Chromosome Research

BackgroundMaize knobs are regions of constitutive heterochromatin that are readily identified in both meiotic and somatic chromosomes. These structures have been characterized as stable throughout the cell cycle, exhibiting late replication during the S-phase, and are composed of two specific families of highly repetitive DNA sequences: K180 and TR-1. Although widely used as cytogenetic markers due to their variability in number and chromosomal position across inbred lines, hybrids, and landraces, little is known about their chromatin structure and dynamics. In this study, we analyzed chromatin accessibility of knobs using DNS-seq data across four maize tissues representing distinct developmental stages. ResultsOur results reveal that K180 knobs exhibit tissue-specific variation in chromatin accessibility, transitioning between open and closed states during development. In contrast, the TR-1 knob of chromosome 4 remained consistently inaccessible across all tissues analyzed. A knob composed of both K180, and TR-1 further supported this observation, with only the K180 region showing dynamic accessibility. To validate these findings, we also analyzed other repetitive regions such as centromeres, which showed a uniformly closed chromatin structure similar to TR-1. These results suggest a unique developmental modulation of chromatin accessibility associated with K180 repeats. While the chromatin accessibility of knobs does not reach the levels observed at Transcription Start Sites (TSS), the comparison among different classes of repetitive DNA within maize constitutive heterochromatin provides compelling evidence for sequence-specific and tissue-specific chromatin dynamics. ConclusionsOur findings uncover a previously unrecognized property of maize knobs and establish a reference for future studies on chromatin organization and epigenetic regulation of repetitive DNA in plant genomes.

Background/ObjectivesRNA polymerase II is a multifunctional complex that is critical for gene regulation and environmental responses. Its POLR2I subunit in human is associated with various pathologies, including cancer chemoresistance. However, much of our understanding of how POLR2I could function indirectly derives from studies of its homologs in yeasts called Rpb9. Here, we endogenously humanized the rpb9 gene of the fission yeast Schizosaccharomyces pombe to examine the functional capabilities of POLR2I. MethodsWe edited the genomic rpb9 locus in S. pombe so that it encodes the human POLR2I protein, and investigated functional and structural conservation. ResultsWith our humanized yeast system, we find widespread functional complementation by human POLR2I of S. pombe rpb9 roles in yeast growth, chronological aging, and stress responses. We also find that POLR2I complements novel roles for yeast rpb9 in facultative heterochromatin assembly, resistance against the chemotherapy 5-fluorouracil, and resistance against the fungicide thiabendazole. In contrast, we find that POLR2I cannot complement the role of rpb9 in resistance against the transcription elongation inhibitor 6-azauracil (6-AU) in our system. Interestingly, POLR2I could complement 6-AU resistance if ectopically expressed. Lastly, we observe extensive structural homology between Rpb9 and POLR2I proteins. ConclusionsOur study establishes an endogenous cross-species gene complementation strategy that uncovers both conserved and rewired functions of fission yeast rpb9 and its human homolog, POLR2I. In addition to validating conserved roles, we also identified conservation of previously unrecognized roles of rpb9 in heterochromatin formation and chemoresistance.

Genomic imprinting is a rare epigenetic phenomenon in plants and animals, defined by parent-of-origin specific gene expression. Its molecular mechanisms and evolutionary significance remain incompletely understood. In this study, we investigated whether genomic imprinting occurs in Scots pine and, by extension, in other conifers to gain insight into the evolutionary origins of imprinting. We performed reciprocal crosses to assess imprinting in seed embryos and applied a unique approach that used exome-capture data from the haploid, maternally inherited megagametophyte tissue to identify maternal alleles, thereby allowing us to infer paternal alleles in the embryos of the same seeds. Our findings show that maternally inherited haploid megagametophyte tissue offers an effective strategy for resolving parental alleles in offspring while simultaneously removing extensive paralogous variation from the dataset. This framework is broadly applicable to other conifer species and to taxa that possess comparable maternally derived haploid tissues. No evidence of genomic imprinting was detected. Although the limited overlap between the exome-capture and RNA-sequencing datasets and the stringent paralog filtering reduced the amount of analyzable data considerably, the absence of detectable imprinting may also reflect genuinely weak or absent imprinting signals in conifers. We identified several limitations in this preliminary study and outline recommendations for future work to overcome them, and additional research will be necessary to determine whether genomic imprinting occurs in conifers

