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1. Paralogs and Off-Target Sequences Improve Phylogenetic Resolution in a Densely Sampled Study of the Breadfruit Genus ( Artocarpus , Moraceae)

   https://doi.org/10.1093/sysbio/syaa073  

   Gardner, Elliot M ; Johnson, Matthew G ; Pereira, Joan T ; Puad, Aida Shafreena ; Arifiani, Deby ; Sahromi, null ; Wickett, Norman J ; Zerega, Nyree J ( September 2020 , Systematic Biology)

   Charleston, Michael (Ed.)

   Abstract We present a 517-gene phylogenetic framework for the breadfruit genus Artocarpus (ca. 70 spp., Moraceae), making use of silica-dried leaves from recent fieldwork and herbarium specimens (some up to 106 years old) to achieve 96% taxon sampling. We explore issues relating to assembly, paralogous loci, partitions, and analysis method to reconstruct a phylogeny that is robust to variation in data and available tools. Although codon partitioning did not result in any substantial topological differences, the inclusion of flanking noncoding sequence in analyses significantly increased the resolution of gene trees. We also found that increasing the size of data sets increased convergence between analysis methods but did not reduce gene-tree conflict. We optimized the HybPiper targeted-enrichment sequence assembly pipeline for short sequences derived from degraded DNA extracted from museum specimens. Although the subgenera of Artocarpus were monophyletic, revision is required at finer scales, particularly with respect to widespread species. We expect our results to provide a basis for further studies in Artocarpus and provide guidelines for future analyses of data sets based on target enrichment data, particularly those using sequences from both fresh and museum material, counseling careful attention to the potential of off-target sequences to improve resolution. [Artocarpus; Moraceae; noncoding sequences; phylogenomics; target enrichment.] 

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2. An evaluation of transcriptome‐based exon capture for frog phylogenomics across multiple scales of divergence (Class: Amphibia, Order: Anura)

   https://doi.org/10.1111/1755-0998.12541  

   Portik, Daniel M. ; Smith, Lydia L. ; Bi, Ke ( June 2016 , Molecular Ecology Resources)

   Custom sequence capture experiments are becoming an efficient approach for gathering large sets of orthologous markers in nonmodel organisms. Transcriptome‐based exon capture utilizes transcript sequences to design capture probes, typically using a reference genome to identify intron–exon boundaries to exclude shorter exons (<200 bp). Here, we test directly using transcript sequences for probe design, which are often composed of multiple exons of varying lengths. Using 1260 orthologous transcripts, we conducted sequence captures across multiple phylogenetic scales for frogs, including outgroups ~100 Myr divergent from the ingroup. We recovered a large phylogenomic data set consisting of sequence alignments for 1047 of the 1260 transcriptome‐based loci (~561 000 bp) and a large quantity of highly variable regions flanking the exons in transcripts (~70 000 bp), the latter improving substantially by only including ingroup species (~797 000 bp). We recovered both shorter (<100 bp) and longer exons (>200 bp), with no major reduction in coverage towards the ends of exons. We observed significant differences in the performance of blocking oligos for target enrichment and nontarget depletion during captures, and differences inPCRduplication rates resulting from the number of individuals pooled for capture reactions. We explicitly tested the effects of phylogenetic distance on capture sensitivity, specificity, and missing data, and provide a baseline estimate of expectations for these metrics based on a priori knowledge of nuclear pairwise differences among samples. We provide recommendations for transcriptome‐based exon capture design based on our results, cost estimates and offer multiple pipelines for data assembly and analysis.

    

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3. Reconstructing Dipsacales phylogeny using Angiosperms353: issues and insights

   https://doi.org/10.1002/ajb2.1695  

   Lee, Aaron K. ; Gilman, Ian S. ; Srivastav, Mansa ; Lerner, Ariel D. ; Donoghue, Michael J. ; Clement, Wendy L. ( July 2021 , American Journal of Botany)

   Phylogenetic relationships within major angiosperm clades are increasingly well resolved, but largely informed by plastid data. Areas of poor resolution persist within the Dipsacales, including placement ofHeptacodiumandZabelia, and relationships within the Caprifolieae and Linnaeeae, hindering our interpretation of morphological evolution. Here, we sampled a significant number of nuclear loci using a Hyb‐Seq approach and used these data to infer the Dipsacales phylogeny and estimate divergence times.

