text:
Resolving Cypriniformes relationships using an anchored enrichment approach
- Carla C. Stout†1Email author,
- Milton Tan†1,
- Alan R. Lemmon2,
- Emily Moriarty Lemmon3 and
- Jonathan W. Armbruster1
DOI: 10.1186/s12862-016-0819-5
© The Author(s). 2016
Received: 22 May 2016
Accepted: 28 October 2016
Published: 9 November 2016
Cypriniformes (minnows, carps, loaches, and suckers) is the largest group of freshwater fishes in the world (~4300 described species). Despite much attention, previous attempts to elucidate relationships using molecular and morphological characters have been incongruent. In this study we present the first phylogenomic analysis using anchored hybrid enrichment for 172 taxa to represent the order (plus three out-group taxa), which is the largest dataset for the order to date (219 loci, 315,288 bp, average locus length of 1011 bp).
Concatenation analysis establishes a robust tree with 97 % of nodes at 100 % bootstrap support. Species tree analysis was highly congruent with the concatenation analysis with only two major differences: monophyly of Cobitoidei and placement of Danionidae.
Most major clades obtained in prior molecular studies were validated as monophyletic, and we provide robust resolution for the relationships among these clades for the first time. These relationships can be used as a framework for addressing a variety of evolutionary questions (e.g. phylogeography, polyploidization, diversification, trait evolution, comparative genomics) for which Cypriniformes is ideally suited.
KeywordsFish High-throughput sequencing Phylogenetics Ostariophysi Cyprinidae
Cypriniformes (minnows, carps, loaches, and suckers) is the largest group of freshwater fishes in the world. Diversity ranges from some of the smallest vertebrates in the world (Paedocypris, 7.9 mm in standard length) to members of Tor (almost 3 m SL) [1]. The number of valid species is currently estimated at around 4300 [2] with as many as 2500 still awaiting description [3]. To place the Cypriniformes into perspective, about one third of freshwater fish species is a cypriniform and about 6 % of all vertebrate species is a cyprinform [2]. Species of Cypriniformes are distributed in freshwater habitats across Asia, Europe, Africa, and North America [4]. Example representatives include the zebrafish (Danio rerio), a model organism used in genomic and developmental biology, important aquaculture species like the common carp (Cyprinus carpio), major invasive species to North America such as Hypophthalmichthys (silver carp), and many popular aquarium species (rasboras and barbs).
For taxonomic clarity, this study follows the proposition by Mayden and Chen [1] that elevates subfamilies within Cyprinidae to the family level based on consistent support of major clades. Superfamilies are elevated to the suborder level to be consistent with the recognition of suborders as the taxonomic level above family and below order in the classification of bony fishes [5, 6]. Other taxonomic assignments follow designations established by Tang et al. [7], Kottelat [8], van der Laan et al. [9] and Yang et al. [10]. Because of the great diversity within Cypriniformes, most phylogenetic studies have focused on smaller groups within the order (for example [11–14]). Approaches used to resolve relationships at these levels have typically included standard methods using PCR to amplify targeted mitochondrial and/or nuclear genes [11–19]. These approaches have had varied success at elucidating relationships at these taxonomic levels, but deeper, all-inclusive studies have resulted in conflicting phylogenies. These major differences in findings even include two publications in the same volume [1, 19] whose results are incongruent. Morphological studies have also been at odds with the molecular hypotheses, particularly concerning placement of the paedomorphic taxa (Danionella, Paedocypris, and Sundadanio) [1, 20–22]. The results of analyses to date mean that this radiation of organisms that is nearly the size of the Mammalia and that is the predominant freshwater order of fishes has an unsettled taxonomy and phylogeny despite the fact that it has been very highly studied. With the vertebrate developmental model (zebrafish) being part of the Cypriniformes, we are currently lacking a basic understanding of the evolutionary context of its characteristics, and it is clear that new approaches to the phylogenetics of this very important group of fishes must be employed.
To date, the only nuclear genomic scale study [23] consisted of 100 genes and was limited to only thirteen individuals, most of which belong to Xenocyprididae within Cyprinoidei. The large number of taxa in Cypriniformes has forced researches to either focus on a small subset of representatives with an increasing number of molecular loci, or focus on large taxonomic representation with relatively fewer numbers of markers.
Evaluating tree topologies from previous large-scale studies has led to moderate consensus supporting monophyly for some clades within the order, including families of loaches (e.g. Botiidae, Cobitidae, Balitoridae, Nemacheilidae), Catostomidae (suckers), Cyprinidae, Xenocyprididae, Gobionidae, Leuciscidae, and Acheilognathidae [1, 19, 24–33]. Despite support for monophyly of many families, clear establishment of the relationships among them still remains elusive. Other families, most notably Danionidae, have been more problematic, with paedomorphic genera like Paedocypris and Sundadanio changing placement across trees employing both morphological and varying molecular data [1, 19–21, 31, 34].
If analyses result in incongruent relationships due to conflict or weak phylogenetic signal among individual genes, the next approach to establishing robust resolution would be to incorporate high-throughput sequencing data that can increase the signal to noise ratio and reduce stochastic error. New methods have been established that have been specifically tailored for use in systematics [35–37] and that address problems typical of transcriptome approaches for phylogenomics. These problems include tissue preservation, orthology assessment, missing data, and resolution capabilities across various taxonomic levels [35–37]. All of these factors make anchored hybrid enrichment an attractive option for addressing the phylogenetic uncertainties still present within Cypriniformes. This study represents the largest dataset developed for Cypriniformes, both in taxonomic representation and genetic data, ameliorating many of the problems associated with resolving the relationships among and within families of this order. Not until these relationships are resolved can researchers begin to take advantage of the size, diversity, and distribution of Cypriniformes to gain insight into various biological facets, such as biogeography, timing of diversifications, morphological and ecological evolution, and comparative genomics.