All cells of a multicellular organism usually share an identical genome, faithfully transmitted through successive divisions. Yet, a number of animal species deviate from this dogma, as parts of their DNA are systematically eliminated in all their somatic nuclei, in a process called Programmed DNA Elimination (PDE). PDE leads to the unexpected reorganisation of the genome at every generation in all somatic cells but its molecular mechanism, evolutionary origins, and functional significance remain unknown. This lack of understanding partially stems from limitations in genetically tractable model species. PDE can target an entire chromosome, or involve chromosome fragmentation followed by selective fragment retention and elimination, raising further questions on genome stability, genome integrity and mechanisms of DNA repair. PDE by chromosome fragmentation has been described in parasitic nematodes in the family Ascarididae, copepods in the genus Cyclops and unicellular ciliates. More recently, PDE has been discovered in three non-parasitic, lab-tractable nematode species from the Rhabditidae family, opening new perspectives. In this study, we used cytological approaches to screen 25 new Rhabditidae species for PDE. We found evidence of PDE in 17 species. Our work reveals that PDE is present in 12 out of 17 tested genera, demonstrating its widespread presence in Rhabditidae nematodes, with the notable exception of C. elegans. Genetic tools have already been established for some species. This work provides a collection of lab-tractable species that can be used to test many aspects of somatic Programmed DNA Elimination by chromosome fragmentation in animals.

The cohesin complex has conserved roles in sister chromatid cohesion, DNA replication, genome organization, and the DNA damage response. We heterologously expressed the human cohesin complex in yeast to probe the behaviour of human cohesin. Human cohesin was unable to complement loss of function mutations in yeast cohesin, either as single subunits or as complexes, including in the context of co-expressing up to 12 human cohesin-associated genes. Heterologous expression of human cohesin in yeast expressing wildtype yeast cohesin resulted in dominant cohesion dysregulation and DNA damage sensitivity phenotypes. We used co-immunoprecipitation to demonstrate that human SMC proteins interact with endogenous yeast cohesin rings creating dominant-negative hybrid complexes that disrupt endogenous cohesin biology.

BackgroundAlthough strict maternal transmission of mitochondria is a general feature of animals and humans for ensuring homogeneity in mitochondrial DNA (mtDNA) across generations, exceptions were reported in the recent past. For example, some extremely rare but spectacular cases of heteroplasmy and paternal transmission in humans have questioned the universal evolutionary principle. Hence, as an alternative, the Mega-NUMT concept was coined to explain this discovery and was thereafter partly proven to exist. This concept expands on the quite common transfer of mtDNA fragments to the nucleus (NUMTs) by considering the existence of multicopy mitochondrial nuclear insertions. Mega-NUMT reports are currently restricted to a few cases in animals, including humans. However, even in humans, their detailed genomic organization, natural prevalence, and potential biological functions remain unclear. Methodology/Principal FindingsHere, we discovered that up to 60 full-sized mitochondrial genomes are integrated into the nuclear genome of the neotropical fruit fly Drosophila paulistorum using long-read sequencing and confirmed their presence by in situ hybridization. The copies are organized in one cluster on chromosome 3, which we, due to its similarity with the Mega-NUMT concept, designated the "Dpau Mega-NUMT". Contrary to the rarity in humans, this Mega-NUMT is found at high prevalence (40%) in both long-term laboratory lines and natural D. paulistorum populations of different semispecies. Additionally, the mitochondrial copies in the Mega-NUMT cluster are phylogenetically separated from the current mitotypes of D. paulistorum. Together, these observations suggest long-term maintenance of the Mega-NUMT in nature. Hence, we propose that the Dpau Mega-NUMT may have been transferred to the nuclear genome before D. paulistorum semispecies radiation and maintained at relatively high prevalence in nature by balancing selection due to yet undetermined functions. Conclusions/SignificanceTo our knowledge, this is the first verified existence and detailed dissection of a Mega-NUMT outside cats and humans. We show that Mega-NUMTs can be persistent in nature, even at high prevalence, potentially due to balancing selection. Our findings strengthen the importance of high-quality long-read sequencing technologies for deciphering complex repeat-rich genomic regions to deepen our understanding of the dynamics of genome evolution within genomic "dark matter".

