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Abstract Horseshoe crabs, considered living fossils with a stable morphotype spanning ∼445 million years, are evolutionarily, ecologically, and biomedically important species experiencing rapid population decline. Of the four extant species of horseshoe crabs, the Atlantic horseshoe crab, Limulus polyphemus, has become an essential component of the modern medicine toolkit. Here, we present the first chromosome-level genome assembly, and the most contiguous and complete assembly to date, for L. polyphemus using nanopore long-read sequencing and chromatin conformation analysis. We find support for three horseshoe crab-specific whole-genome duplications, but none shared with Arachnopulmonata (spiders and scorpions). Moreover, we discovered tandem duplicates of endotoxin detection pathway components Factors C and G, identify candidate centromeres consisting of Gypsy retroelements, and classify the ZW sex chromosome system for this species and a sister taxon, Carcinoscorpius rotundicauda. Finally, we revealed this species has been experiencing a steep population decline over the last 5 million years, highlighting the need for international conservation interventions and fisheries-based management for this critical species. 

Abstract PremiseRobust standards to evaluate quality and completeness are lacking in eukaryotic structural genome annotation, as genome annotation software is developed using model organisms and typically lacks benchmarking to comprehensively evaluate the quality and accuracy of the final predictions. The annotation of plant genomes is particularly challenging due to their large sizes, abundant transposable elements, and variable ploidies. This study investigates the impact of genome quality, complexity, sequence read input, and method on protein‐coding gene predictions. MethodsThe impact of repeat masking, long‐read and short‐read inputs, and de novo and genome‐guided protein evidence was examined in the context of the popular BRAKER and MAKER workflows for five plant genomes. The annotations were benchmarked for structural traits and sequence similarity. ResultsBenchmarks that reflect gene structures, reciprocal similarity search alignments, and mono‐exonic/multi‐exonic gene counts provide a more complete view of annotation accuracy. Transcripts derived from RNA‐read alignments alone are not sufficient for genome annotation. Gene prediction workflows that combine evidence‐based and ab initio approaches are recommended, and a combination of short and long reads can improve genome annotation. Adding protein evidence from de novo assemblies, genome‐guided transcriptome assemblies, or full‐length proteins from OrthoDB generates more putative false positives as implemented in the current workflows. Post‐processing with functional and structural filters is highly recommended. DiscussionWhile the annotation of non‐model plant genomes remains complex, this study provides recommendations for inputs and methodological approaches. We discuss a set of best practices to generate an optimal plant genome annotation and present a more robust set of metrics to evaluate the resulting predictions. 

Abstract DNA methylation is critical to the regulation of transposable elements and gene expression and can play an important role in the adaptation of stress response mechanisms in plants. Traditional methods of methylation quantification rely on bisulfite conversion that can compromise accuracy. Recent advances in long‐read sequencing technologies allow for methylation detection in real time. The associated algorithms that interpret these modifications have evolved from strictly statistical approaches to Hidden Markov Models and, recently, deep learning approaches. Much of the existing software focuses on methylation in the CG context, but methylation in other contexts is important to quantify, as it is extensively leveraged in plants. Here, we present methylation profiles for two maple species across the full range of 5mC sequence contexts using Oxford Nanopore Technologies (ONT) long‐reads. Hybrid and reference‐guided assemblies were generated for two newAceraccessions:Acer negundo(box elder; 65x ONT and 111X Illumina) andAcer saccharum(sugar maple; 93x ONT and 148X Illumina). The ONT reads generated for these assemblies were re‐basecalled, and methylation detection was conducted in a custom pipeline with the publishedAcerreferences (PacBio assemblies) and hybrid assemblies reported herein to generate four epigenomes. Examination of the transposable element landscape revealed the dominance ofLTR Copiaelements and patterns of methylation associated with different classes of TEs. Methylation distributions were examined at high resolution across gene and repeat density and described within the broader angiosperm context, and more narrowly in the context of gene family dynamics and candidate nutrient stress genes. 

SUMMARY Maples (the genusAcer) represent important and beloved forest, urban, and ornamental trees distributed throughout the Northern hemisphere. They exist in a diverse array of native ranges and distributions, across spectrums of tolerance or decline, and have varying levels of susceptibility to biotic and abiotic stress. AmongAcerspecies, several stand out in their importance to economic interest. Here we report the first two chromosome‐scale genomes for North American species,Acer negundoandAcer saccharum. Both assembled genomes contain scaffolds corresponding to 13 chromosomes, withA. negundoat a length of 442 Mb, an N50 of 32 Mb, and 30 491 genes, andA. saccharumat a length of 626 Mb, an N50 of 46 Mb, and 40 074 genes. No recent whole genome duplications were detected, thoughA. saccharumhas local gene duplication and more recent bursts of transposable elements, as well as a large‐scale translocation between two chromosomes. Genomic comparison revealed thatA. negundohas a smaller genome with recent gene family evolution that is predominantly contracted and expansions that are potentially related to invasive tendencies and tolerance to abiotic stress. Examination of RNA sequencing data obtained fromA. saccharumgiven long‐term aluminum and calcium soil treatments at the Hubbard Brook Experimental Forest provided insights into genes involved in the aluminum stress response at the systemic level, as well as signs of compromised processes upon calcium deficiency, a condition contributing to maple decline.