Taxon selection and tissue preparation
The 172 taxa selected for this study (Additional file 1: Table S1) represent almost all families within the order. Families not represented in this study are: Psilorhynchidae (26 species), Barbuccidae (two species), Tincidae (13 species), Serpenticobitidae (three species), Ellopostomidae (two species) and Leptobarbidae (five species). Species were chosen based on tissue availability and because of their incorporation in recent studies that will allow for direct comparisons [11, 13, 14, 26, 30, 38, 39]. Type genera for each of the families were included if available. Exceptions include Botiidae, Balitoridae, Gastromyzontidae, and Xenocyprididae, but in these cases other representatives were chosen based on their supported inclusion within their respected families according to previous studies [8, 40]. Three outgroup taxa were chosen to represent the three other ostariophysan orders: Siluriformes, Gymnotiformes, and Characiformes.
Whole genomic DNA was prepared using the Omegabiotek E.Z.N.A. animal tissue extraction kit (product #D3396-02) and verified for quality and quantity using gel electrophoresis and nanodrop, respectively.
Locus selection and probe design
Although the Anchored Hybrid Enrichment kit developed for vertebrates by Lemmon et al. [36] contains a fish reference (Danio) and has been utilized in teleosts with moderate success [41], we desired an enrichment tool more efficient and appropriate for phylogenomics in teleosts. Because of the complex nature of teleost genome evolution, which involved multiple whole-genome duplications and lineage-specific gene losses [42], it is impractical to identify a set of loci that are truly single-copy across all of Teleostei. Previous studies claiming to have identified single-copy loci in teleosts (e.g. [43]) likely only identified loci that were single-copy in the species they considered; evaluation of those loci in additional teleost lineages suggests that these loci are not universally single-copy (see below). Consequently, we aimed to target loci containing up to four gene copies in each of three diverse lineages of teleosts: zebrafish, platyfish, and cichlids.
Candidate target regions for Teleostei were derived by combining the 394 Vertebrate Anchor (v2) loci of Prum et al. [44] and the 135 loci identified as Fugu-Danio single-copy orthologs by Li [43]. For the vertebrate anchor loci, teleost orthologs were obtained for Danio rerio (danRer7) using the human (hg19) coordinates and the USCS genome browser batch-coordinate (liftover) tool [45]. For the Fugu-Danio orthologs, orthologous human (hg19) and chicken (galGal3) coordinates were obtained using the USCS liftover tool and the Danio coordinates identified by Li [43]. Once the coordinates for Danio, Homo, and Gallus were obtained for all 529 candidate target regions, sequences corresponding to those regions [plus sufficient flanking region to obtain up to 3000 base pairs (bp) total] were extracted from the genomes and aligned by locus using MAFFT [46], v7.023b with "–genafpair" and "–maxiterate 1000" flags. The alignments were then used to generate a Danio-specific reference database containing spaced 20-mers. The Danio reference was then used to identify homologous regions in the genomes of zebrafish (Cypriniformes: Cyprinidae: Danio rerio; danRer7), platyfish (Cyprinodontiformes: Poeciliidae: Xiphophorus maculatus [47], and cichlid (Perciformes, Cichlidae: Maylandia zebra; [48]).
As expected, we obtained multiple homologs for many of the candidate loci (only 64 loci were single copy in all three species). Consequently, only 277 loci had fewer than five homologs per species and were considered further. We aligned with MAFFT [46], v7.023b with "–genafpair" and "–maxiterate 1000" flags) all homolog sequences (up to 12 per locus) for each of the 277 candidates together with the homologous human probe region sequence from the Vertebrate Anchor (v2) design. Alignments were then manually inspected for misplaced and grossly misaligned sequences, which were removed. Finally, alignments were trimmed to include regions best suited for Anchored Hybrid Enrichment (conserved, low-gap, high taxon representation), taking care that the chosen region contained the human probe region. A total of 260 loci were retained.
Finally, in order to ensure efficient enrichment, we checked for high-copy regions (e.g. microsatellites and transposable elements) in each of the three teleost references as follows. First, a database was constructed for each species using all 15-mers found in the trimmed alignments for that species. We also added to the database all 15-mers that were 1 bp removed from the observed 15-mers. The genome for the species was then exhaustively scanned for the presence of these 15-mers and matches were tallied at the alignment positions at which the 15-mer was found. Alignment regions containing > 100,000 counts in any of the three species were masked to prevent probe tiling across these regions. Probes of 120 bp were tiled uniformly at 5.5× tiling density.
Data collection
Multilocus sequence data were collected at the Center for Anchored Phylogenomics at Florida State University (www.anchoredphylogeny.com) following Lemmon et al. [41] with some adjustments. Each genomic DNA sample was sonicated to a fragment size of ~175–300 bp using a Covaris E220 Focused-ultrasonicator with Covaris microTUBES. Library preparation and indexing followed Meyer and Kircher [49]. Indexed libraries were pooled at equal quantities (12 pools of 16 samples each), and the library pools were enriched using a custom Agilent Custom SureSelect kit (Agilent Technologies), with probes designed as described above. The 12 enriched library pools were pooled with equal quantities for sequencing on 4 PE150 Illumina HiSeq2000 lanes with 8 bp indexing. Sequencing was performed at Florida State University in the College of Medicine Translational Science Laboratory.