The actin polymerization machinery, comprising the ARP2/3 complex and its activators, the WASP family proteins, has been implicated in regulating a broad spectrum of nuclear processes, such as transcriptional regulation and nuclear organization. Here, using clustered protocadherin (cPcdh) and {beta}-globin genes as model systems, we showed that WAVE2, a member of the WASP family, regulates chromatin organization by maintaining heterochromatin dynamics. Specifically, by CRISPR DNA-fragment editing, in conjunction with integrated analyses of ChIP-seq, MeDIP-seq, ATAC-seq, 4C-seq, and RNA-seq, we showed that deposition of H3K9me3, a key heterochromatin mark, is significantly decreased at the cPcdh locus upon WAVE2 deletion, concurrent with aberrant accumulation of CTCF/cohesin complex at promoter regions and spatial reorganization of chromatin architecture around nucleolus. In addition, REST/NRSF exerts a similar heterochromatindependent effect on the cPcdh locus. Finally, genetic and genomic data showed that WAVE2 regulates {beta}-globin gene expression by maintaining heterochromatin status. Together our data suggested that WAVE2 and REST/NRSF regulate clustered gene expression in a heterochromatin-dependent manner.

Enhancer RNAs (eRNAs) are non-coding transcripts produced at enhancer regions, which appear to be involved in transcriptional regulation. Up to date, these have been primarily investigated using labor-and cost-intensive genomic techniques. However, the precise mechanisms by which eRNA transcription or the eRNA transcripts themselves mediate transcriptional regulation remain unclear. Here, we present a novel experimental approach that allows us to analyze the characteristics of eRNA transcription in fixed and live whole Drosophila melanogaster embryos. We employ the anterior-posterior patterning genes as a model system to investigate the dynamics of eRNA expression, utilizing an imaging-based approach. We combined high-sensitivity fluorescence in situ hybridization (FISH) chain reaction (HCR) with high-resolution confocal microscopy to detect eRNA and mRNA molecules. Through this experimental assay, we identified foci of elevated transcriptional activity that generate eRNA transcripts correlated with mRNA production at the same gene locus. We could show that this eRNA transcription is independent of promoter activity. Additionally, we demonstrate that insulators can influence eRNA transcription, resulting in loss of eRNA transcription. Moreover, we observe that eRNAs can originate both within classical enhancer regions and outside of them, including from foreign bacterial sequences when these are placed near enhancer sequences, underscoring the strong influence of local regulatory context on eRNA initiation. In live embryos using MS2-MCP live imaging, our analysis of insulators showed a modest reduction in mRNA burst intensity accompanied by a slight increase in burst frequency. Overall, our imaging-based approach offers a novel platform for dissecting enhancer-eRNA interactions and could be adapted for wider applications.

The cockroach Blattella germanica possesses panoistic ovaries, in which oocytes lack nurse cells and therefore need to rely on their own transcriptional activity to support embryogenesis. Ovarian development in this species involves the development of a single basal ovarian follicle (BOF) per gonadotropic cycle, a process strictly regulated by endocrine signals, primarily juvenile hormone and ecdysone, which act at both the transcriptional and translational levels. In addition, transcriptional activity in these ovaries is necessary for both regulating and genome protection, and at this level, PIWI-interacting RNAs (piRNAs) play an essential role. Although insect ovaries are known to be particularly rich in piRNAs, their function in ovary maturation is still not well defined. For this purpose, we characterize the piRNA expression dynamics across seven key developmental and reproductive stages, ranging from late nymphal instars to post-vitellogenic adults. piRNA expression in B. germanica shows coordinated fluctuations. Expression remains stable in previtellogenic ovaries, whereas vitellogenic ovaries show pronounced changes. Moreover, vitellogenic ovaries exhibit reduced piRNA diversity due to strong enrichment of a subset of highly expressed piRNAs. Our data show that although piRNAs predominantly map to transposable elements, particularly LINEs, there is a notable increase in gene-derived piRNAs toward the end of the cycle. Our results suggest regulatory roles of piRNAs in modulating both TEs and mRNAs during BOF maturation, likely related to changes in the follicular cell program.

