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  1. Premise

    The ability to sequence genome‐scale data from herbarium specimens would allow for the economical development of data sets with broad taxonomic and geographic sampling that would otherwise not be possible. Here, we evaluate the utility of a basic double‐digest restriction site–associatedDNAsequencing (ddRADseq) protocol usingDNAs from four genera extracted from both silica‐dried and herbarium tissue.


    DNAs fromDraba,Boechera,Solidago, andIlexwere processed with a ddRADseq protocol. The effects ofDNAdegradation, taxon, and specimen age were assessed.


    Although taxon, preservation method, and specimen age affected data recovery, large phylogenetically informative data sets were obtained from the majority of samples.


    These results suggest that herbarium samples can be incorporated into ddRADseq project designs, and that specimen age can be used as a rapid on‐site guide for sample choice. The detailed protocol we provide will allow users to pursue herbarium‐based ddRADseq projects that minimize the expenses associated with fieldwork and sample evaluation.

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  2. Abstract

    The capability to generate densely sampled single nucleotide polymorphism (SNP) data is essential in diverse subdisciplines of biology, including crop breeding, pathology, forensics, forestry, ecology, evolution and conservation. However, the wet‐laboratory expertise and bioinformatics training required to conduct genome‐scale variant discovery remain limiting factors for investigators with limited resources.

    Here we present ISSRseq, a PCR‐based method for reduced representation of genomic variation using simple sequence repeats as priming sites to sequence inter simple sequence repeat (ISSR) regions. Briefly, ISSR regions are amplified with single primers, pooled, used to construct sequencing libraries with a commercially available kit, and sequenced on the Illumina platform. We also present a flexible bioinformatic pipeline that assembles ISSR loci, calls and hard filters variants, outputs data matrices in common formats, and conducts population analyses using R.

    Using three angiosperm species as case studies, we demonstrate that ISSRseq is highly repeatable, necessitates only simple wet‐laboratory skills and commonplace instrumentation, is flexible in terms of the number of single primers used, and can generate genomic‐scale variant discovery on par with existing RRS methods which require more complex wet‐laboratory procedures.

    ISSRseq represents a straightforward approach to SNP genotyping in any organism, and we predict that this method will be particularly useful for those studying population genomics and phylogeography of non‐model organisms. Furthermore, the ease of ISSRseq relative to other RRS methods should prove useful to those lacking advanced expertise in wet‐laboratory methods or bioinformatics.

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  3. Abstract

    Whole‐genome duplication is considered an important speciation mechanism in plants. However, its effect on reproductive isolation between higher cytotypes is not well understood. We used backcrosses between different ploidy levels and surveys of mixed‐ploidy contact zones to determine how reproductive barriers differed with cytotype across a polyploid complex. We backcrossed F1 hybrids derived from 2X‐4X and 4X‐6X crosses in theCampanula rotundifoliaautopolyploid complex, measured backcross fitness, and estimated backcross DNA cytotype. We then sampled four natural mixed‐ploidy contact zones (two 2X‐4X and two 4X‐6X), estimated ploidy, and genotyped individuals across each contact zone. Reproductive success and capacity for gene flow was markedly lower for 2X‐4X than 4X‐6X hybrids. In fact, 3X hybrids could not backcross; all 2X‐4X backcross progeny resulted from neotetraploid F1 hybrids. Further, no 3X individuals were found in 2X‐4X contact zones, and 2X and 4X individuals were genetically distinct. By contrast, backcrosses of 5X hybrids were relatively successful, particularly when crossed to 6X individuals. In 4X‐6X contact zones, 5X individuals and aneuploids were common and all cytotypes were largely genetically similar and spatially intermixed. Taken together, these results provide strong evidence that reproduction is low between 2X and 4X cytotypes, primarily occurring via unreduced gamete production, but that reproduction and gene flow are ongoing between 4X and 6X cytotypes. Further, it suggests whole‐genome duplication can result in speciation between diploids and polyploids, but is less likely to create reproductive barriers between different polyploid cytotypes, resulting in two fundamentally different potentials for speciation across polyploid complexes.

