Optimal Data Partitioning and a Test Case for Ray-Finned Fishes (Actinopterygii) Based on Ten Nuclear Loci

Data partitioning, the combined phylogenetic analysis of homogeneous blocks of data, is a common strategy used to accommodate heterogeneities in complex multilocus data sets. Variation in evolutionary rates and substitution patterns among sites are typically addressed by partitioning data by gene, codon position, or both. Excessive partitioning of the data, however, could lead to overparameterization; therefore, it seems critical to define the minimum numbers of partitions necessary to improve the overall fit of the model. We propose a new method, based on cluster analysis, to find an optimal partitioning strategy for multilocus protein-coding data sets. A heuristic exploration of alternative partitioning schemes, based on Bayesian and maximum likelihood (ML) criteria, is shown here to produce an optimal number of partitions. We tested this method using sequence data of 10 nuclear genes collected from 52 ray-finned fish (Actinopterygii) and four tetrapods. The concatenated sequences included 7995 nucleotide sites maximally split into 30 partitions defined a priori based on gene and codon position. Our results show that a model based on only 10 partitions defined by cluster analysis performed better than partitioning by both gene and codon position. Alternative data partitioning schemes also are shown to affect the topologies resulting from phylogenetic analysis, especially when Bayesian methods are used, suggesting that overpartitioning may be of major concern. The phylogenetic relationships among the major clades of ray-finned fish were assessed using the best data-partitioning schemes under ML and Bayesian methods. Some significant results include the monophyly of “Holostei” (Amia and Lepisosteus), the sister-group relationships between (1) esociforms and salmoniforms and (2) osmeriforms and stomiiforms, the polyphyly of Perciformes, and a close relationship of cichlids and atherinomorphs

Exon Primed, Intron Crossing (EPIC) markers for evolutionary studies of Ficus and other taxa in the fig family (Moraceae)

• Premise of the study: The genus Ficus (fi g trees) comprises ca. 750 species of trees, vines, and stranglers found in tropical forests throughout the world. Fig trees are keystone species in many tropical forests, and their relationship with host-specific wasp pollinators has received much attention, although many questions remain unresolved regarding the levels of host specificity,cospeciation, and the role of hybridization in fig and wasp speciation. We developed exon-primed intron-crossing (EPIC)markers to obtain phylogenetic resolution needed to address these questions. • Methods and Results: Expressed sequence tags (ESTs) from F. elastica were compared to Arabidopsis and Populus genomes to locate introns and to design primers in flanking exons. Primer pairs for 80 EPIC markers were tested in samples from divergent clades within Ficus and the outgroup Poulsenia (Moraceae). • Conclusions: Thirty-one EPIC markers were successfully sequenced across Ficus , and 29 of the markers also amplified in Poulsenia , indicating broad transferability within Moraceae. All of the EPIC markers were polymorphic and showed levels of polymorphism similar to that of the widely used internal transcribed spacer (ITS).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/100185/1/Yao2013.pd

A Practical Approach to Phylogenomics: The Phylogeny of Ray-Finned Fish (Actinopterygii) as a Case Study

Background: Molecular systematics occupies one of the central stages in biology in the genomic era, ushered in by unprecedented progress in DNA technology. The inference of organismal phylogeny is now based on many independent genetic loci, a widely accepted approach to assemble the tree of life. Surprisingly, this approach is hindered by lack of appropriate nuclear gene markers for many taxonomic groups especially at high taxonomic level, partially due to the lack of tools for efficiently developing new phylogenetic makers. We report here a genome-comparison strategy to identifying nuclear gene markers for phylogenetic inference and apply it to the ray-finned fishes – the largest vertebrate clade in need of phylogenetic resolution. Results: A total of 154 candidate molecular markers – relatively well conserved, putatively single-copy gene fragments with long, uninterrupted exons – were obtained by comparing whole genome sequences of two model organisms, Danio rerio and Takifugu rubripes. Experimental tests of 15 of these (randomly picked) markers on 36 taxa (representing two-thirds of the ray-finned fish orders) demonstrate the feasibility of amplifying by PCR and directly sequencing most of these candidates from whole genomic DNA in a vast diversity of fish species. Preliminary phylogenetic analyses of sequence data obtained for 14 taxa and 10 markers (total of 7,872 bp for each species) are encouraging, suggesting that the markers obtained will make significant contributions to future fish phylogenetic studies. Conclusion: We present a practical approach that systematically compares whole genome sequences to identify single-copy nuclear gene markers for inferring phylogeny. Our method is an improvement over traditional approaches (e.g., manually picking genes for testing) because it uses genomic information and automates the process to identify large numbers of candidate makers. This approach is shown here to be successful for fishes, but also could be applied to other groups of organisms for which two or more complete genome sequences exist, which has important implications for assembling the tree of life