Data analysis
Reads were quality filtered using Illumina's Casava software with the chastity filter set to high. In order to increase read length and accuracy overlapping reads were then merged following Rokyta et al. [50]. Non-overlapping read pairs were kept separate but still used in the assembly. All reads were then assembled into contigs following Prum et al. [44] using mapping references derived from the zebrafish, platyfish, and cichlid sequences used for probe design. This assembler produces separate contigs for gene copies differing by more than 5 % sequence divergence. To reduce errors caused by low-level indexing errors during sequencing, contigs were then filtered by removing those derived from fewer than 50 reads. Additional file 2: Table S2 provides a summary of the sequence data collected and assemblies that resulted.
Sets of homologs were produced by grouping by target locus (across individuals) and the filtered consensus sequences. Orthology was then determined for each target locus as follows. First, a pairwise distance measure was computed for pairs of homologs, with distance being computed as the percentage of 20-mers observed in the two sequences that were found in both sequences. A neighbor-joining clustering algorithm was then used to cluster the consensus sequences in to orthologous sets, with at most one sequence per species in each orthologous set (see [44] for details). In order to minimize the effects of missing data, clusters containing fewer than 130 (72 %) of the species were removed from downstream processing.
Sequences in each orthologous set were aligned using MAFFT v7.023b [46] with --genafpair and --maxiterate 1000 flags. In order to remove poorly aligned regions raw alignments were then trimmed and masked following Prum et al. [44], with the following adjustments: sites with > 50 % similarity were identified as good, 20 bp regions containing < 14 good sites were masked, and sites with fewer than 30 unmasked bases were removed from the alignment.
For all phylogenetic analyses, sequences from the gymnotiform, siluriform, and characiform species were used as the outgroup. For the concatenated dataset, the alignment was partitioned by locus and the phylogeny estimated using RAxML using GTR+ Γ model with 500 bootstrap replicates. For the species tree analysis, a maximum likelihood phylogeny was estimated with 100 bootstrap replicates for each of the separate loci using RAxML with GTR+ Γ model assumed. We then used the RAxML bootstrap trees to estimate a species tree using STAR [51] with default parameters using STRAW [52]. ASTRAL-II (v4.10.2) [53] was also used for species tree inference using the gene trees and their 100 bootstrap replicates. We performed 100 replicates of multi-locus bootstrapping.
To test our analyses against previous morphological hypotheses, we re-examined the datasets in Conway [54] and Britz et al. [21] by running 1000 replicates of a heuristic search in PAUP* [55]. We traced the characters in Mesquite v.3.04 [56]. We also performed Bayesian analyses on these morphological datasets under the Mk + Γ model in mrBayes 3.2 [57], which has been demonstrated to perform better than parsimony due to rate heterogeneity in character evolution [58]. Estimating rate heterogeneity can be biased by sampling only variable or parsimony-informative characters, so we analyzed the data with correction for parsimony-informative characters for the Conway [54] dataset and variable characters for the Britz et al. [21] datasets (one character in these datasets was not parsimony-informative). For each dataset, we ran MCMC with two runs of four chains for 1,000,000 generations, sampling every 1,000. We assessed convergence using Tracer v1.5 [59].
Concatenated tree vs. species tree
We find only a few major differences between our maximum likelihood concatenated tree (CT) and the species trees (ST; Additional file 4: Figure S2 and Additional file 5: Figure S3). These include support for monophyly of Cobitoidei in the ST but not in the CT, and a different placement for the Danionidae between the two trees. Other minor differences are found among a few shallow sister relationships that had lower support values in both trees. Other studies have shown that concatenation methods may perform better over coalescent species tree methods, especially at deeper nodes, and our discussion of clades will focus on the CT tree [44, 60, 61].
Reanalysis of Cobitoidei morphological datasets
The most robust morphological phylogenies putatively supporting a monophyletic Cobitoidei is that of Conway [54]; however, when we reanalyzed the characters using parsimony in PAUP* [55], we achieved different results. We ran the analysis according to Conway [54] with the exception that we ran 1000 replicates of a heuristic search; it appears Conway [54] only ran a single replicate of a heuristic search, and that search settled on a tree island of 14 most parsimonious trees. We found one additional tree island with an additional 56 trees, which was found nearly as often as the 14-tree island (515 times vs. 485). The strict consensus of the 70 trees showed a polytomy at the base of the Cypriniformes with the gyrinocheilids, catostomids, loaches, and cyprinoids. The analyses in Britz et al. [21] did use 10 replicates of the heuristic search and are more accurate (we found more trees for their Morphological Dataset 3), and always found a monophyletic Cobitoidei, but this was weakly supported. Conway [54] lists seven characters supporting Cobitoidei, but our analysis showed that two of these (characters 32:1 and 99:1) were not listed as changed along the branch leading to the Cobitoidea and only one (character 19:1) is actually present in all families of cobitoids. All the remaining derived character states are absent in one of the three lineages (gyrinocheilids, catostomids, or loaches) meaning morphological support for a monophyletic group containing these three clades is poor. Support was stronger for a sister group relationship between gyrinocheilids plus catostomids (seven characters in [54], six in our analysis); however, we found seven characters supporting loaches plus cyprinoids (characters 7:0, 18:0, 46:1, 76:0, 83:2, 100:0, and 111:2) and seven characters supporting catostomids plus loaches plus cyprinoids (characters 11:0, 31:1, 36:1, 53:1, 68:1, 69:1, and 77:1) indicating roughly equal morphological support for the two hypotheses. Considerable homoplasy is found in most of the characters under all arrangements; however, characters 53, 83, and 77 provide unambiguous support for the relationships presented in this study.