In their recent study entitled "Ancient DNA reveals the co-existence of domestic horses, donkeys and their hybrids in the prehistorical northwestern China", Li and colleagues (2026) report the genetic identification of three horses, three donkeys and four first-generation hinny hybrids dating to 400-160 BCE from the Mazongshan jade mining site in northwestern China. While a re-analysis of their ancient DNA sequence data confirms the horse and donkey identifications, it indicates that the four putative hinny specimens were, in fact, donkeys. This revision removes the primary evidence originally shown for the presence of hinnies at this site. Therefore, new data from the Mazongshan bone assemblage are required to support the proposed role of hinny hybrids as integral components of trans-regional trade networks during the Late Warring States and Early Han periods.

BackgroundGene expression changes in response to developmental and environmental cues rely on cis-regulatory sequence elements (cREs). BRG1/BRM-Associated Factors (BAF) chromatin remodeling complexes maintain chromatin accessibility at many cREs, enabling binding by transcription factors (TFs). However, cREs exhibit a broad range of sensitivity to loss of BAF function, and the basis of this variability remains unknown. ResultsTo identify the characteristics of BAF-dependent cREs, we mapped chromatin accessibility changes following acute pharmacologic BAF inhibition in GM12878 lymphoblastoid cells. We integrated these results with over 100 TF and histone modification ChIP-seq datasets and used machine learning to identify features that predict chromatin accessibility changes. We found that Activator Protein 1 (AP-1) factors and lymphoid lineage-defining TFs including RUNX3 and PU.1 predicted BAF-dependence. Strikingly, we found that cREs bearing the chromatin signature of "primed" enhancers - enriched for H3K4me1 but lacking H3K27ac - were significantly more sensitive to BAF inhibition than typical active enhancers. As primed enhancers are known to facilitate transcriptional responses to stimuli, we tested the requirement of BAF activity in these responses. Acute BAF inhibition was sufficient to prevent both chromatin and transcriptional responses to interferon gamma and dexamethasone. cREs which normally gained accessibility in response to stimulation failed to do so with BAF inhibition, and these cREs were linked to genes with suppressed transcriptional induction. ConclusionsCollectively, our results demonstrate a requirement for continuous BAF activity to enable stimulus response and suggest that defective signal responsiveness may be a pathogenic mechanism in disease states caused by loss-of-function mutations in BAF subunits.

BackgroundConventional models of human ribosomal DNA (rDNA) array organization have historically depended on transcription-centric boundaries, partitioning the unit into a [~]13 kb rDNA transcription region and a monolithic [~]31 kb intergenic spacer (IGS). While our previous identification of Duplication Segment Units (DSUs) mapped these arrays based on an intuitive analysis of the microsatellite density landscape of the complete reference human genome, our present deep mining of this landscape has revealed a more accurate rDNA Gene Unit Pattern. Methods & ResultsIn this study, we conducted a deep mining analysis of our previously established microsatellite density landscape of the T2T-CHM13 assembly, focusing specifically on nucleolar organizing regions (NORs). We suggest a more accurate rDNA Gene Unit Pattern containing a (CTTT)n microsatellite aggregation ahead of the rDNA gene and a (CT)n microsatellite aggregation behind the gene, rather than a pattern featuring an IGS region inserted between two rDNA genes. ConclusionsA correct rDNA gene pattern of the human genome probably includes a (CTTT)n microsatellite aggregation ahead of the gene and a (CT)n microsatellite aggregation behind it, which possibly constitute cis- and trans-regulating regions; the (CTTT)n and (CT)n microsatellite aggregations may provide two different local stable DNA structures for regulatory protein binding.

