In vivo CAR T-cell generation in non-human primates using lentiviral vectors displaying a multi-domain fusion ligand.

Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high cost and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVecTM platform, a lentiviral vector capable of generating CAR T-cells in vivo. Here we describe the incorporation of T cell activation and costimulatory signals onto the surface of VivoVecTM particles (VVPs) in the form of a multi-domain fusion protein and show enhanced in vivo transduction and improved CAR-T cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into non-human primates resulted in the robust generation of anti-CD20 CAR T-cells and the complete depletion of B cells for more than 10 weeks. These data validate the VivoVecTM platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.

Dense sampling of bird diversity increases power of comparative genomics.

Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity1-4. Sparse taxon sampling has previously been proposed to confound phylogenetic inference5, and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families-including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species.

Efficacy of Wolbachia-Infected Mosquito Deployments for the Control of Dengue.

BACKGROUND: Aedes aegypti mosquitoes infected with the wMel strain of Wolbachia pipientis are less susceptible than wild-type A. aegypti to dengue virus infection. METHODS: We conducted a cluster-randomized trial involving releases of wMel-infected A. aegypti mosquitoes for the control of dengue in Yogyakarta, Indonesia. We randomly assigned 12 geographic clusters to receive deployments of wMel-infected A. aegypti (intervention clusters) and 12 clusters to receive no deployments (control clusters). All clusters practiced local mosquito-control measures as usual. A test-negative design was used to assess the efficacy of the intervention. Patients with acute undifferentiated fever who presented to local primary care clinics and were 3 to 45 years of age were recruited. Laboratory testing was used to identify participants who had virologically confirmed dengue (VCD) and those who were test-negative controls. The primary end point was symptomatic VCD of any severity caused by any dengue virus serotype. RESULTS: After successful introgression of wMel into the intervention clusters, 8144 participants were enrolled; 3721 lived in intervention clusters, and 4423 lived in control clusters. In the intention-to-treat analysis, VCD occurred in 67 of 2905 participants (2.3%) in the intervention clusters and in 318 of 3401 (9.4%) in the control clusters (aggregate odds ratio for VCD, 0.23; 95% confidence interval [CI], 0.15 to 0.35; P = 0.004). The protective efficacy of the intervention was 77.1% (95% CI, 65.3 to 84.9) and was similar against the four dengue virus serotypes. The incidence of hospitalization for VCD was lower among participants who lived in intervention clusters (13 of 2905 participants [0.4%]) than among those who lived in control clusters (102 of 3401 [3.0%]) (protective efficacy, 86.2%; 95% CI, 66.2 to 94.3). CONCLUSIONS: Introgression of wMel into A. aegypti populations was effective in reducing the incidence of symptomatic dengue and resulted in fewer hospitalizations for dengue among the participants. (Funded by the Tahija Foundation and others; AWED ClinicalTrials.gov number, NCT03055585; Indonesia Registry number, INA-A7OB6TW.).

Expression of the wheat multipathogen resistance hexose transporter Lr67res is associated with anion fluxes.

Many disease resistance genes in wheat (Triticum aestivum L.) confer strong resistance to specific pathogen races or strains, and only a small number of genes confer multipathogen resistance. The Leaf rust resistance 67 (Lr67) gene fits into the latter category as it confers partial resistance to multiple biotrophic fungal pathogens in wheat and encodes a Sugar Transport Protein 13 (STP13) family hexose-proton symporter variant. Two mutations (G144R, V387L) in the resistant variant, Lr67res, differentiate it from the susceptible Lr67sus variant. The molecular function of the Lr67res protein is not understood, and this study aimed to broaden our knowledge on this topic. Biophysical analysis of the wheat Lr67sus and Lr67res protein variants was performed using Xenopus laevis oocytes as a heterologous expression system. Oocytes injected with Lr67sus displayed properties typically associated with proton-coupled sugar transport proteins-glucose-dependent inward currents, a Km of 110 ± 10 µM glucose, and a substrate selectivity permitting the transport of pentoses and hexoses. By contrast, Lr67res induced much larger sugar-independent inward currents in oocytes, implicating an alternative function. Since Lr67res is a mutated hexose-proton symporter, the possibility of protons underlying these currents was investigated but rejected. Instead, currents in Lr67res oocytes appeared to be dominated by anions. This conclusion was supported by electrophysiology and 36Cl- uptake studies and the similarities with oocytes expressing the known chloride channel from Torpedo marmorata, TmClC-0. This study provides insights into the function of an important disease resistance gene in wheat, which can be used to determine how this gene variant underpins disease resistance in planta.