In addition, the Bayesian analysis of the morphological characters resulted in only poor support (<.95 posterior probability, following Alfaro & Holder [62]) for monophyly of the Cobitoidei. In the analysis of the Conway [54] dataset, the catostmoids, gyrinocheilids, loaches, and cyprinoids form an unresolved polytomy in the consensus tree; this differs from the support present in Conway [54] for this node (.5–.9 pp). In the analyses of the Britz et al. [21] datasets, support ranged from .57 to.63 posterior probability across datasets, indicating low levels of support.
We have presented the first order-wide, phylogenomic analysis of the Cypriniformes, and we demonstrate the utility of anchored enrichment at assessing the relationships of fishes from deep to more recent divergences. Our analyses demonstrate conflict in the relationships of the Cobitoidei, the placement of Paedocypris as sister to all other cyprinoids, and a validation of the previously well-supported monophyly of many major cypriniform families. Although the wide variety of different hypotheses for the cypriniforms has been called the "Cypriniformes tree of confusion" [22, 63], the anchored enrichment phylogenomic tree that we present provides the most robust phylogenetic analysis to date, supporting many of the previous hypotheses of relationships and providing new ideas that will require further scrutiny.
Non-monophyly of Cobitoidei
Phylogenetic reanalysis of available morphological characters does not provide strong evidence for a monophyletic Cobitoidei, and morphological characters provide at least equally strong support for the relationships presented here. We restrict Cobitoidei to the loaches, and erect new suborders for the Gyrinocheilidae (Gyrinocheiloidei) and the Catostomidae (Catostomoidei).
For the remaining members of Cyprinidae, we find resolution for clades similar to those by Yang et al. [10] although none of the AE relationships among these clades are consistent with their results. For example, we resolve Labeoninae as sister to remaining members of Cyprinidae as opposed to Probarbinae as presented in Yang et al. [10]. Of particular interest is Chagunius chagunio, which Yang et al. [10] placed in the Smiliogastrinae. We obtain it as sister to a clade comprised of Spinibarbinae, Acrossocheilinae, Schizopygopsinae, Schizothoracinae, Torinae and Barbinae, with other Smiliogastrinae species more closely related to "Poropuntiinae" than to Chagunius. Yang et al. [10] had 0.80 posterior probability support for their placement based on mitogenome data, but less than 0.50 in their nuclear analysis (RAG1). Yang et al. [10] found numerous inter-clade hybridization events leading to allopolyploidy, which greatly complicates phylogenetic analysis within the Cyprinidae. We leave Chagunius as incertae sedis within Cyprinidae.
Danionidae, Paedocyprididae, Sundadanionidae
Differing from previous studies, we find the genus Esomus as a separate lineage sister to all remaining members of the Danionidae. The placement of Esomus has been contentious [67]. Esomus has been placed as closely related to Danionella or Sundadanio within Danioninae with poor support [19, 31, 66]. Because of poorly supported nodes, molecular phylogenies are ambiguous on the placement of Esomus among the clades of Danionidae. Liao et al. [67] remark that Esomus has a long branch in molecular phylogenetic analyses, and this may attract this branch towards other long branches such as Danionella and Sundadanio. Using morphological characters, Liao et al. [67] establish Esomus as sister to all other members of Chedrinae based on four characters, including two acquired states and two homoplasious states. In a subsequent paper, they admit this topology is never recovered in molecular analysis [68]. Both of the acquired character states relate to the postcleithrum: first its presence, and secondly its orientation. In Esomus, the postcleithrum is absent, and the postcleithrum orientation was coded as missing, and thus may not be informative on its placement relative to the Chedrinae. Additionally, although postcleithrum absence within Danionidae is only found in the Chedrinae, postcleithrum absence is also found in disparate genera from multiple cyprinoid groups including leuciscids, cyprinids, and gobionids [67]. Morphological homoplasy, long branch attraction, and short intervening branch lengths between danionid clades may have all contributed to the varying placement of Esomus between molecular and morphological studies.
We do not find support for Paedocypris and Sundadanio within Danionidae. This conflicts with multiple phylogenetic studies [11, 31, 34, 66]. Prior molecular analyses placing Paedocypris and Sundadanio within Danionidae have poor to moderate support. Rüber et al. [34] obtain Paedocypris and Sundadanio as sister to Danionidae with 0.86 posterior probability (pp), less than the 0.95 pp required to be considered strong support [62]. Fang et al. [31] place Paedocypris and Sundadanio sister to other Danionidae, but with only 0.76 pp. Tang et al. [1] find Paedocypris and Sundadanio in a polytomy with Rasborinae and Danioninae in a moderately supported clade (76 % bootstrap in CT). Mayden and Chen [1] proposed the exclusion of these two genera from Danionidae, which resolve Paedocypris as either sister to Cypriniformes or Cyprinoidei, and Sundadanio as sister to a clade comprised of Xenocyprididae, Acheilognathidae, Tincidae, Tanichthyidae, Gobionidae, and Leuciscidae. Our results are congruent with their placement of Sundadanio, but we resolve Paedocypris as a lineage sister to the remainder of Cyprinoidei. This is incongruent with the published topology of Mayden and Chen [1], but is congruent with an equally supported topology and an unpublished mitogenome analysis as discussed in their study. Difficulty in obtaining consistent placement of Paedocypris and Sundadanio may be due to several factors. Britz et al. [21] reanalyzed the Rüber et al. [34] and Mayden and Chen [1] datasets and demonstrated that the phylogenetic signal in most previously sequenced genes are equivocal on the placement of Paedocypris. Additionally, the branches for Paedocypris and Sundadanio are quite long, potentially contributing to spurious results with limited datasets prior to high-throughput sequencing. Our results robustly support the recognition of Paedocyprididae and Sundadanionidae based on their independent lineages from the remaining members of Danionidae.
