Background and Aims Plant volatile organic compounds (VOCs) induced by herbivory boost defences in neighbouring plants. These effects have been shown primarily for direct plant defences and are often stronger when emitter and receiver plants are genetically related. However, we know much less about how plant indirect defence is affected by VOC signalling. To address this, we conducted field experiments controlling for plant relatedness, testing the effects of VOC signalling on extrafloral nectar (EFN) production, a key indirect defence, and its impact on ant recruitment and attacks on herbivores of wild cotton (Gossypium hirsutum) plants.Methods Experiments consisted of plant triplets, in which one individual acted as an emitter of VOCs and two as receivers. One receiver shared the same mother plant as the emitter, and the other was descended from a different mother. Half of the emitter plants were induced using the specialist caterpillar Alabama argillacea, and VOCs were collected. We then induced receivers and measured their EFN production, in addition to ant abundance and attack on sentinel caterpillars. We also subsequently excluded ants from half of the receivers to test for ant-mediated effects on natural herbivory occurring over the following weeks.Key Results Receivers exposed to VOCs of damaged emitters produced a greater volume and concentration of EFN in response to herbivory relative to those exposed to undamaged emitters and, accordingly, showed higher rates of ant attack on sentinel caterpillars (albeit no differences in ant abundance). These effects were not contingent on emitter-receiver relatedness. In addition, we found no effect of ant exclusion on natural herbivory levels on receiver plants, although damage was low overall.Conclusions These findings provide insight into inter-plant VOC signalling effects on multitrophic interactions by revealing indirect defence induction that leads to herbivore reduction by ants but that such effect occurs independently of the degree of emitter-receiver relatedness.

Gossypium hirsutum is the world's most important source of cotton fibre, yet the diversity and population structure of its wild forms remain largely unexplored. The complex domestication history of G. hirsutum combined with reciprocal introgression with a second domesticated species, G. barbadense, has generated a wealth of morphological forms and feral derivatives of both species and their interspecies recombinants, which collectively are scattered across a large geographic range in arid regions of the Caribbean basin. Here we assessed genetic diversity within and among populations from two Caribbean islands, Puerto Rico ( n  = 43, five sites) and Guadeloupe ( n  = 25, one site), which contain putative wild or introgressed forms. Using whole-genome resequencing data and a phylogenomic framework derived from a broader genomic survey, we parsed individuals into feral derivatives and truly wild forms. Feral cottons display uneven levels of genetic and morphological resemblance to domesticated cottons, with diverse patterns of genetic variation and heterozygosity. These patterns are inferred to reflect a complex history of interspecific and intraspecific gene flow that is spatially highly variable in its effects. Wild cottons in both Caribbean islands appear to be relatively inbred, especially the Guadeloupe samples. Our results highlight the dynamics of population demographics in relictual wild cottons that experienced profound genetic bottlenecks associated with repeated habitat destruction superimposed on a natural ecogeographical distribution comprising widely scattered populations. These results have implications for conservation and utilisation of wild diversity in G. hirsutum .

Cellulose microfibrils in plant cell walls are essential for mechanical strength and overall quality of cotton fibers. This study quantified and compared the nano-omics: nanoscale structural, and mechanical properties of cellulose microfibrils such as microfibril dimensions, crossover count and angles, roughness, and Young’s modulus for two popular cotton species: Gossypium hirsutum (Gh) and Gossypium barbadense (Gb) fibers across four stages of growth (8, 12, 18, and 22 days post-anthesis) using atomic force microscopy (AFM). Our results revealed that Gb fibers exhibited better alignment, finer surface, and higher stiffness compared to Gh fibers at the nanoscale, resulting in smoother fiber surfaces and improved quality at the macroscale. We have also developed an integrative machine learning (ML) framework combining nanoscale AFM-derived features with gene expression data to model variation in key fiber traits. This framework was used to compare feature modalities and assess their relative contributions across developmental stages. To further characterize feature importance, we applied Shapley Additive exPlanations (SHAP), which highlighted nanoscale and transcriptomic variables associated with model behavior at different growth stages. Overall, this study demonstrates that nanoscale mechanical properties provide complementary information to transcriptomics and establishes a framework for integrating nanoscale and molecular features to generate mechanistic hypotheses linking gene expression to cotton fiber structure and quality.