Dissecting the causal polymorphism of the Lr67res multipathogen resistance gene.

Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by a variant of the Lr67 gene, which encodes a hexose-proton symporter. Two mutations (G144R, V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). Lr67res lacks sugar transport capability and was associated with anion transporter-like properties when expressed in Xenopus laevis oocytes. Here, we extended this functional characterization to include yeast and in planta studies. The Lr67res allele, but not Lr67sus, induced sensitivity to ions in yeast (including NaCl, LiCl, KI), which is consistent with our previous observations that Lr67res expression in oocytes induces novel ion fluxes. We demonstrate that another naturally occurring single amino acid variant in wheat, containing only the Lr67G144R mutation, confers rust resistance. Transgenic barley plants expressing the orthologous HvSTP13 gene carrying the G144R and V387L mutations were also more resistant to Puccinia hordei infection. NaCl treatment of pot-grown adult wheat plants with the Lr67res allele induced leaf tip necrosis and partial leaf rust resistance. An Lr67res-like function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the strong possibility of generating disease resistance in other crops, especially with gene editing.

A conditional mutation in a wheat (Triticum aestivum L.) gene regulating root morphology.

KEY MESSAGE: Characterisation and genetic mapping of a key gene defining root morphology in bread wheat. Root morphology is central to plants for the efficient uptake up of soil water and mineral nutrients. Here we describe a conditional mutant of hexaploid wheat (Triticum aestivum L.) that when grown in soil with high Ca2+ develops a larger rhizosheath accompanied with shorter roots than the wild type. In wheat, rhizosheath size is a reliable surrogate for root hair length and this was verified in the mutant which possessed longer root hairs than the wild type when grown in high Ca2+ soil. We named the mutant Stumpy and showed it to be due to a single semi-dominant mutation. The short root phenotype at high Ca2+ was due to reduced cellular elongation which might also explain the long root hair phenotype. Analysis of root cell walls showed that the polysaccharide composition of Stumpy roots is remodelled when grown at non-permissive (high) Ca2+ concentrations. The mutation mapped to chromosome 7B and sequencing of the 7B chromosomes in both wild type and Stumpy identified a candidate gene underlying the Stumpy mutation. As part of the process to determine whether the candidate gene was causative, we identified wheat lines in a Cadenza TILLING population with large rhizosheaths but accompanied with normal root length. This finding illustrates the potential of manipulating the gene to disconnect root length from root hair length as a means of developing wheat lines with improved efficiency of nutrient and water uptake. The Stumpy mutant will be valuable for understanding the mechanisms that regulate root morphology in wheat.

Reduced tillering and dwarfing genes alter root traits and rhizo-economics in wheat.

The tiller inhibition (tin) and Reduced height (Rht) genes strongly influence the carbon partitioning and architecture of wheat shoots, but their effects on the energy economy of roots have not been examined in detail. We examined multiple root traits in three sets of near-isogenic wheat lines (NILs) that differ in the tin gene or various dwarfing gene alleles (Rht-B1b, Rht-D1b, Rht-B1c and Rht-B1b + Rht-D1b) to determine their effects on root structure, anatomy and carbon allocation. The tin gene resulted in fewer tillers but more costly roots in an extreme tin phenotype with a Banks genetic background due to increases in root-to-shoot ratio, total root length, and whole root respiration. However, this effect depended on the genetic background as tin caused both smaller shoots and roots in a different genetic background. The semi-dwarf gene Rht-B1b caused few changes to the root structure, whereas Rht-D1b, Rht-B1c and the double dwarf (Rht-B1b + Rht-D1b) decreased the root biomass. Rht-B1c reduced the energy cost of roots by increasing specific root length, increasing the volume of cortical aerenchyma and by reducing root length, number, and biomass without affecting the root-to-shoot ratio. This work informs researchers using tin and Rht genes how to modify root system architecture to suit specific environments.

