Low-Copy Nuclear Genes Reveal New Evidence of Incongruence in Relationships Within Malvaceae s. l.

Abstract— The family Malvaceae s. l. is a clade that comprises nine subfamilies. Phylogenetic relationships among them are not completely resolved and are inconsistent among studies, probably due to low phylogenetic informativeness of conventional molecular markers. In the present study, we provide new phylogenetic information for Malvaceae s. l. derived from newly-designed group-specific nuclear markers. By mining transcriptome data from the One Thousand Plants Project (1KP) and publicly available genome information from cotton, cacao, and Arabidopsis, we designed a set of molecular markers of potentially single- or low-copy nuclear genes for Malvaceae s. l. Phylogenetic potential of these new loci was compared to previously applied conventional markers (i.e. plastid trnK-matK region and rbcL gene and the nrDNA ITS region) using the phylogenetic informativeness method. The results show that, when the mined nuclear regions are used in combination, it is possible to resolve relationships at different taxonomic levels within the phylogeny. However, incongruence among nuclear loci is frequent in the group, explaining the prevalence of unresolved phylogenetic relationships.

The mitochondrial genomes of Tortricidae: nucleotide composition, gene variation and phylogenetic performance

Background Mitochondrial genomes (mitogenomes) have greatly improved our understanding of the backbone phylogeny of Lepidoptera, but few studies on comparative mitogenomics below the family level have been conducted. Here, we generated 13 mitogenomes of eight tortricid species, reannotated 27 previously reported mitogenomes, and systematically performed a comparative analysis of nucleotide composition, gene variation and phylogenetic performance. Results The lengths of completely sequenced mitogenomes ranged from 15,440 bp to 15,778 bp, and the gene content and organization were conserved in Tortricidae and typical for Lepidoptera. Analyses of AT-skew and GC-skew, the effective number of codons and the codon bias index all show a base bias in Tortricidae, with little heterogeneity among the major tortricid groups. Variations in the divergence rates among 13 protein-coding genes of the same tortricid subgroup and of the same PCG among tortricid subgroups were detected. The secondary structures of 22 transfer RNA genes and two ribosomal RNA genes were predicted and comparatively illustrated, showing evolutionary heterogeneity among different RNAs or different regions of the same RNA. The phylogenetic uncertainty of Enarmoniini in Tortricidae was confirmed. The synonymy of Bactrini and Olethreutini was confirmed for the first time, with the representative Bactrini consistently nesting in the Olethreutini clade. Nad6 exhibits the highest phylogenetic informativeness from the root to the tip of the resulting tree, and the combination of the third coding positions of 13 protein-coding genes shows extremely high phylogenetic informativeness. Conclusions This study presents 13 mitogenomes of eight tortricid species and represents the first detailed comparative mitogenomics study of Tortricidae. The results further our understanding of the evolutionary architectures of tortricid mitogenomes and provide a basis for future studies of population genetics and phylogenetic investigations in this group.

Hundreds of nuclear and plastid loci yield insights into orchid relationships

ABSTRACTPremise of the studyEvolutionary relationships in the species-rich Orchidaceae have historically relied on organellar DNA sequences and limited taxon sampling. Previous studies provided a robust plastid-maternal phylogenetic framework, from which multiple hypotheses on the drivers of orchid diversification have been derived. However, the extent to which the maternal evolutionary history of orchids is congruent with that of the nuclear genome has remained uninvestigated.MethodsWe inferred phylogenetic relationships from 294 low-copy nuclear genes sequenced/obtained using the Angiosperms353 universal probe set from 75 species representing 69 genera, 16 tribes and 24 subtribes. To test for topological incongruence between nuclear and plastid genomes, we constructed a tree from 78 plastid genes, representing 117 genera, 18 tribes and 28 subtribes and compared them using a co-phylogenetic approach. The phylogenetic informativeness and support of the Angiosperms353 loci were compared with those of the 78 plastid genes.Key ResultsPhylogenetic inferences of nuclear datasets produced highly congruent and robustly supported orchid relationships. Comparisons of nuclear gene trees and plastid gene trees using the latest co-phylogenetic tools revealed strongly supported phylogenetic incongruence in both shallow and deep time. Phylogenetic informativeness analyses showed that the Angiosperms353 genes were in general more informative than most plastid genes.ConclusionsOur study provides the first robust nuclear phylogenomic framework for Orchidaceae plus an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely documented: nuclear and plastid phylogenetic trees are not fully congruent and therefore should not be considered interchangeable.

homologizer: Phylogenetic phasing of gene copies into polyploid subgenomes

SummaryOrganisms such as allopolyploids and F1 hybrids contain multiple subgenomes, each potentially with its own evolutionary history. These organisms present a challenge for multilocus phylogenetic inference and other analyses since it is not apparent which gene copies from different loci are from the same subgenome.Here we introduce homologizer, a flexible Bayesian approach that uses a phylogenetic framework to infer the phasing of gene copies across loci into polyploid subgenomes.Through the use of simulation tests we demonstrate that homologizer is robust to a wide range of factors, such as the phylogenetic informativeness of loci and incomplete lineage sorting. Furthermore, we establish the utility of homologizer on real data, by analyzing a multilocus dataset consisting of nine diploids and 19 tetraploids from the fern family Cystopteridaceae.Finally, we describe how homologizer may potentially be used beyond its core phasing functionality to identify non-homologous sequences, such as hidden paralogs, contaminants, or allelic variation that was erroneously modelled as homeologous.

