineages that are geographically structured. Phylogenetic and population genetic analyses provide clear evidence that individuals of P. gracilis are distributed north and west of the Isthmus of Tehuantepec in both Pacific and Atlantic river systems in Mexico, whereas individuals of P. pleurospilus are distributed in both Atlantic and Pacific river systems south and east of the Isthmus of Tehuantepec, from southern Mexico to Honduras.

Phylogenetic relationships of the North American cyprinid subgenus Hydrophlox.

Notropis is one of the largest genera of North American fishes and is composed of a number of morphologically diagnosed subgroups; however, the validity of many has not been tested in a phylogenetic framework. One such subgroup is the subgenus Hydrophlox, which is composed of brilliantly colored species that engage in the symbiotic reproductive behavior of nest association. Although they have long been recognized as a cohesive group due to their nuptial coloration and fin tuberculation, very little is known about the relationships of species within Hydrophlox. We tested the monophyly of Hydrophlox using a mitochondrial marker (ND2) and two nuclear markers (ITS1 and RH), with Maximum Parsimony and Bayesian inference approaches. A well supported clade of "core"Hydrophlox was recovered and is composed of five taxa: Notropis chiliticus, Notropis rubricroceus, Notropis lutipinnis, Notropis chlorocephalus, and Notropis chrosomus. Hydrophlox s.l. is paraphyletic with respect to three taxa: Notropis baileyi, Notropis leuciodus and Notropis nubilus. While there was some discordance among the individual marker topologies, a combined evidence analysis recovered a topology that incorporated elements from all single-gene trees. Our analyses suggest that Hydrophlox is composed of five nominal species and additional undescribed diversity exists within this clade.

Molecular systematics of the enigmatic Middle American genus Vieja (Teleostei: Cichlidae).

The genus Vieja represents a group of heroine cichlids (Teleostei: Cichlidae) distributed on the Atlantic and Pacific slopes of North and Central America from southern Mexico to Panama. Sixteen species of Vieja are presently recognized; however, based on long-standing taxonomic problems, the genus itself appears to be weakly defined. A number of different generic designations have been proposed for members of Vieja, and recent systematic studies of heroine cichlids have not specifically addressed the validity of the grouping and have not included all species in the genus. Therefore, the purpose of this study was to assess the monophyly of the genus Vieja by including all nominal species in the genus using the mitochondrial encoded cytochrome b gene and nuclear S7-1 intron. Results of Maximum Parsimony, Bayesian inference, and topology tests (constraint tree searches and post-burn-in Bayesian filtering) indicate that the genus is not monophyletic as it is currently recognized. The genus Herichthys was recovered as sister to a clade consisting of a number of Vieja species (V. fenestrata, V. guttulata, V. zonata, V. hartwegi, V. bifasciata, V. breidohri, V. argentea, V. regani, V. melanura, V. synspila, and V. maculicauda, as well as Paraneetroplusbulleri). A clade consisting of V. intermedia, V. godmanni, and V. microphthalma was recovered sister to Theraps. Additionally, V. heterospila and V. tuyrensis were recovered outside of Vieja and Herichthys clades. Based on the results of this comprehensive study, we suggest a revised classification of Vieja species.

Discordant molecular and morphological evolution in buffalofishes (Actinopterygii: Catostomidae).

Buffalofishes (Genus Ictiobus) are large, robust-bodied suckers adapted to large rivers and lakes of North America. Currently recognized species are readily diagnosed by morphological characters, and the group is known from fossils dating back to the Miocene. However, sympatrically occurring species in the Mississippi River Basin are known to hybridize in nature and in the laboratory. Here we describe patterns of morphological (morphometric) and DNA sequence variation (mitochondrial and nuclear genes) across the geographic ranges of extant species of genus Ictiobus. We show that Ictiobus species form more of less discrete entities based on body morphometry, consistent with current taxonomy. However, except for I. labiosus, there is extensive sharing of alleles of nuclear and mitochondrial genes among species, and the species do not form reciprocally monophyletic groups in nuclear or mitochondrial gene trees. Moreover, the pattern is not confined to the broad area of sympatry in the Mississippi River Basin. We attribute this to a long history of introgressive hybridization and gene flow among species inhabiting the present-day Mississippi River Basin, and recent colonization of the Great Lakes, Hudson Bay drainage and gulf coastal rivers east and west of the Mississippi River by introgressed Mississippi River Basin stocks.

Distribution and current conservation status of the Mexican Goodeidae (Actinopterygii, Cyprinodontiformes).

The current distribution and abundance of the 40 species of Goodeidae fishes known from Mexico are described, and a total of 84 Evolutionarily Significant Units (ESUs) is designated within these species. Two species and four ESUs are likely extinct with no captive populations, and three species and eight ESUs are probably extinct in the wild but have at least one captive population in Mexico, the United States, or Europe. Of the 35 extant species, the analyses indicate that nine should be considered as critically endangered, 14 as endangered, nine as vulnerable, and only three as least concern. Twenty-seven of these species have experienced substantial declines in distribution or abundance or both since 2000, and only eight appear to have remained relatively stable. Of the 72 extant ESUs, our analyses indicate that 29 should be considered as critically endangered, 21 as endangered, 18 as vulnerable, and only four as least concern. Brief summaries of the historic and current distributions and abundance of each species are provided, as well as ESU. Three strategies are recommended to conserve Mexican goodeids: protect the best-quality remaining habitats where goodeids still persist, restore degraded habitat and re-introduce species or ESUs where practical, and establish captive populations to ensure continued survival of the many species and ESUs that will almost inevitably go extinct in the coming years. Limited resources require cooperation and collaboration between scientists, conservationists, and aquarium hobbyists for successful captive maintenance.