DNA repair in biochemical and genetic experimental systems permits a precise definition of enzyme requirements and mechanistic steps. Comparing these findings to repair events at naturally occurring damage sites in multicellular organisms is essential for confirming and expanding these insights into a physiologic context. However, heterogeneity in any normal cell population increases with each cell division, and the reliable detection of replication-independent DNA damage sites and their repair has been a major barrier. Here, we examine single human colon crypts, which harbor natural cell clones, using a novel whole-genome sequencing (WGS) method to identify complex insertion-deletion (indel) in the crypt stem cells. Analysis of complex indel events likely repaired by non-homologous end joining occurring in crypt stem cells permits inferences about the in vivo repair of naturally occurring DNA damage within physiologically-relevant chromatin in normal human cells.

Stem cell technologies have become a vital component of conservation efforts around the globe. Biobanks and pluripotent stem cell lines help to ensure species and their genetic diversity are preserved. These efforts have however, focussed mostly on mammals and birds, and the cryopreservation protocols for embryos and cells were developed decades ago laying the basis for artificial reproductive techniques for species conservation. With over 20% of non-avian reptile species facing extinction, it is imperative to establish protocols for reptiles to ensure species preservation and also to facilitate the establishment of new reptile model organisms to match the standard of mammals. Here, we have generated a cryopreservation method for preserving early gastrulating veiled chameleon embryos as a representative squamate species. To this end, we first developed a tissue culture method for maintaining cells extracted from peri-gastrulation chameleon embryos and then tested different cryopreservation methods altering the concentration of the penetrating cryoprotectant DMSO and assessing the effect of the addition of non-penetrating cryoprotectants Trehalose and Sucrose. We then optimised a protocol for whole embryo vitrification in 20% DMSO with added Trehalose or Sucrose that can easily be adapted for fieldwork. Taken together, our method not only provides a protocol for conservation efforts but also lays the basis for mechanistic studies of early squamate embryo development by enabling cryopreservation of whole embryos in a fieldwork setting, which facilitates their live transport back to a laboratory for functional experiments or molecular analyses.

Text AbstractIn this study, we characterized the genetic structure and reconstructed the demographic history of cactus wrens (Campylorhynchus brunneicapillus), an endemic species of desert regions of North America, that shows a clear phenotypic and genotypic variation. We evaluated the effects of historical climate change on the structure and population dynamics of desert species using genomic data through genotyping by sequencing (GBS) and applied a population structure analysis (FST and ADMIXTURE), revealing two genetically differentiated groups: one continental and another peninsular in Baja California. Subsequently, we implemented the MSMC2 coalescent model on data divided into autosomal regions and the Z sex chromosome to estimate changes in effective population size (Ne) through evolutionary time. Additionally, we developed ecological niche models (ENMs) projected to the Last Glacial Maximum (LGM), Last Interglacial (LIG), Present times, and Future (2060 - 2080). Results indicate that both populations maintained moderated Nes before the LGM, experienced severe bottlenecks (Ne [~] 102-103), followed by a sustained expansion. However, recovery was limited to the Z chromosome of the peninsular population. These findings reveal how glaciations and interglacials shaped the evolutionary history of desert species and provide genomic evidence of the splitting of C. affinis from C. brunneicapillus. Article summaryThis research examines how climate changes shaped genetic diversity of cactus wrens across North American warm deserts. Using coalescent methods, researchers tracked effective population size changes over 100,000 years, using ecological niche modeling they predicted habitat suitability across climate periods. Results showed that continental and peninsular populations experienced bottlenecks during the Last Glacial Maximum, followed by demographic recovery on warm periods. However, the sex chromosome (Z) revealed male-biased demographic patterns in peninsular populations. Future projections indicated habitat suitability reductions for peninsular populations, highlighting conservation concerns. These findings demonstrate that past climate shaped genetic diversity of cactus wrens.