Britz et al. [21] provide considerable morphological support for the paedomorphs forming a monophyletic clade, even when using the dataset of Conway [54] that did not include characters specific to paedomorphs. We reanalyzed the dataset of Britz et al. [21], and found that even with their morphological dataset 3 (that of Conway [54], plus some additional taxa and only one species of Psilorhynchus), we noted three character changes supporting all paedomorphs as monophyletic and nine character changes uniting Paedocypris and Danionella. Adding in characters specific to the paedomorphs [morphological datasets 4 and 5 from Britz et al. [21] only increases the level of support. Under Bayesian analysis, the support for paedomorphic taxa forming a clade is weak in morphological dataset 3 (0.76 pp) but increases dramatically with addition of the paedomorphic-specific characters of datasets 4 and 5 (1.00 pp). We believe the weak support for the relationships of the various cyprinoids in the original dataset [54] explains the disparity between the morphological and molecular hypotheses. In both the parsimony and Bayesian reanalyses of Britz et al.'s [21] morphological dataset 3, the basal relationships of the cyprinoids are an almost complete comb. Without strong support for relationships within the Cyprinoidei, the dataset is insufficient for distinguishing synapomorphy from convergence among the paedomorphs, and adding characters specific to paedomorphs will only decrease the ability of the morphology to detect convergence. Conway's [54] dataset already contains considerable homoplasy before the addition of the paedomorphs, indicating that morphological evolution within Cypriniformes was rapid. The support in our dataset for three separate transitions to paedomorphism is strong, corroborating Mayden and Chen's [1] suggestion of convergence in morphology, and we find at least five character changes in the Britz et al.'s [21] morphological dataset 3 that support monophyly of the cyprinoids minus Paedocypris (21:1, 24:1, 34:1, 82:1, 101:0).
Xenocyprididae, Acheilognathidae, Gobionidae, Tanichthyidae, and Leuciscidae
The Cypriniformes is among the most important clades of freshwater fishes and among the most studied with phylogenetic inference. This great deal of work makes them a key group in understanding the various pit-falls of phylogenetic studies, and they exemplify the phylogenetic conflicts from the varying analyses of morphological, mitochondrial, and nuclear data. While many major clades of Cypriniformes have been long-supported, relationships within and among them have proven difficult to resolve across the entire order. Varying markers and morphological data have given different results and have been difficult to apply across such a large and diverse group. With the development of phylogenomic techniques, researchers can now acquire a substantial amount of highly informative, quality data for resolving dynamic relationships, and we demonstrate the efficacy of the approach using the very complex cypriniforms. Robust phylogenies are not only a prerequisite for a stable taxonomy, but are needed to address important evolutionary questions such as the timing of diversification, the geographic origins of clades, and the evolution of morphological and ecological novelty. For example, according to our results, Cypriniformes appear to have invaded North America at least twice and Africa several times from Eurasia, with these transcontinental migrations resulting in very diverse clades. With the robust phylogeny we present here, we provide a framework for studying the consequences of these transcontinental migrations and how clades can diversify from within established ecosystems. Such studies will have broad consequences in studies on the evolution of diversity.
The great diversity of Cypriniformes and the inclusion of perhaps the most important vertebrate model organism (Zebra Danio) make Cypriniformes an ideal group for comparative analyses. Considerable insight into the functioning of genes within vertebrate organisms has been obtained from the analysis of the Zebra Danio including forced mutations that often result in unviable larvae. By comparing the genome of the Zebra Danio with close relatives, the role of mutations and gene expression can be determined. Comparative genomic studies within Cypriniformes have already benefited from the foundation and annotation of the Zebra Danio genome sequence to generate insights into the functional evolution of various adaptations including adaptation to harsh environments such as caves and high altitude streams [82, 83]. With a robust phylogeny, we can get a much better understanding of the function of genes by treating relatives of the Zebra Danio as natural mutants screened by natural selection [1]. As the Cypriniformes continues to become a more genome-enabled clade, with several new genomes published in the last few years [83–86], we expect our phylogeny to provide a useful framework for comparative genomics.
- Bp:
Base pairs
Creek chub – plagopterin
Concatenated tree
Open posterior myodome
- pp:
Posterior probability
Species tree
Western North America
We would like to thank the following people and affiliations for providing tissues: David C. Werneke at Auburn University Museum of Natural History, Drs. Susana Schonhuth and Richard Mayden at St. Louis University, Gabriela M. Hogue at North Carolina Museum of Natural Sciences, and Frank's Aquarium and Wet Spot Tropical Fish. We also thank Nicole Garrison for biocomputing assistance. We also thank Hannah Ralicki, Michelle Kortyna, and Alyssa Bigelow for assistance with collection of the Anchored Phylogenomic data. We are grateful for fish images provided by Fishes of Mainland Southeast Asia (ffish.asia, various photographers) and BOLD (Rupert Collins) through creative commons licenses, and with permission from David Neely. This work was supported by Auburn University Cellular and Molecular Biosciences Peaks of Excellence Research Fellowships to CCS and MT, and by National Science Foundation DEB-0315963, DEB-0107751, DEB-1022720 to JWA, NSF IIP 1313554 to ARL, and NSF DEB 1120516 to EML.
Availability of data and materials
The data set supporting the results of this article are available in the Dryad repository, accession number http://dx.doi.org/10.5061/dryad.b3d03.