Baobab trees (Adansonia) are iconic members of Madagascar's biota that serve as emblems for the island's unique biodiversity. Despite this recognition, many questions remain regarding evolutionary relationships and species limits within the genus. We used custom-designed targeted sequence capture data to obtain phased allelic sequences for hundreds of nuclear loci and plastomes. Using broad geographic sampling, representing all species in the genus, and including a few possible hybrid individuals, we reconstructed the population history of Malagasy baobabs using network inference methods to detect reticulation. We obtained a species tree that is well supported, but with clear cases of reticulation, most notably admixture between Adansonia rubrostipa and A. madagascariensis in northwestern Madagascar, and instances of introgression involving the widespread species, A. za. Analysis of exclusivity factors suggests elevating one variety of A. za to the species rank, and also raises the possibility, pending additional sampling, of splitting A. rubrostipa into two species. We discuss the implications of gene flow on species delimitation and diversification in the context of conservation efforts for these iconic and threatened trees. Given our results, we formally resurrect the species A. bozy from A. za var. bozy, thus raising the number of endemic Malagasy baobab species from six to seven.

The shapes and material properties of cotton (Gossypium spp.) seed coat trichoblasts form the basis of a multibillion-dollar natural fiber industry. As such, these highly specialized cells are low-hanging fruit for intentional trait engineering. However, broad success will require more mechanistic knowledge of their systems-level cellular controls. This time-series study integrates daily measurements of purified fiber transcriptomes and proteomes with multiscale fiber phenotyping datasets that span the same developmental interval. Abundance profiles of the subcellular proteomes are the foundation of the analyses. This resource article provides direct information about which homoeologs operate and offers informative depictions of how compartmentalized cellular systems change during developmental transitions. Prediction accuracy was partially validated by analyzing protein expression group 11, which contained multiple known secondary cell wall (CW) cellulose synthases together with dozens of unknown proteins, and displayed an averaged expression profile that strongly correlated with a sharp state transition in cellulose microfibril alignment and increased cellulose content. The dataset as a whole can serve as a hypothesis-generating tool to guide future experiments related to CW glycome remodeling, morphogenesis, reversible tissue formation, and growth rate control. Integration of mRNA and protein abundance revealed widespread evidence of post-transcriptional control. In addition, there were hundreds of transcriptionally controlled genes with different time points of transition. This latter gene set can be used to more reliably analyze transcriptional control networks and to generate collections of gene expression drivers for cotton fiber research. The protein and transcript abundance profiles are organized into user-friendly tables and a web interface that can be searched using any plant ortholog of interest based on developmental time, abundance, annotations, or phenotypic association.

ABSTRACT Island endemic species are particularly vulnerable to extinction due to their limited geographic ranges and small population sizes. Kokia is a genus found exclusively in the Hawaiian Islands, whose species were once major components of local forests but have experienced significant population reductions due to habitat destruction and the consequences of invasive species. Although conservation of Kokia species has been an ongoing topic for over a century, records regarding historical efforts are sparse. Recently generated genomes for each of the three extant species provide the foundation for understanding genetic diversity and population structure for future conservation work. Whole genome resequencing of K. cookei (n = 23 samples), K. drynarioides (n = 92), and K. kauaiensis (n = 45) suggests that K. drynarioides has the lowest overall (nucleotide) diversity, reflecting propagation from a limited part of the remaining gene pool, whereas K. kauaiensis exhibits the most diversity. Diversity in the primarily graft‐propagated K. cookei is higher than expected and slightly higher than in the free‐living K. drynarioides. Notably, our analyses identified a source of novel variation in K. cookei in a cultivated plant historically labeled K. drynarioides. Population structure analyses reveal a single population for K. cookei, but three groups for each of the other two species. Importantly, our analyses identify clusters of related individuals, reflected in genetic distance and clustering metrics, which provide valuable information for increasing diversity in managed populations and in ex situ conservation collections. These results provide a genomic framework for ongoing efforts in restoring and maintaining diversity in these critically endangered Hawaiian species.