A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.

Potential for redistribution of post-moult habitat for Eudyptes penguins in the Southern Ocean under future climate conditions.

Anthropogenic climate change is resulting in spatial redistributions of many species. We assessed the potential effects of climate change on an abundant and widely distributed group of diving birds, Eudyptes penguins, which are the main avian consumers in the Southern Ocean in terms of biomass consumption. Despite their abundance, several of these species have undergone population declines over the past century, potentially due to changing oceanography and prey availability over the important winter months. We used light-based geolocation tracking data for 485 individuals deployed between 2006 and 2020 across 10 of the major breeding locations for five taxa of Eudyptes penguins. We used boosted regression tree modelling to quantify post-moult habitat preference for southern rockhopper (E. chrysocome), eastern rockhopper (E. filholi), northern rockhopper (E. moseleyi) and macaroni/royal (E. chrysolophus and E. schlegeli) penguins. We then modelled their redistribution under two climate change scenarios, representative concentration pathways RCP4.5 and RCP8.5 (for the end of the century, 2071-2100). As climate forcings differ regionally, we quantified redistribution in the Atlantic, Central Indian, East Indian, West Pacific and East Pacific regions. We found sea surface temperature and sea surface height to be the most important predictors of current habitat for these penguins; physical features that are changing rapidly in the Southern Ocean. Our results indicated that the less severe RCP4.5 would lead to less habitat loss than the more severe RCP8.5. The five taxa of penguin may experience a general poleward redistribution of their preferred habitat, but with contrasting effects in the (i) change in total area of preferred habitat under climate change (ii) according to geographic region and (iii) the species (macaroni/royal vs. rockhopper populations). Our results provide further understanding on the regional impacts and vulnerability of species to climate change.

Characterization of Extracellular Vesicle-Coupled miRNA Profiles in Seminal Plasma of Boars with Divergent Semen Quality Status.

Sperm heterogeneity creates challenges for successful artificial insemination. Seminal plasma (SP) surrounding sperm is an excellent source for detecting reliable non-invasive biomarkers of sperm quality. Here, we isolated microRNAs (miRNAs) from SP-derived extracellular vesicles (SP-EV) of boars with divergent sperm quality statuses. Raw semen from sexually mature boars was collected for eight weeks. Sperm motility and normal morphology were analyzed, and the sperm was classified as poor- or good-quality based on standard cutoffs of 70% for the parameters measured. SP-EVs were isolated by ultracentrifugation and confirmed by electron microscopy, dynamic light scattering, and Western immunoblotting. The SP-EVs were subjected to total exosome RNA isolation, miRNA sequencing, and bioinformatics analysis. The isolated SP-EVs were round spherical structures approximately 30-400 nm in diameter expressing specific molecular markers. miRNAs were detected in both poor- (n = 281) and good (n = 271)-quality sperm, with fifteen being differentially expressed. Only three (ssc-miR-205, ssc-miR-493-5p, and ssc-miR-378b-3p) allowed gene targeting associated with cellular localization (nuclear and cytosol) and molecular functions (acetylation, Ubl conjugation, and protein kinase binding), potentially impairing sperm quality. PTEN and YWHAZ emerged as essential proteins for protein kinase binding. We conclude that SP-EV-derived miRNAs reflect boar sperm quality to enable therapeutic strategies to improve fertility.

Physical Mapping of QTLs for Root Traits in a Population of Recombinant Inbred Lines of Hexaploid Wheat.