Authors' contributions
CCS, MT, and JWA conceived of the study. ARL and EML provided reagents and collected sequence data. All authors contributed to data analysis and writing of the manuscript. JWA, ARL, and EML provided funding for the study. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
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Authors' Affiliations
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|Dr. E.S.L. Narasimhan, Governor of Andhra Pradesh
On behalf of all the people of Andhra Pradesh I offer prostrations to His Holiness Sri Swamiji. Many great people are gathered here. Since all names are God's names, when I say the name, Sri Ganapati Sachchidananda it includes everyone.
Today this Raga Ragini Nada Yogamu book is released. The main objective of Sri Swamiji is to rid our society of all ailments. We are under a lot of stress, pressure and tension. Health is affected. When we listen to Sri Swamiji's healing music the roga (disease) of society is removed and it becomes Raga (melody). It is the purity of Sri Swamiji's heart that brings bliss to the music. Listening to beautiful music is healing. Sri Swamiji told me, "What I want is not applause. I want my music to touch their hearts and transform them".
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Sri Swamiji with his pure heart gives happiness away. He transfers his bliss to us. We are greatly benefited by his presence, his music, and his blessings.
Eight crores (80 million) of population of Andhra Pradesh state have received your blessings today. We will ever remain in a state of happiness.


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Arizona Massacre Prompts Political 'Cheap Shots'
When Arizona Rep. Gabrielle Giffords collapsed outside the Safeway in Tucson Saturday morning, felled by a hail of bullets that killed six and wounded another 13 innocent people that had come to see her, some were quick to claim that the carnage was the product not merely of the tortured mind and trigger-happy fingers of the alleged shooter, 22-year-old Jared Lee Loughner.
Rather, many on the American Left said the horror could be traced to the malign influence of American conservatives; members of the Tea Party; right-wing pundits Rush Limbaugh and Glenn Beck; former Alaska Governor Sarah Palin; and Fox News.
That was the narrative of culpability spun in the immediate aftermath of the shootings by some leading liberal commentators and Democratic politicians — despite warnings from religious leaders, lawyers, academics, ethicists, reporters and historians that such a rush to judgment only further deepens the partisan divide in America, and further poisons its discourse.
Within minutes after the attempted assassination of Giffords — indeed, at a point when it was still erroneously believed in many quarters that she was dead, and the identity of her shooter was not publicly known — some commentators, absent any credible evidence, were already busily laying blame for the atrocity in political terms. Nobel Prize-winning economist and New York Times columnist Paul Krugman blogged at 3:22 p.m. ET Saturday: "We don't have proof yet that this was political, but the odds are that it was."
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|Price (USD)
|Customer Support and Order Management Specialist for GourmetFoodStore.com
|JOB DESCRIPTION: [url removed, login to view] is an online retailer of high-end specialty food products within the United States. We are looking for a customer support and order management specialist for [url removed, login to view] in a work-from-home position. Overall, your responsibility is to make sure customers are happy and orders are being fulfilled and processed properly. Some of your pr...
|Accounting, Customer Support, Phone Support, Order Processing, Call Center
|Apr 27, 2017
|Apr 27, 20175d 13h
|Need Shipping API for Fedex and UPS
|Need Shipping API for fedex, and UPS Need to validate address, compare rate and generate labels and be able to print (thermal labels) with tracking numbers. .net mvc5 only Please contact me if you have any question. Please share deom if you have one for immedidate selection.
|.NET, C# Programming, MVC, Web Services, ASP.NET
|Apr 26, 2017
|Apr 26, 20175d 6h
|Shopify - Shipwire integration
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|Software Architecture, node.js
|Apr 25, 2017
|Apr 25, 20173d 23h
|Magento Troubleshooting
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|PHP, Magento, PayPal API
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|Website design/development
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|PHP, Website Design, Graphic Design
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|Opencart Fedex plugin
|Help me fix the opencart Fedex plugin, migrate server, has to be done within today Mac user preferable, immediately availability preferable
|PHP, Website Design, Graphic Design, HTML
|Apr 20, 2017
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|Install Sugar CRM with Simple (Custom) Lead Workflow for the Pharmaceutical Industry
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|PHP, MySQL, SugarCRM
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|Modify existing Joomla Virtuemart e-commerce website
|1. Existing e-commerce web optimization: page requests, page speed, browser caching and render blocking. 2. Make checkout process more user friendly. 3. Configure FedEx shipping with account. 4. Add related products to product pages. 5. Correct duplicated products code. 6. Create RSS feed for existing blog. 7. Fix Search module. 8. Fix errors.
|PHP, Joomla, CSS, HTML, Virtuemart
|Apr 19, 2017
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|Address Verification to CMS
|Need to have the address verification feature added to my CMS to check the validity of the shipping addresses from my customer. It MUST verify customer's addresses prior to checkout. Using FEDEX, USPS, UPS API documentation. The shipping modules for FEDEX, USPS, UPS are ALREADY in my CMS, and I just need the address verification part done......
|PHP, Javascript, XML, HTML, BigCommerce
|Apr 19, 2017
|Apr 19, 2017Ended
|Data Entry
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|Hi mkvaish10, I'm looking to add a simple extension to a Ruby script I'm writing. I have a set of customer details that I'd like to parse to FedEx via their API so that the customer is saved in the address book. This will then allow me to "skip a step" when finalizing the FedEx order as I do not manually have to enter in the customer details in the address book. would th...
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|hi this is my drupal website [url removed, login to view] shipping cost is calculated based on the amount purchased I want to change it to calculate based on weight and dimensions with Canada Post please tell me the cost of doing this ... what if I also want to add fedex and have my special fedex rate ? what the cost for this the website has paypal payment and buyers can use credit ca...