Exon-primed intron-crossing (EPIC) markers for non-model teleost fishes

Background: Exon-primed intron-crossing (EPIC) markers have three advantages over anonymous genomic sequences in studying evolution of natural populations. First, the universal primers designed in exon regions can be applied across a broad taxonomic range. Second, the homology of EPIC-amplified sequences can be easily determined by comparing either their exon or intron portion depending on the genetic distance between the taxa. Third, having both the exon and intron fragments could help in examining genetic variation at the intraspecific and interspecific level simultaneously, particularly helpful when studying species complex. However, the paucity of EPIC markers has hindered multilocus studies using nuclear gene sequences, particularly in teleost fishes. Results: We introduce a bioinformatics pipeline for developing EPIC markers by comparing the whole genome sequences between two or more species. By applying this approach on five teleost fishes whose genomes were available in the Ensembl database http://www.ensembl.org, we identified 210 EPIC markers that have single-copy and conserved exon regions with identity greater than 85% among the five teleost fishes. We tested 12 randomly chosen EPIC markers in nine teleost species having a wide phylogenetic range. The success rate of amplifying and sequencing those markers varied from 44% to 100% in different species. We analyzed the exon sequences of the 12 EPIC markers from 13 teleosts. The resulting phylogeny contains many traditionally well-supported clades, indicating the usefulness of the exon portion of EPIC markers in reconstructing species phylogeny, in addition to the value of the intron portion of EPIC markers in interrogating the population history. Conclusions: This study illustrated an effective approach to develop EPIC markers in a taxonomic group, where two or more genome sequences are available. The markers identified could be amplified across a broad taxonomic range of teleost fishes. The phylogenetic utility of individual markers varied according to intron size and amplifiability. The bioinformatics pipelines developed are readily adapted to other taxonomic groups

Multiple freshwater invasions of the tapertail anchovy (Clupeiformes: Engraulidae) of the Yangtze River.

Freshwater fish evolved from anadromous ancestors can be found in almost all continents. The roles of paleogeographic events and nature selection in speciation process often are under focus of research. We studied genetic diversity of anadromous and resident tapertail anchovies (Coilia nasus species complex) in the Yangtze River Basin using 4,434 nuclear loci, and tested the history of freshwater invasion of C. nasus. We found that both C. brachygnathus and C. nasus were valid species, but the resident C. nasus taihuensis and the anadromous C. nasus were not different genetically based on Bayes factor species delimitation (BFD*). Maximum likelihood tree, Network, PCA and STRUCTURE analyses all corroborated the results of BFD*. Two independent freshwater invasion events of C. nasus were supported, with the first event occurring around 4.07 Ma and the second happened around 3.2 Ka. The time of the two freshwater invasions is consistent with different paleogeographic events. Estimation showed that gene flow was higher within ecotypes than between different ecotypes. F-DIST analyses identified 120 disruptive outliers by comparing C. brachygnathus to anadromous C. nasus, and 21 disruptive outliers by comparing resident C. nasus to anadromous C. nasus. Nine outliers were found to be common between the two comparisons, indicating that independent freshwater invasion of C. nasus might involve similar molecular pathways. The results of this study suggest that adaptation to landlocked freshwater environment of migratory fish can evolve multiple times independently, and morphology of landlocked ecotypes may cause confusion in their taxonomy

A practical approach to phylogenomics: the phylogeny of ray-finned fish (Actinopterygii) as a case study