Root architecture is key in determining how effective plants are at intercepting and absorbing nutrients and water. Previously, the wheat (Triticum aestivum) cultivars Spica and Maringa were shown to have contrasting root morphologies. These cultivars were crossed to generate an F6:1 population of recombinant inbred lines (RILs) which was genotyped using a 90 K single nucleotide polymorphisms (SNP) chip. A total of 227 recombinant inbred lines (RILs) were grown in soil for 21 days in replicated trials under controlled conditions. At harvest, the plants were scored for seven root traits and two shoot traits. An average of 7.5 quantitative trait loci (QTL) were associated with each trait and, for each of these, physical locations of the flanking markers were identified using the Chinese Spring reference genome. We also compiled a list of genes from wheat and other monocotyledons that have previously been associated with root growth and morphology to determine their physical locations on the Chinese Spring reference genome. This allowed us to determine whether the QTL discovered in our study encompassed genes previously associated with root morphology in wheat or other monocotyledons. Furthermore, it allowed us to establish if the QTL were co-located with the QTL identified from previously published studies. The parental lines together with the genetic markers generated here will enable specific root traits to be introgressed into elite wheat lines. Moreover, the comprehensive list of genes associated with root development, and their physical locations, will be a useful resource for researchers investigating the genetics of root morphology in cereals.

Independent evolution of intermediate bill widths in a seabird clade.

Interspecific introgression can occur between species that evolve rapidly within an adaptive radiation. Pachyptila petrels differ in bill size and are characterised by incomplete reproductive isolation, leading to interspecific gene flow. Salvin's prion (Pachyptila salvini), whose bill width is intermediate between broad-billed (P. vittata) and Antarctic (P. desolata) prions, evolved through homoploid hybrid speciation. MacGillivray's prion (P. macgillivrayi), known from a single population on St Paul (Indian Ocean), has a bill width intermediate between salvini and vittata and could also be the product of interspecies introgression or hybrid speciation. Recently, another prion population phenotypically similar to macgillivrayi was discovered on Gough (Atlantic Ocean), where it breeds 3 months later than vittata. The similarity in bill width between the medium-billed birds on Gough and macgillivrayi suggest that they could be closely related. In this study, we used genetic and morphological data to infer the phylogenetic position and evolutionary history of P. macgillivrayi and the Gough medium-billed prion relative other Pachyptila taxa, to determine whether species with medium bill widths evolved through common ancestry or convergence. We found that Gough medium-billed prions belong to the same evolutionary lineage as macgillivrayi, representing a new population of MacGillivray's prion that originated through a colonisation event from St Paul. We show that macgillivrayi's medium bill width evolved through divergence (genetic drift) and independently from that of salvini, which evolved through hybridisation (gene flow). This represents the independent convergence towards a similarly medium-billed phenotype. The newly discovered MacGillivray's prion population on Gough is of utmost conservation relevance, as the relict macgillivrayi population in the Indian Ocean is very small.

Manipulating exudate composition from root apices shapes the microbiome throughout the root system.

Certain soil microorganisms can improve plant growth, and practices that encourage their proliferation around the roots can boost production and reduce reliance on agrochemicals. The beneficial effects of the microbial inoculants currently used in agriculture are inconsistent or short-lived because their persistence in soil and on roots is often poor. A complementary approach could use root exudates to recruit beneficial microbes directly from the soil and encourage inoculant proliferation. However, it is unclear whether the release of common organic metabolites can alter the root microbiome in a consistent manner and if so, how those changes vary throughout the whole root system. In this study, we altered the expression of transporters from the ALUMINUM-ACTIVATED MALATE TRANSPORTER and the MULTIDRUG AND TOXIC COMPOUND EXTRUSION families in rice (Oryza sativa L.) and wheat (Triticum aestivum L.) and tested how the subsequent release of their substrates (simple organic anions, including malate, citrate, and γ-amino butyric acid) from root apices affected the root microbiomes. We demonstrate that these exudate compounds, separately and in combination, significantly altered microbiome composition throughout the root system. However, the root type (seminal or nodal), position along the roots (apex or base), and soil type had a greater influence on microbiome structure than the exudates. These results reveal that the root microbiomes of important cereal species can be manipulated by altering the composition of root exudates, and support ongoing attempts to improve plant production by manipulating the root microbiome.

Selection for early shoot vigour in wheat increases root hair length but reduces epidermal cell size of roots and leaves.