Among the two allopolyploid cultivated species of cotton, Gossypium barbadense is known for its superior quality fiber compared to Gossypium hirsutum. Length and strength are key determinants of the fiber quality. Although mature fibers are composed of dried cell walls that mainly consist of cellulose, the dynamic remodeling of pectin, xyloglucan, and xylan polysaccharides during fiber growth significantly impacts the final fiber quality. Comprehensive knowledge of polysaccharides and their biosynthesis during fiber development in cultivated species is crucial for improving fiber quality. In this study, comparative large-scale glycome, transcriptome and proteome profiling were conducted daily on fibers of both cotton species, covering critical stages of fiber development spanning primary cell wall synthesis and the transition to secondary cell wall synthesis. Interspecific comparisons revealed that a delayed deposition of cellulose content, as well as the occurrence of lower levels and differential compositions of non-fucosylated/fucosylated xyloglucans, homogalacturonans, and highly branched rhamnogalacturonan-I polysaccharides, possibly contribute to longer elongation time and longer fiber phenotypes of G. barbadense relative to G. hirsutum. Our study also suggests that differential temporal compositions of arabinoxylans and glucuronoxylans might contribute to the variation in cellulose microfibril arrangement and the strength of fiber that exists between the two species of cotton. Comparative transcriptomic analysis identified differentially accumulated polysaccharide-synthesizing glycosyltransferases that may underlie differences in fiber quality between the two species. Transcripts encoding many cell wall-localized expansins were found to be more abundant in G. barbadense than in G. hirsutum, which could be a contributing factor for the longer fibers of G. barbadense. Overall, these findings expand our understanding of the molecular factors that contribute to fiber quality and provide insights for targeted cotton fiber improvement.

Gossypium hirsutum is the leading fiber crop globally, but its origin as a domesticated plant and patterns of diversity in the wild remain to be elucidated. Here, we use extensive sampling of wild populations and comparative genome sequence data to illuminate the scope and patterning of wild cotton diversity across its native range. Analyses confirm the hypothesis that the Yucatán Peninsula (México) is the center of domestication, from which the original perennial forms and later modern annualized cultivars were derived. Population structure and phylogenomic analyses indicate that northwestern Yucatán harbors greater genetic diversity relative to smaller, geographically dispersed populations in northeastern Yucatán and the Caribbean basin. Genetic load and transposable element burden also are the lowest in northwestern Yucatán relative to other regions, consistent with its greater diversity and reflecting the effects of historical genetic bottlenecks in other populations. Populations from Florida and elsewhere in the Caribbean basin maintain unique pockets of diversity. Analyses of selection suggest that cotton domestication entailed long-term accumulation of mutations with relatively minor phenotypic effects, as opposed to a more punctuated process involving major domestication genes. Our study quantifies the scope and scale of genomic diversity in wild cotton, the origin of the cultivated gene pool, and the likely ecological and anthropogenic processes that shaped extant diversity and modern geographic patterning.

Cotton (Gossypium hirsutum L.) is a key allopolyploid crop with global economic importance. Here we present a telomere-to-telomere assembly of the elite variety Zhongmian 113. Leveraging technologies including PacBio HiFi, Oxford Nanopore Technology (ONT) ultralong-read sequencing and Hi-C, our assembly surpasses previous genomes in contiguity and completeness, resolving 26 centromeric and 52 telomeric regions, 5S rDNA clusters and nucleolar organizer regions. A phylogenetically recent centromere repositioning on chromosome D08 was discovered specific to G. hirsutum, involving deactivation of an ancestral centromere and the formation of a unique, satellite repeat-based centromere. Genomic analyses evaluated favorable allele aggregation for key agronomic traits and uncovered an early-maturing haplotype derived from an 11 Mb pericentric inversion that evolved early during G. hirsutum domestication. Our study sheds light on the genomic origins of short-season adaptation, potentially involving introgression of an inversion from primitively domesticated forms, followed by subsequent haplotype differentiation in modern breeding programs.