BACKGROUND: Molecular systematics occupies one of the central stages in biology in the genomic era, ushered in by unprecedented progress in DNA technology. The inference of organismal phylogeny is now based on many independent genetic loci, a widely accepted approach to assemble the tree of life. Surprisingly, this approach is hindered by lack of appropriate nuclear gene markers for many taxonomic groups especially at high taxonomic level, partially due to the lack of tools for efficiently developing new phylogenetic makers. We report here a genome-comparison strategy to identifying nuclear gene markers for phylogenetic inference and apply it to the ray-finned fishes – the largest vertebrate clade in need of phylogenetic resolution. RESULTS: A total of 154 candidate molecular markers – relatively well conserved, putatively single-copy gene fragments with long, uninterrupted exons – were obtained by comparing whole genome sequences of two model organisms, Danio rerio and Takifugu rubripes. Experimental tests of 15 of these (randomly picked) markers on 36 taxa (representing two-thirds of the ray-finned fish orders) demonstrate the feasibility of amplifying by PCR and directly sequencing most of these candidates from whole genomic DNA in a vast diversity of fish species. Preliminary phylogenetic analyses of sequence data obtained for 14 taxa and 10 markers (total of 7,872 bp for each species) are encouraging, suggesting that the markers obtained will make significant contributions to future fish phylogenetic studies. CONCLUSION: We present a practical approach that systematically compares whole genome sequences to identify single-copy nuclear gene markers for inferring phylogeny. Our method is an improvement over traditional approaches (e.g., manually picking genes for testing) because it uses genomic information and automates the process to identify large numbers of candidate makers. This approach is shown here to be successful for fishes, but also could be applied to other groups of organisms for which two or more complete genome sequences exist, which has important implications for assembling the tree of life

Towards a compact soliton microcomb fully referenced on atomic reference

A fully stabilized soliton microcomb is critical for many applications of optical frequency comb based on microresonators. However, the current approaches for full frequency stabilization require either external acousto-optic or electro-optic devices or auxiliary lasers and multiple phase-locked loops, which compromises the convenience of the system. This study explores a compact atomic referenced fully stabilized soliton microcomb that directly uses a rubidium atomic optical frequency reference as the pump source, and complements the repetition rate (7.3 GHz) of the soliton microcomb was phase-locked to an atomic-clock-stabilized radio frequency (RF) reference by mechanically tuning the resonance of the optical resonator. The results demonstrate that the stability of the comb line (0.66 THz away from the pump line) is consistent with that of the Rb87 optical reference, attaining a level of approximately 4 Hz @100 s, corresponding to the frequency stability of 2E-14 @100 s. Furthermore,the frequency reproducibility of the comb line was evaluated over six days and it was discovered that the standard deviation (SD) of the frequency of the comb line is 10 kHz, resulting in a corresponding absolute deviation uncertainty of 1.3E-10, which is technically limited by the locking range of the soliton repetition rate. The proposed method gives a low-power and compact solution for fully stabilized soliton micorcombs.Comment: 6 pages, 5 figure

Introgressive hybridization between two close species Siniperca chuatsi and Siniperca kneri (Percomorpharia: Sinipercidae) in the Middle Reaches of the Yangtze River

Siniperca chuatsi and Siniperca kneri are two economically important freshwater fishes endemic to East Asia. Recently, some Siniperca specimens collected from Lake Poyang and Lake Dongting in the middle reaches of the Yangtze River couldn't be clearly identified as they showed intermediate morphological characteristics between S. chuatsi and S. kneri, and some inter-species hybrids were detected by microsatellite loci. To further verify genetic composition of these intermediate individuals, and determine the prevalence and degree of introgression between the two Siniperca species, a large set of high-quality, independent, diagnostic genetic markers were necessarily required. Based on enrichment and sequencing of target genes in sinipercid fishes, 463 loci (FST = 1) between S. chuatsi and S. kneri were selected and verified for species diagnosis. A total of 349 loci with 458 diagnostic SNPs were identified for discriminating S. chuatsi and S. kneri. From those markers, 224 diagnostic SNPs (only one SNP per locus) were selected to identify and categorize 48 specimens with intermediate morphological characters. The results showed that there were 8 specimens identified as hybrids, 8 specimens as S. chuatsi and 32 specimens as S. kneri. NEWHYBRIDS analysis showed that the hybrid offsprings were composed of the first-generation hybrid (2 individuals), first-generation backcross (1 individual), second-generation backcross (1 individual) and fourth-generation backcross (4 individuals), and the backcrossing could happen to both S. chuatsi and S. kneri. These hybrids could occur naturally, or escaped from farmed fish, due to extensive artificial breeding practice in these regions. However, the origin of the introgressive hybridization can't be easily traced. Therefore, some measures for protecting genetic resource of Siniperca speies in the Yangtze River should be enforced, such as assessing genetic background of the cultured stocks, reducing the escapement from farmed fish, and monitoring the trend of introgressive hybridization between Siniperca species in the future