Let's jump in: A phylogenetic study of the great basin springfishes and poolfishes, Crenichthys and Empetrichthys (Cyprinodontiformes: Goodeidae).

North America's Great Basin has long been of interest to biologists due to its high level of organismal endemicity throughout its endorheic watersheds. One example of such a group is the subfamily Empetricthyinae. In this paper, we analyzed the relationships of the Empetrichtyinae and assessed the validity of the subspecies designations given by Williams and Wilde within the group using concatenated phylogenetic tree estimation and species tree estimation. Samples from 19 populations were included covering the entire distribution of the three extant species of Empetricthyinae-Crenichthys nevadae, Crenichthys baileyi and Empetricthys latos. Three nuclear introns (S8 intron 4, S7 intron 1, and P0 intron 1) and one mitochondrial gene (Cytb) were sequenced for phylogenetic analysis. Using these sequences, we generated two separate hypotheses of the evolutionary relationships of Empetrichtyinae- one based on the mitochondrial data and one based on the nuclear data using Bayesian phylogenetics. Haplotype networks were also generated to look at the relationships of the populations within Empetrichthyinae. After comparing the two phylogenetic hypotheses, species trees were generated using *BEAST with the nuclear data to further test the validity of the subspecies within Empetrichthyinae. The mitochondrial analyses supported four lineages within C. baileyi and 2 within C. nevadae. The concatenated nuclear tree was more conserved, supporting one clade and an unresolved polytomy in both species. The species tree analysis supported the presence of two species within both C. baileyi and C. nevadae. Based on the results of these analyses, the subspecies designations of Williams and Wilde are not valid, rather a conservative approach suggests there are two species within C. nevadae and two species within C. baileyi. No structure was found for E. latos or the populations of Empetricthyinae. This study represents one of many demonstrating the invalidity of subspecies and their detriment to species identification, conservation, and understanding.

Two new species of the genus Xenotoca Hubbs and Turner, 1939 (Teleostei, Goodeidae) from central-western Mexico.

The subfamily Goodeinae (Goodeidae) is one of the most representative and well-studied group of fishes from central Mexico, with around 18 genera and 40 species. Recent phylogenetic studies have documented a high degree of genetic diversity and divergences among populations, suggesting that the diversity of the group may be underestimated. The species Xenotoca eiseni has had several taxonomic changes since its description. Xenotoca eiseni is considered a widespread species along the Central Pacific Coastal drainages of Mexico, inhabiting six independent drainages. Recent molecular phylogenetic studies suggest that X. eiseni is a species complex, represented by at least three independent evolutionary lineages. We carried out a meristic and morphometric study in order to evaluate the morphological differences among these genetically divergent populations and describe two new species. The new species of goodeines, Xenotoca doadrioi and X. lyonsi, are described from the Etzatlan endorheic drainage and upper Coahuayana basin respectively.

Black liquor and the hangover effect: fish assemblage recovery dynamics following a pulse disturbance.

Anthropogenic perturbations impact aquatic systems causing wide-ranging responses, from assemblage restructuring to assemblage recovery. Previous studies indicate the duration and intensity of disturbances play a role in the dynamics of assemblage recovery. In August 2011, the Pearl River, United States, was subjected to a weak black liquor spill from a paper mill which resulted in substantial loss of fish in a large stretch of the main channel. We quantified resilience and recovery of fish assemblage structure in the impacted area following the event. We compared downstream (impacted) assemblages to upstream (unimpacted) assemblages to determine initial impacts on structure. Additionally, we incorporated historic fish collections (1988-2011) to examine impacts on assemblage structure across broad temporal scales. Based on NMDS, upstream and downstream sites generally showed similar assemblage structure across sample periods with the exception of the 2 months postdischarge, where upstream and downstream sites visually differed. Multivariate analysis of variance (PERMANOVA) indicated significant seasonal variation among samples, but found no significant interaction between impacted and unimpacted assemblages following the discharge event. However, multivariate dispersion (MVDISP) showed greater variance among assemblage structure following the discharge event. These results suggest that 2 months following the disturbance represent a time period of stochasticity in regard to assemblage structure dynamics, and this was followed by rapid recovery. We term this dynamic the "hangover effect" as it represents the time frame from the cessation of the perturbation to the assemblage's return to predisturbance conditions. The availability and proximity of tributaries and upstream refugia, which were not affected by the disturbance, as well as the rapid recovery of abiotic parameters likely played a substantial role in assemblage recovery. This study not only demonstrates rapid recovery in an aquatic system, but further demonstrates the value of continuous, long-term, data collections which enhance our understanding of assemblage dynamics.

Taxonomy and systematics of the herichthyins (Cichlidae: Tribe Heroini), with the description of eight new Middle American Genera.

In recent years great strides have been made for improving our understanding of the evolutionary relationships among neotropical cichlids, particularly within the clade Heroini and its crown clade the herichthyins. Most phylogenetic studies have largely converged on congruent topologies for relationships among species and major lineages within the herichthyins. One major aspect missing from previous studies of these cichlids is a formal taxonomic revision, including the redefining of genera. Based on analysis of  52 species and three mitochondrial and two nuclear loci, we generate a Bayesian phylogeny for the herichthyin cichlids, and formally revise the taxonomy for genera within this clade using morphological features. Eight new genera are recognized and a key to all 16 genera of herichthyin cichlids is also presented.