Transposable elements are major components of plant genomes and major drivers of plant genome evolution. The cotton genus (Gossypium) is an excellent evolutionary model for polyploidization, speciation, domestication, and crop improvement. Here, we implement genome and pangenome analyses to study in detail the dynamics of LTR-retrotransposons during the cotton evolution. We show that some LTR-retrotransposon lineages amplified in tetraploid cotton compared to their diploid progenitors, whereas others stayed stable or amplified but were removed through solo-LTR formation. Using species-level pangenomes we show that only a few lineages (CRM, Tekay, Ivana, and Tork) remained active after polyploidization and are still transposing. Tekay and CRM elements have re-shaped the centromeric and pericentromeric regions of tetraploid cottons in a subgenome specific manner, through new insertions but also selective eliminations through solo-LTR formation. On the other hand, Ivana and Tork have actively inserted within or close to genes affecting their expression. Finally, population-level analyses using the two pangenomes and data from 283 and 223 varieties of G. hirsutum and G. barbadense reveal changes in Transposon Insertion Polymorphism frequencies accompanying domestication and improvement of both species, suggesting the possibility of selection on linked regions. Our findings reveal that LTR-retrotransposon lineages followed differential dynamics during cotton evolution, displaying differences among species and the two coresident genomes of allopolyploid cotton. A handful of the LTR-retrotransposon lineages that expanded after polyploidization helped shape the genomes of both G. hirsutum and G. barbadense, impacting their centromere and pericentromeric regions as well as protein-coding genes.

Cotton fiber development entails complex genome-wide gene regulatory networks (GRNs) that remain insufficiently resolved. Here, we present integrative analyses of fiber GRNs using public RNA-seq datasets, integrated with genomic, transcriptomic, and cistromic data. We detail the fiber co-expression dynamics and regulatory connections, validating findings with external datasets and transcription factor (TF) binding site data. We elucidate previously uncharacterized TFs that regulate genes involved in fiber-related functions and cellulose synthesis, and identify the regulatory role of two homoeologous G2-like TFs on fiber length. Analysis of duplicated gene expression and network relationships in allopolyploid cotton, which has two co-resident genomes (A, D), revealed novel aspects of asymmetric subgenomic developmental contributions. Whereas D-biased homoeolog pairs drive higher overall gene expression from the D subgenome, TFs from the A subgenome play a preferential regulatory role in the fiber GRN. Following allopolyploid formation, it appears that the trans-regulatory roles of TFs diversified more rapidly between homoeologs than did the cis-regulatory elements of their target genes. Our approach underscores the utility of network analysis for detecting master regulators and provides fresh perspectives on fiber development and polyploid functional genomics through the lens of co-expression and GRN dynamics.

Polyploidy or whole-genome duplication (WGD) is a significant evolutionary force. However, the mechanisms governing polyploid genome evolution remain unclear, limited largely by a lack of functional analysis tools in organisms that best exemplify the earliest stages of WGD. Tragopogon (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy. In this study, we significantly improved the transformation system and obtained genome-edited T. porrifolius (2x) and T. mirus (4x) primary generation (T0) individuals. Using CRISPR/Cas9, we knocked out the dihydroflavonol 4-reductase (DFR) gene, which controls anthocyanin synthesis, in both species. All transgenic allotetraploid T. mirus individuals had at least one mutant DFR allele, and 71.4% had all four DFR alleles edited. The resulting mutants lacked anthocyanin, and these mutations were inherited in the T1 generation. This study demonstrates a highly efficient CRISPR platform, producing genome-edited Tragopogon individuals that have completed the life cycle. The approaches used and challenges faced in building the CRISPR system in Tragopogon provide a framework for building similar systems in other non-genetic models. Genome editing in Tragopogon paves the way for novel functional biology studies of polyploid genome evolution and the consequences of WGD on complex traits, holding enormous potential for both basic and applied research.

The common bottlenose dolphin (Tursiops truncatus) is a key marine mammal species in the Gulf of Mexico, playing an essential role as a top predator. This study investigates the genetic diversity and population structure of bottlenose dolphins stranded in the Mississippi Sound from 2010 to 2021. Tissue samples (muscle, liver, lung, kidney, and brain) were collected from 511 stranded dolphins, and mitochondrial DNAs (mtDNA) were extracted for analysis. A total of 417 samples were successfully amplified and sequenced using high throughput sequencing, yielding 386 complete mitogenomes. Genetic diversity metrics, such as nucleotide and haplotype diversity, were calculated, and population structure was inferred for both mitochondrial control region (mtCR) and whole mitogenome sequences. Using the whole mitogenome, the study identified four genetically distinct populations within the Mississippi Sound, demonstrating regional variation in dolphin populations. Notably, two stranded individuals likely originated from populations outside the sampled area. The use of whole mitogenomes allowed for improved resolution of genetic diversity and population differentiation compared to previous studies using partial mtDNA sequences. These findings enhance our understanding of bottlenose dolphin population structure in the region and underscore the value of stranded animals for population genetic studies.