Novel Molecular Resources to Facilitate Future Genetics Research on Freshwater Mussels (Bivalvia: Unionidae)

Molecular data have been an integral tool in the resolution of the evolutionary relationships and systematics of freshwater mussels, despite the limited number of nuclear markers available for Sanger sequencing. To facilitate future studies, we evaluated the phylogenetic informativeness of loci from the recently published anchored hybrid enrichment (AHE) probe set Unioverse and developed novel Sanger primer sets to amplify two protein-coding nuclear loci with high net phylogenetic informativeness scores: fem-1 homolog C (FEM1) and UbiA prenyltransferase domain-containing protein 1 (UbiA). We report the methods used for marker development, along with the primer sequences and optimized PCR and thermal cycling conditions. To demonstrate the utility of these markers, we provide haplotype networks, DNA alignments, and summary statistics regarding the sequence variation for the two protein-coding nuclear loci (FEM1 and UbiA). Additionally, we compare the DNA sequence variation of FEM1 and UbiA to three loci commonly used in freshwater mussel genetic studies: the mitochondrial genes cytochrome c oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1), and the nuclear internal transcribed spacer 1 (ITS1). All five loci distinguish among the three focal species (Potamilus fragilis, Potamilus inflatus, and Potamilus purpuratus), and the sequence variation was highest for ND1, followed by CO1, ITS1, UbiA, and FEM1, respectively. The newly developed Sanger PCR primers and methodologies for extracting additional loci from AHE probe sets have great potential to facilitate molecular investigations targeting supraspecific relationships in freshwater mussels, but may be of limited utility at shallow taxonomic scales.

First mitochondrial genomes of five hoverfly species of the genus Eristalinus (Diptera: Syrphidae)

The hoverfly genus Eristalinus (Diptera, Syrphidae) contains many widespread pollinators. The majority of the species of Eristalinus occur in the Afrotropics and their molecular systematics still needs to be investigated. This study presents the first complete and annotated mitochondrial genomes for five species of Eristalinus. They were obtained by high-throughput sequencing of total genomic DNA. The total length of the mitogenomes varied between 15 757 and 16 245 base pairs. Gene composition, positions, and orientation were shared across species, and were identical to those observed for other Diptera. Phylogenetic analyses (maximum likelihood and Bayesian inference) based on the 13 protein coding and both rRNA genes suggested that the subgenus Eristalinus was paraphyletic with respect to the subgenus Eristalodes. An analysis of the phylogenetic informativeness of all protein coding and rRNA genes suggested that NADH dehydrogenase subunit 5 (nad5), cytochrome c oxidase subunit 1, nad4, nad2, cytochrome b, and 16S rRNA genes are the most promising mitochondrial molecular markers to result in supported phylogenetic hypotheses of the genus. In addition to the five complete mitogenomes currently available for hoverflies, the five mitogenomes published here will be useful for broader molecular phylogenetic analyses among hoverflies.

Interrogating Genomic-Scale Data for Squamata (Lizards, Snakes, and Amphisbaenians) Shows no Support for Key Traditional Morphological Relationships

Genomics is narrowing uncertainty in the phylogenetic structure for many amniote groups. For one of the most diverse and species-rich groups, the squamate reptiles (lizards, snakes, and amphisbaenians), an inverse correlation between the number of taxa and loci sampled still persists across all publications using DNA sequence data and reaching a consensus on the relationships among them has been highly problematic. In this study, we use high-throughput sequence data from 289 samples covering 75 families of squamates to address phylogenetic affinities, estimate divergence times, and characterize residual topological uncertainty in the presence of genome-scale data. Importantly, we address genomic support for the traditional taxonomic groupings Scleroglossa and Macrostomata using novel machine-learning techniques. We interrogate genes using various metrics inherent to these loci, including parsimony-informative sites (PIS), phylogenetic informativeness, length, gaps, number of substitutions, and site concordance to understand why certain loci fail to find previously well-supported molecular clades and how they fail to support species-tree estimates. We show that both incomplete lineage sorting and poor gene-tree estimation (due to a few undesirable gene properties, such as an insufficient number of PIS), may account for most gene and species-tree discordance. We find overwhelming signal for Toxicofera, and also show that none of the loci included in this study supports Scleroglossa or Macrostomata. We comment on the origins and diversification of Squamata throughout the Mesozoic and underscore remaining uncertainties that persist in both deeper parts of the tree (e.g., relationships between Dibamia, Gekkota, and remaining squamates; among the three toxicoferan clades Iguania, Serpentes, and Anguiformes) and within specific clades (e.g., affinities among gekkotan, pleurodont iguanians, and colubroid families).