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  4. Abstract Premise Annual plants often exhibit drought‐escape and avoidance strategies to cope with limited water availability. Determining the extent of variation and factors underlying the evolution of divergent strategies is necessary for determining population responses to more frequent and severe droughts. Methods We leveraged five Mimulus guttatus populations collected across an aridity gradient within manipulative drought and quantitative genetics experiments to examine constitutive and terminal‐drought induced responses in drought resistance traits. Results Populations varied considerably in drought‐escape‐ and drought‐avoidance‐associated traits. The most mesic population demonstrated a unique resource conservative strategy. Xeric populations exhibited extreme plasticity when exposed to terminal drought that included flowering earlier at shorter heights, increasing water‐use efficiency, and shifting C:N ratios. However, plasticity responses also differed between populations, with two populations slowing growth rates and flowering at earlier nodes and another population increasing growth rate. While nearly all traits were heritable, phenotypic correlations differed substantially between treatments and often, populations. Conclusions Our results suggest drought resistance strategies of populations may be finely adapted to local patterns of water availability. Substantial plastic responses suggest that xeric populations can already acclimate to drought through plasticity, but populations not frequently exposed to drought may be more vulnerable. 
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    Free, publicly-accessible full text available August 1, 2024
  5. Abstract— Like many fern lineages comprising reticulate species complexes, Polypodium s.s. (Polypodiacaeae) has a history shaped by rapid diversification, hybridization, and polyploidy that poses substantial challenges for phylogenetic inference with plastid and single-locus nuclear markers. Using target capture probes for 408 nuclear loci developed by the GoFlag project and a custom bioinformatic pipeline, SORTER, we constructed multi-locus nuclear datasets for diploid temperate and Mesoamerican species of Polypodium and five allotetraploid species belonging to the well-studied Polypodium vulgare complex. SORTER employs a clustering approach to separate putatively paralogous copies of targeted loci into orthologous matrices and haplotype phasing to infer allopolyploid haplotypes across loci, resulting in datasets amenable to both concatenated maximum likelihood and multi-species coalescent phylogenetic analyses. By comparing phylogenies derived from maximum likelihood and multi-species coalescent analyses of unphased and phased datasets, as well as evaluating discordance among gene trees and species trees, we recover support for incomplete lineage sorting within Polypodium s.s., novel relationships among diploid taxa of the Polypodium vulgare complex and its Mesoamerican sister clade, and the placement of several Polypodium species within other genera. Additionally, we were able to infer well-supported phylogenies that identified the hypothesized progenitors of the allotetraploid species, indicating that SORTER is an effective and accurate tool for reconstructing homeolog haplotypes of allopolyploids in fern taxa and other non-model organisms from target capture data. 
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  6. Abstract— The genus Solidago represents a taxonomically challenging group due to its sheer number of species, putative hybridization, polyploidy, and shallow genetic divergence among species. Here we use a dataset obtained exclusively from herbarium specimens to evaluate the status of Solidago ulmifolia var. palmeri , a morphologically subtle taxon potentially confined to Alabama, Arkansas, Mississippi, and Missouri. A multivariate analysis of both discrete and continuous morphological data revealed no clear distinction between S. ulmifolia var. palmeri and Solidago ulmifolia var. ulmifolia . Solidago ulmifolia var. palmeri ’s status was also assessed with a phylogenomic and SNP clustering analysis of data generated with the “Angiosperms353” probe kit. Neither analysis supported Solidago ulmifolia var. palmeri as a distinct taxon, and we suggest that this name should be discarded. The status of Solidago delicatula (formerly known as Solidago ulmifolia var. microphylla ) was also assessed. Both morphological and phylogenetic analyses supported the species status of S. delicatula and we suggest maintaining this species at its current rank. These results highlight the utility of the Angiosperms353 probe kit, both with herbarium tissue and at lower taxonomic levels. Indeed, this is the first study to utilize this kit to identify genetic groups within a species. 
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  7. Eyre-Walker, Adam (Ed.)
    Abstract The invasive Japanese stiltgrass (Microstegium vimineum) affects a wide range of ecosystems and threatens biodiversity across the eastern USA. However, the mechanisms underlying rapid adaptation, plasticity, and epigenetics in the invasive range are largely unknown. We present a chromosome-level assembly for M. vimineum to investigate genome dynamics, evolution, adaptation, and the genomics of phenotypic plasticity. We generated a 1.12-Gb genome with scaffold N50 length of 53.44 Mb respectively, taking a de novo assembly approach that combined PacBio and Dovetail Genomics Omni-C sequencing. The assembly contains 23 pseudochromosomes, representing 99.96% of the genome. BUSCO assessment indicated that 80.3% of Poales gene groups are present in the assembly. The genome is predicted to contain 39,604 protein-coding genes, of which 26,288 are functionally annotated. Furthermore, 66.68% of the genome is repetitive, of which unclassified (35.63%) and long-terminal repeat (LTR) retrotransposons (26.90%) are predominant. Similar to other grasses, Gypsy (41.07%) and Copia (32%) are the most abundant LTR-retrotransposon families. The majority of LTR-retrotransposons are derived from a significant expansion in the past 1–2 Myr, suggesting the presence of relatively young LTR-retrotransposon lineages. We find corroborating evidence from Ks plots for a stiltgrass-specific duplication event, distinct from the more ancient grass-specific duplication event. The assembly and annotation of M. vimineum will serve as an essential genomic resource facilitating studies of the invasion process, the history and consequences of polyploidy in grasses, and provides a crucial tool for natural resource managers. 
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