Cotton fiber development relies on complex and intricate biological processes to transform newly differentiated fiber initials into the mature, extravagantly elongated cellulosic cells that are the foundation of this economically important cash crop. Here we extend previous research into cotton fiber development by employing controlled conditions to minimize variability and utilizing time-series sampling and analyses to capture daily transcriptomic changes from early elongation through the early stages of secondary wall synthesis (6 to 24 days post anthesis; DPA). A majority of genes are expressed in fiber, largely partitioned into two major coexpression modules that represent genes whose expression generally increases or decreases during development. Differential gene expression reveals a massive transcriptomic shift between 16 and 17 DPA, corresponding to the onset of the transition phase that leads to secondary wall synthesis. Subtle gene expression changes are captured by the daily sampling, which are discussed in the context of fiber development. Coexpression and gene regulatory networks are constructed and associated with phenotypic aspects of fiber development, including turgor and cellulose production. Key genes are considered in the broader context of plant secondary wall synthesis, noting their known and putative roles in cotton fiber development. The analyses presented here highlight the importance of fine-scale temporal sampling on understanding developmental processes and offer insight into genes and regulatory networks that may be important in conferring the unique fiber phenotype.

The cotton textile industry is based on the material properties of terminally differentiated trichoblasts. Tens of thousands epidermal trichoblasts emerge from the seed coat and march through a reproducible developmental program that includes interconversion between cell-autonomous and tissue-based morphogenesis. Proteomic analyses of purified fibers provide a way to associate molecules with cellular processes that directly affect morphological transitions and material properties of the harvested cells. However, insufficient temporal sampling and poor protein coverage have limited the extent to which proteomic data predict control mechanisms. Here we quantified the subcellular proteomes of purified fibers daily over a 20-day interval that includes diverse modes of elongation and cell wall remodeling. Thousands of reliable protein abundance profiles were assigned to expression groups and tested for associations with a broad array of phenotypes. Integration of proteomic and RNA-sequencing data revealed distinct modes of transcriptional and post-transcriptional control. Collectively these datasets provide large-scale gene function predictions that can serve as a toolkit to engineer cotton material properties. ### Competing Interest Statement The authors have declared no competing interest.

Gossypium barbadense is renowned for its superior fiber quality, particularly its extra-long fibers, although its fiber yield is lower compared to G. hirsutum. Here, to further reveal fiber-related genomic variants of G. barbadense, we de novo assemble 12 genomes of G. barbadense that span the wild-to-domesticated continuum, and construct a graph-based pangenome by integrating these assemblies and 17 publicly available tetraploid cotton genome assemblies. We uncover the divergent evolutionary trajectories and subsequent exchanges between G. barbadense and G. hirsutum through investigation of structural variants (SVs). We perform the SV-based GWAS analysis in G. barbadense and identify four, three, and seven candidate SVs for fiber length, fiber strength, and lint percentage, respectively. Furthermore, we detect the underlying candidate genes and uncover the origin and distribution of favorable alleles, and reveal the tradeoff between lint percentage and fiber quality. These pangenome and trait-associated SVs provide insights into and resources for improving cotton fiber.

The economic value of cotton is based on long, thin, strong, and twisted trichoblast cells that emerge from the ovule epidermis. The mature dried cell reflects the final outcome of a complicated morphogenesis process that includes rapid tapering of the nascent trichoblast, weeks of polarized diffuse growth, followed by a transition to persistent secondary cell wall synthesis. Cellulose microfibril-based anistropic growth control is central to this process. It is widely assumed that the transition to secondary wall synthesis causes a reduced growth rate. However, the biomechanical details that directly link cell wall properties, with fiber morphogenesis is very poorly understood. In this paper, we developed novel imaging quantitative phenotyping, and computational modeling pipelines to analyze fiber growth at a daily resolution. We uncovered unexpected variability in growth rate, cell wall properties, and cell geometry across a broad developmental window. Finite element computational modeling of fiber growth was used to predict how spatial gradients of fiber and matrix material properties can interact to dictate the patterns of shape change. Cellulose patterning is central to the developmental process, and as an initial step toward gaining insight into the molecular control the expression profiles of a broad set of genes known to orchestrate cellulose biosynthesis were quantified across the same developmental timeline and correlated with fiber phenotypes. This analysis identified specific Gossypium hirsutum targets for fiber quality improvement. ### Competing Interest Statement The authors have declared no competing interest.