The spatiotemporal organisation of chromatin in the eukaryotic nucleus is fundamental for genome regulation. Chromatin undergoes rapid remodelling and rearrangements within minutes, altering its diffusion properties. Considering the tight coupling between genome function and nuclear architecture, a key question is how chromatin dynamics adapt to or promote nuclear processes. To elucidate the underlying physical principles, we employed High-resolution Diffusion mapping (Hi-D) to track chromatin motion throughout interphase in live human cells. Our analysis, that considers both diffusive motion and drift generated by active forces, revealed that chromatin dynamics are heterogeneous, with distinct behaviours in different subnuclear zones. Notably, both diffusive and processive contributions to chromatin motion progressively decrease from G1 to G2 phase, with this reduction occurring uniformly across all subzones. This suggests a global mechanism driving the observed decrease in chromatin mobility during cell cycle progression. By combining genetic knockout experiments and polymer modelling, we demonstrate that the doubling of DNA content, rather than cohesin-mediated sister chromatid entrapment, is responsible for the gradual decrease in chromatin motion during the cell cycle in human nuclei. These findings provide new insights into the physical and functional organisation of chromatin and its regulation during cellular proliferation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712877v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@75c654org.highwire.dtl.DTLVardef@2fbd3dorg.highwire.dtl.DTLVardef@31025aorg.highwire.dtl.DTLVardef@191808e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Methods to visualize and quantify the molecular responses of cells to local forces exerted at adhesions are crucial to elucidate how physical forces control cellular behavior. Of the many proteins involved in focal adhesions, vinculin plays a key role in mediating force-sensitive processes. Here, we combined optical tweezers and Forster resonance energy transfer (FRET) microscopy to measure the intensity and FRET efficiency of the vinculin tension sensor, VinTS, in response to a force. Fibroblasts expressing VinTS formed adhesions on fibronectin-coated, 3m-diameter, polystyrene beads. As the beads were displaced by the cell, we applied an optical trap to counteract this movement and increase the traction force required by the cell to maintain the bead displacement. The optical trap stiffness varied from zero (no laser) up to 0.26 pN/nm. In this range, the median bead displacement after 5 min was ~200nm in all trapping conditions inducing counteracting forces in the 10-100pN range. To maintain this displacement, vinculin recruitment increased (up to 35% in relative intensity at high stiffness) while tension increased but more moderately (1-2% decrease in absolute FRET efficiency). For higher trap stiffness, the main response was an increase in vinculin recruitment, while the tension did not increase significantly. The increase in vinculin intensity was correlated with the decrease in FRET efficiency at 0.26 pN/nm but not at lower stiffness. Thus, the presence of the high stiffness optical trap over 5 min appears to induce a positive correlation between vinculin recruitment and vinculin tension. In a few instances, vinculin puncta migrated a few microns away from the bead exceeding the bead movement speed while experiencing an increase in both vinculin intensity and tension. Taken together, the results suggest that combining an optical trap with vinculin tension measurements uncovers novel vinculin dynamics in the presence of a force.

Concerns over climate change have intensified the demand for stress resistant crops like hybrid wheatgrass (HWG; Elymus hoffmannii, StStStStHH), a perennial forage species known for its exceptional salt and drought tolerance. However, hexaploidy and high heterozygosity have complicated efforts to resolve its genomic structure and evolutionary history. Here, we present high-quality, haplotype-resolved, chromosome-level genome assemblies for HWG (CDC Saltking) and its putative progenitor, bluebunch wheatgrass (Pseudoroegneria spicata). By integrating PacBio HiFi and ultra-long Oxford Nanopore sequencing with Hi-C scaffolding, we assembled the 10.7 Gb HWG genome into 21 pseudochromosomes per haplotype. Our phylogenomic analysis redefines the origin of the H subgenome, positioning it as an intermediate between Old-World Hordeum marinum (sea barley) and Hordeum brevisubulatum. Notably, we identified significant chromosomal rearrangements, including a unique duplication on St chromosome 4. Transcriptome analysis across multiple tissues revealed a pronounced expression dominance of the H subgenome. This dominance was not associated with reduced LTR density, suggesting that selective pressures for rapid adaptation of the latest subgenome entrant may drive its dominance. Finally, using the f-branch statistic, population genomic analysis of 189 accessions representing eight Elymus and Pseudoroegneria species revealed extensive reticulate evolutionary relationships and identified P. spicata as a major, asymmetric genetic donor within the wheatgrass complex. These resources provide a foundational framework for future genomic research and genetic improvement in grasses and for the introgression of stress-tolerance traits into cereal crops such as wheat. Key MessagesDevelopment of world-first high-quality chromosomal-level haplotype-resolved genome assemblies of hexaploid HWG and diploid progenitor, Pseudoroegneria spicata, enabled the identification of the subgenome origins. This study resolved the evolutionary placement of the St genome and clarified the history of polyploidization and hybridization in HWG. Homeolog expression bias in the H subgenome likely reflects selective pressure favoring greater gene retention and upregulation of functionally important genes, thereby enhancing hybrid fitness. Population structure analysis distinctly differentiates P. spicata, E. repens, E. hoffmannii from other European Pseudoroegneria species. The findings reveal the complex patterns of interspecific gene flow and population dynamics within the Elymus and Pseudoroegneria species.