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|Apr 8, 2017
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|I would like to hire a Website Designer
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|Change features in an existing Online store with FedEx, UPS and USPS API
|Hi, I want to fix few things related to Shipping APIs like FedEx, UPS and USPS.
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This from the FT yesterday:
"Europe's banks 'not really investable' says Credit Suisse's (NYSE:CS) Thiam
"European banks are in a "very fragile situation" and are "not really investable as a sector", Credit Suisse chief executive Tidjane Thiam said in an interview in London.
"You get extreme movements on the basis of relatively minor piece of news because there's a lot of uncertainty," he said, citing "regulatory uncertainty" about future capital requirements and concerns about "potential fines like you've seen on Deutsche Bank this week."
"All that together creates a very fragile situation," he told a Bloomberg conference on Wednesday, reports Laura Noonan in London.
"I think there is also a lot of doubt, a fundamental doubt, is there a viable business model that covers its cost of equity?" Mr Thiam added.
"That's the big big question," he said, describing it as something that "makes banks not really investable as a sector."
- FT, September 28
My first reaction to this report was to say something along the lines of, "Speak for yourself Mr Thiam". True, by saying banks are not really investable as a sector, the CS CEO might have merely meant not all banks are easy to invest in, so as a sector you can't buy indiscriminately. Still, this seems a sweeping statement and investors should be aware of the qualifications.
By far the greatest problem for the sector this year has not been the "regulatory uncertainty" cited by Mr Thiam. Nor are business models generally unviable - anything retail deposit funded has a good model.
In the case of Credit Suisse, the problem has been it blowing up every seven years or so, which puts a question over the viability of its business model more than regulatory overstretch does now. Here is the Credit Suisse experience from the point of view of its long term shareholders since 2001:
Source: Company data
While regulatory uncertainties persist, they are relatively minor in comparison to what the sector faced previously with most banks over their capital thresholds it seems further increases will be incremental. It also appears that the EU commission is satisfied with capital levels and feels they should not go much higher.
The main problem faced by European banks is the super low rate environment and therefore the macro drivers behind this (China, Commodities, spare capacity, deleveraging and internal adjustment in Europe). It is this environment that has led to >20% downgrades in the estimated sector EPS this year.
In this respect, a possible new stance towards production levels from OPEC and the growing acceptance that fiscal expansion is needed among G20 policy makers are positive signs although it is still too early for a reflation trade.
And some banks of course are coping well with the environment. The Scandinavian banks are in good shape, as are Benelux and main French banks. The chart below shows a selection of large European banks with stable core revenue alongside U.S. Bancorp (NYSE:USB) - a very steady performer in the U.S. and a useful yardstick for stability. Note the presence of Deutsche Bank (NYSE:DB) in this sample.
Source: Company data
German banks are suffering. Deutsche because of over expansion combined with truly abysmal management practices and Commerzbank (OTCPK:CRZBY) because of the brutally competitive landscape in the domestic banking space, which in turn is a function of government policy whereby state backed land and savings banks provide cheap banking to the mittelstand. The Bundestag's political guns are trained on Mario Draghi - and yes, negative rates make the hyper-competitive German banking system even harder to make money in - but the guns are not trained on the regulator. By the way, the German government could help the cause of higher rates by spending more money on infrastructure.
Italian banks are suffering too: because of a massive NPL problem. If anything, I suspect there has been some regulatory forbearance there. Investors should not think that the models are "unviable". There is also a little good news: there is evidence that the rate of NPL accrual is slowing, and while the sector faces many questions over how to finance its NPLs, investors can still find banks that can work there way through them. The flattening out of NPL growth can be seen in the chart below. This will allow future provision charges to reduce net balances further, rather than just maintaining coverage levels, or to increase shareholder returns.
Source: Bank of Italy
Credit Suisse is working to restructure and find its "viable business model". The European banks universe is replete with well run institutions, coping with the difficult conditions and paying healthy dividends. Say what you like about German cost/income, Italian NPLs, Spanish pricing strategies and indeed Credit Suisse's search for an identity. But leave ING (NYSE:ING), Danske (OTCPK:DNSKY), BNP (OTCQX:BNPQY), KBC (OTCPK:KBCSY), and Intesa San Paulo (OTCPK:ISNPY) alone!
Disclosure: I/we have no positions in any stocks mentioned, and no plans to initiate any positions within the next 72 hours.
I wrote this article myself, and it expresses my own opinions. I am not receiving compensation for it (other than from Seeking Alpha). I have no business relationship with any company whose stock is mentioned in this article.
Editor's Note: This article discusses one or more securities that do not trade on a major U.S. exchange. Please be aware of the risks associated with these stocks.


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Doctor insights on:
Stomach Pain After Endoscopy
What to do if I have bad stomach pain after painful endoscopy?
Time frame ?: In the immediate period after endoscopy, the pain could be from gaseous distention but this usually passes once the gas has been passed. If however the pain symptoms are severe after this period or even in the immediate period following the procedure, i would check right away with your GI doctor so as to assure that all is well. ...Read more
Endoscopy (Definition)
Endoscopy broadly refers to the direct visualization of the intestinal tract by way of a flexible, fiberoptic scope. Generally, the term is used to describe evaluation of the esophagus, stomach and duodenum (upper endoscopy), or the ...Read more
Bad stomach pain after painful endoscopy, what do you recommend?
Call physician: Who did the endoscopy to be safe. If pain is not going way, getting worse, or you start noticing other symptoms, stay safe and go to er. Endoscopy, as long as it is not along with something to help with a stricture, should not be painful hours later. If it was painful at time, may have caused some inflammation, but check to be safe. ...Read more
Stomach pain normal a day after endoscopy?