PREMISE:A complicating factor in analyzing allopolyploid genomes is the possibility of physical interactions between homoeologous chromosomes during meiosis, resulting in either crossover (homoeologous exchanges) or non-crossover products (homoeologous gene conversion). Homoeologous gene conversion was first described in cotton by comparing SNP patterns in sequences from two diploid progenitors with those from the allopolyploid subgenomes. These analyses, however, did not explicitly consider other evolutionary scenarios that may give rise to similar SNP patterns as homoeologous gene conversion, creating uncertainties about the reality of the inferred gene conversion events. METHODS:Here, we use an expanded phylogenetic sampling of high-quality genome assemblies from seven allopolyploid Gossypium species (all derived from the same polyploidy event), four diploid species (two closely related to each subgenome), and a diploid outgroup to derive a robust method for identifying potential genomic regions of gene conversion and homoeologous exchange. RESULTS:We found little evidence for homoeologous gene conversion in allopolyploid cottons, and that only two of the 40 best-supported events were shared by more than one species. We did, however, reveal a single, shared homoeologous exchange event at one end of chromosome 1, which occurred shortly after allopolyploidization but prior to divergence of the descendant species. CONCLUSIONS:Overall, our analyses demonstrated that homoeologous gene conversion and homoeologous exchanges are uncommon in Gossypium, affecting between zero and 24 genes per subgenome (0.0-0.065%) across the seven species. More generally, we highlighted the potential problems of using simple four-taxon tests to investigate patterns of homoeologous gene conversion in established allopolyploids.

Reniform and root-knot nematode are two of the most destructive pests of conventional upland cotton, Gossypium hirsutum L., and continue to be a major threat to cotton fiber production in semiarid regions of the Southern United States and Central America. Fortunately, naturally occurring tolerance to these nematodes has been identified in the Pima cotton species (Gossypium barbadense) and several upland cotton varieties (G. hirsutum), which has led to a robust breeding program that has successfully introgressed and stacked these independent resistant traits into several upland cotton lineages with superior agronomic traits, e.g. BAR 32-30 and BARBREN-713. This work identifies the genomic variations of these nematode-tolerant accessions by comparing their respective genomes to the susceptible, high-quality fiber-producing parental line of this lineage: Phytogen 355 (PSC355). We discover several large genomic differences within marker regions that harbor putative resistance genes as well as expression mechanisms shared by the two resistant lines, with respect to the susceptible PSC355 parental line. This work emphasizes the utility of whole-genome comparisons as a means of elucidating large and small nuclear differences by lineage and phenotype.

Polyploidy is a prominent mechanism of plant speciation and adaptation, yet the mechanistic understandings of duplicated gene regulation remain elusive. Chromatin structure dynamics are suggested to govern gene regulatory control. Here we characterized genome-wide nucleosome organization and chromatin accessibility in allotetraploid cotton, Gossypium hirsutum (AADD, 2n=4X=52), relative to its two diploid parents (AA or DD genome) and their synthetic diploid hybrid (AD), using DNS-seq. The larger A-genome exhibited wider average nucleosome spacing in diploids, and this inter-genomic difference diminished in the allopolyploid but not hybrid. Allopolyploidization also exhibited increased accessibility at promoters genome-wide and synchronized cis-regulatory motifs between subgenomes. A prominent cis-acting control was inferred for chromatin dynamics and demonstrated by transposable element removal from promoters. Linking accessibility to gene expression patterns, we found distinct regulatory effects for hybridization and later allopolyploid stages, including nuanced establishment of homoeolog expression bias and expression level dominance. Histone gene expression and nucleosome organization are coordinated through chromatin accessibility. Our study demonstrates the capability to track high resolution chromatin structure dynamics and reveals their role in the evolution of cis-regulatory landscapes and duplicate gene expression in polyploids, illuminating regulatory ties to subgenomic asymmetry and dominance.