   Sampling all major clades within the Dipsacales, we applied the Angiosperms353 probe set to 96 species. Data were filtered based on locus completeness and taxon recovery per locus, and trees were inferred using RAxML and ASTRAL. Plastid loci were assembled from off‐target reads, and 10 fossils were used to calibrate dated trees.

   Varying numbers of targeted loci and off‐target plastomes were recovered from most taxa. Nuclear and plastid data confidently placeHeptacodiumwith Caprifolieae, implying homoplasy in calyx morphology, ovary development, and fruit type. Placement ofZabelia, and relationships within the Caprifolieae and Linnaeeae, remain uncertain. Dipsacales diversification began earlier than suggested by previous angiosperm‐wide dating analyses, but many major splitting events date to the Eocene.

   The Angiosperms353 probe set facilitated the assembly of a large, single‐copy nuclear dataset for the Dipsacales. Nevertheless, many relationships remain unresolved, and resolution was poor for woody clades with low rates of molecular evolution. We favor expanding the Angiosperms353 probe set to include more variable loci and loci of special interest, such as developmental genes, within particular clades.

    

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4. A two‐tier bioinformatic pipeline to develop probes for target capture of nuclear loci with applications in Melastomataceae

   https://doi.org/10.1002/aps3.11345  

   Jantzen, Johanna R. ; Amarasinghe, Prabha ; Folk, Ryan A. ; Reginato, Marcelo ; Michelangeli, Fabian A. ; Soltis, Douglas E. ; Cellinese, Nico ; Soltis, Pamela S. ( May 2020 , Applications in Plant Sciences)

   Putatively single‐copy nuclear (SCN) loci, which are identified using genomic resources of closely related species, are ideal for phylogenomic inference. However, suitable genomic resources are not available for many clades, including Melastomataceae. We introduce a versatile approach to identify SCN loci for clades with few genomic resources and use it to develop probes for target enrichment in the distantly relatedMemecylonandTibouchina(Melastomataceae).

   We present a two‐tiered pipeline. First, we identified putatively SCN loci using MarkerMiner and transcriptomes from distantly related species in Melastomataceae. Published loci and genes of functional significance were then added (384 total loci). Second, using HybPiper, we retrieved 689 homologous template sequences for these loci using genome‐skimming data from within the focal clades.

   We sequenced 193 loci common toMemecylonandTibouchina. Probes designed from 56 template sequences successfully targeted sequences in both clades. Probes designed from genome‐skimming data within a focal clade were more successful than probes designed from other sources.

   Our pipeline successfully identified and targeted SCN loci inMemecylonandTibouchina, enabling phylogenomic studies in both clades and potentially across Melastomataceae. This pipeline could be easily applied to other clades with few genomic resources.

    

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5. DNA methylation signatures of duplicate gene evolution in angiosperms

   https://doi.org/10.1093/plphys/kiad220  

   Kenchanmane Raju, Sunil K. ; Ledford, Marshall ; Niederhuth, Chad E. ( April 2023 , Plant Physiology)

   Gene duplication is a source of evolutionary novelty. DNA methylation may play a role in the evolution of duplicate genes (paralogs) through its association with gene expression. While this relationship has been examined to varying extents in a few individual species, the generalizability of these results at either a broad phylogenetic scale with species of differing duplication histories or across a population remains unknown. We applied a comparative epigenomic approach to 43 angiosperm species across the phylogeny and a population of 928 Arabidopsis (Arabidopsis thaliana) accessions, examining the association of DNA methylation with paralog evolution. Genic DNA methylation was differentially associated with duplication type, the age of duplication, sequence evolution, and gene expression. Whole-genome duplicates were typically enriched for CG-only gene body methylated or unmethylated genes, while single-gene duplications were typically enriched for non-CG methylated or unmethylated genes. Non-CG methylation, in particular, was a characteristic of more recent single-gene duplicates. Core angiosperm gene families were differentiated into those which preferentially retain paralogs and “duplication-resistant” families, which convergently reverted to singletons following duplication. Duplication-resistant families that still have paralogous copies were, uncharacteristically for core angiosperm genes, enriched for non-CG methylation. Non-CG methylated paralogs had higher rates of sequence evolution, higher frequency of presence–absence variation, and more limited expression. This suggests that silencing by non-CG methylation may be important to maintaining dosage following duplication and be a precursor to fractionation. Our results indicate that genic methylation marks differing evolutionary trajectories and fates between paralogous genes and have a role in maintaining dosage following duplication.

    

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