Yes: See your health care provider if it persists or worsens. ...Read more
I got my endoscopy today for stomach pain and acidity and they found that I have oesophagitis grade a. What is it?
Inflammation.: You esophagus is inflamed and this could be from any number of reasons including acidity or infection from eg fungus. I am sure your GI doctor will guide you accordingly with regards to appropriate therapy. Be well. ...Read more
3-4x a year I get chronic stomach pain lasting for 6-10 weeks. Not triggered or impacted by stress or food. Endoscopy was inconclusive ?
Functional: 20's male with chronic recurrent intestinal dysfunctions, each of several weeks duration, would seem to be having IBS; a functional disorder probably mediated by parasympathetic intestinal hyperactivity. In such cases, therapeutic benefits provided by Bentyl (dicyclomine) or Cannabis would be diagnostically enlightening. Associated chronic low back and pelvic pains are often seen in males with IBS. ...Read more
Despite using 40 mg of prilosec daily still have stomach pain.any need for endoscopy?
Yes: Undiagnosed abdominal pain that doesn't respond to high dose proton pump inhibitor needs to be further evaluated. If we're thinking along lines of acid reflux or gastroesophageal reflux disease, then upper endoscopy is diagnostic procedure of choice. If pain related to fatty meals, we might recommend ultrasound looking at gall bladder. So work closely w/Family Doc & Gastroenterologist. ...Read more
Hey doc I had an endoscopy done two days ago and I'm having mild stomach pain is this normal?
Post endoscopy: this could be normal after the procedure but to be safe i would place a call to your gastroenterologist ...Read more
1 week ago, I had got a upper endoscopy, they found bleeding and stopped it. But I have been feeling horrible stomach pain, coughing continuously.
Well : A person should not have horrible stomach pain and constant coughing afternoon diascopy. Recommend you seek urgent medical attention. ...Read more
I am suffering in a stomach pain .I have did endoscopy also doctor said there is no problem but the pain is continue what can I do?
Lots of reasons...: "stomach pain" can be caused by lots more than an endoscopy can find & may originate outside the GI tract. Further evaluation is in order before anyone can answer "what to do." your doctor will want to re-examine you as well as know the quality, timing, duration, location, & associations (eating, position, activity, stooling, urinating) of your pain to better direct the next appropriate testing. ...Read more
I had some stomach pain so dr did a FIT occult blood test which was positive. Both upper and lower endoscopy were normal. Am I in the clear?
Repeat test.: Granted, there are reasons for falsely positive stool occult blood tests. But if you repeat this test, properly performed, and it's still positive, then you should have another look. How long ago were the endoscopy and colonoscopy? You may need a small bowel study as well as repeat endoscopy/colonoscopy. ...Read more
Stomach Pain (Definition)
Most people will describe pain in any part of abdomen as stomach pain, it is what many patients say but in medical terms it is pain in the stomach which is located in middle of upper abdomen, so real stomach pain is the pain ...Read more
Stomach Discomfort (Definition)
Stomach discomfort or pain can be due to problems in digestion or irritation to the stomach. Some people use the term to describe discomfort ...Read more
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|Reviews for Born From Magic
| grither55 chapter 5 . 12/13/2015
I love this story! It is very amusing and well written. Please update. I cannot wait for Mozenrath to meet Taiah.
| jaminjess8 chapter 6 . 10/30/2015
Please continue this story it was really good!
| MisstyX0007 chapter 4 . 10/26/2015
Awe she's funny kid
| MisstyX0007 chapter 2 . 10/26/2015
Ah very nice
| bzwman chapter 6 . 8/12/2014
This is sad. RIP Robin Williams
| Carlisle Fan 22 chapter 6 . 8/11/2014
Yeah I heard about that. :'( RIP Robin Williams. I am watching a show where he first started acting called Mork and Mindy. It is an old show but he played an alien from a planet called Ork and was sent to observe life here on Earth and meets a girl called Mindy. It was a pretty funny show. Later I plan to watch all the movies I have at home with him in them. Including the Aladdin movies. So sad that happened to him, seems like such a waste to me. (Sigh)
| Carlisle Fan 22 chapter 5 . 8/10/2014
I like your story. It is really funny. Excellent work.
| Himeno Kazehito chapter 5 . 12/16/2013
Cant wait to see whats next
| FantasyFangirl13 chapter 5 . 11/23/2013
| KeepCalmAndLove chapter 5 . 9/6/2013
Update soon please!
| nightmaster000 chapter 5 . 8/30/2013
This is pretty good. The chapter's are short but still good. I look forward to seeing how the Aladdin gang react's to Taiah.
| Guest chapter 4 . 5/26/2013
This is really good! I hope you can update soon! I really want to see how the rest of the story unfolds!
| Idara chapter 4 . 4/2/2013
I like the story so far!
Taiah is a very interesting little girl, cute and stll acts a bit like Mozenrath nwn!
I hope you update soon, because I really, really, reaaaaally want to know what happens to Taiah :3


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Early Sunday morning is a special time in our house. My husband is home, so he can play with the baby while I make a little bit more extravagant breakfast than cereal. I'll make things like cinnamon roll, pumpkin, or banana pancakes, bacon, and eggs. Sometimes I make homemade waffles with whipped cream and fresh strawberries. It's like having strawberry shortcake for breakfast. The kids love it. And this? I know this'll put big smiles on a certain 7 year old girl's face and, soon, a tiny princess in training.
Walmart has two different Waffle Makers, Disney Princess or Frozen, clearance priced at $5.97 (reg. $14.96) with free in store pickup or free shipping with a $50 purchase. Goodness, this price is so good. I'm picking up an extra one to give as a gift.