The evolution of visual systems has compelled numerous investigations of developmental processes underlying eye patterning across Bilateria. It is well-established that homologs of the transcription factor Pax6 play a highly conserved role in eye fate specification and are at the top of the retinal determination gene network (RDGN) hierarchy. In insects, the two Pax6 homologs eyeless (ey) and twin of eyeless (toy) are required for the development of the two visual systems broadly found within the phylum (i.e., median and lateral eyes). Curiously, Pax6 homologs do not appear to maintain this function in well-studied chelicerate models, with emphasis on spiders, a lineage of arachnids with great diversity of eye form and acuity. It was recently proposed that the gene Pax2 (shaven; sv) may have subsumed the role of eye fate specification in chelicerates, a hypothesis predicated upon the observation that one of two spider Pax2 copies is strongly expressed in the developing lateral eyes during embryogenesis. However, no functional data are available for any Pax homologs across Chelicerata. We examined the incidence of Pax family genes across Chelicerata, as well as interrogated the expression and function of Pax2 and Pax6 homologs in the daddy-longlegs Phalangium opilio, an arachnid recently discovered to bear a highly plesiomorphic arrangement of visual systems. Here, we show that ey and toy are expressed early in the developing head lobes of P. opilio, whereas sv is not expressed until well after stages when downstream RDGN members (eyes absent and sine oculis) are already activated. Gene silencing of ey, toy, and sv individually had no discernible effect on eye development. By contrast, double knockdown of ey and toy resulted in an array of median eye defects, spanning loss of some cells of the eye to total loss of the median eyes. Gene expression assays also showed that depletion of the two Pax6 copies resulted in failure of the vestigial median and vestigial lateral eyes. These data are consistent with a conserved role for Pax6 homologs in patterning both visual systems and all three eye pairs in the daddy-longlegs. Our results comprise the first functional data for Pax6 genes in any chelicerate and suggest that heterochronic shifts in expression, rather than changes in function, underlie the atypical dynamics of Pax genes in derived arachnid groups such as spiders.

The geographical and familial origins of Christopher Columbus have remained a subject of intense historiographical debate for over five centuries. Despite numerous hypotheses, empirical genetic evidence capable of resolving his ancestral history or place of birth has been absent from the literature until now. This study presents the third stage of the first forensic genetic analysis performed on skeletal remains belonging to several direct descendants of Columbus, spanning the 16th to 18th centuries. By applying Massively Parallel Sequencing (MPS) to analyse autosomal, X- and Y- chromosome DNA markers, and integrating the results with multidisciplinary evidence from historical, genealogical, archaeological, and anthropological research implicated in this project, the identification of several individuals founded in the Crypt of Santa Maria de Gracia located in Gelves (Sevilla, Spain) has been achieved. The analysis of their biological relatedness enabled the reconstruction of kinship networks among the individuals interred in the crypt, which, when interpreted in the context of documented genealogical lineages, provides indirect but consistent evidence pointing toward the debated origin of the discoverer.