Six cycles of recurrent selection for early shoot vigour in wheat resulted in significant increases in leaf width and shoot biomass. Here, in replicated controlled-environment studies, the effect of early shoot vigour on root biomass, rhizosheath size, root hair length, and cell size in the roots and leaves was examined across different cycles of selection. Increased shoot vigour was associated with greater root biomass, larger rhizosheath size, and longer root hairs. Our findings demonstrate that rhizosheath size was a reliable surrogate for root hair length in this germplasm. Examination of the root epidermis revealed that the 'cell body' of the trichoblasts (hair-forming cells) and the atrichoblasts (non-hair-forming cells) decreased in size as shoot vigour increased. Therefore, in higher vigour germplasm, longer root hairs emerged from smaller trichoblasts so that total trichoblast volume (root hair plus cell body) was generally similar regardless of shoot vigour. Similarly, the sizes of the four main cell types on the leaf epidermis became progressively smaller as shoot vigour increased, which also increased stomatal density. The relationship between shoot vigour and root traits is considered, and the potential contribution of below-ground root traits to performance and competitiveness of high vigour germplasm is discussed.

Follicular-fluid proteomics during equine follicle development.

The complex composition of the follicular fluid (FF), the intimate proximity to the oocyte, and the continual changes in their composition have a major effect on folliculogenesis and oogenesis. To date, the profiling of FF proteomes during follicle selection, development, and ovulation has not been comprehensively investigated. Therefore, a shotgun proteomics approach and bioinformatics analyses were used to profile the proteomes of equine FF harvested in vivo from follicles at the following development stages: predeviation (18-20 mm), deviation (22-25 mm), postdeviation (26-29 mm), preovulatory (30-35 mm), and impending ovulation. A total of 294 proteins were detected in FF (FDR <1%), corresponding to 65 common proteins and 124, 142, 167, 132, and 142 proteins in the predeviation, deviation, postdeviation, preovulatory, and impending ovulation groups, respectively. The higher expression of properdin and several other proteins belonging to the complement system during the deviation time and ovulation suggested their contribution in the selection of the future dominant follicle and ovulation. Apolipoprotein A-1 and antithrombin-III appeared to be important throughout folliculogenesis. The "complement and coagulation cascades" was the major KEGG pathway across all stages of follicle development. The significant expression of several proteins belonging to the serine-type endopeptidase indicated their likely contribution to follicle and oocyte development. Our data provide an extensive description and functional analyses of the equine FF proteome during follicle selection, development, and ovulation. This information will help improve understanding of the ovarian function and ovulatory dysfunctions and might serve as a reference for future biomarker discovery for oocyte quality assessment.

Rapid increase in Asian bottles in the South Atlantic Ocean indicates major debris inputs from ships.

Most plastic debris floating at sea is thought to come from land-based sources, but there is little direct evidence to support this assumption. Since 1984, stranded debris has been recorded along the west coast of Inaccessible Island, a remote, uninhabited island in the central South Atlantic Ocean that has a very high macrodebris load (∼5 kg·m-1). Plastic drink bottles show the fastest growth rate, increasing at 15% per year compared with 7% per year for other debris types. In 2018, we examined 2,580 plastic bottles and other containers (one-third of all debris items) that had accumulated on the coast, and a further 174 bottles that washed ashore during regular monitoring over the course of 72 d (equivalent to 800 bottles·km-1·y-1). The oldest container was a high-density polyethylene canister made in 1971, but most were polyethylene terephthalate drink bottles of recent manufacture. Of the bottles that washed up during our survey, 90% were date-stamped within 2 y of stranding. In the 1980s, two-thirds of bottles derived from South America, carried 3,000 km by the west wind drift. By 2009, Asia had surpassed South America as the major source of bottles, and by 2018, Asian bottles comprised 73% of accumulated and 83% of newly arrived bottles, with most made in China. The rapid growth in Asian debris, mainly from China, coupled with the recent manufacture of these items, indicates that ships are responsible for most of the bottles floating in the central South Atlantic Ocean, in contravention of International Convention for the Prevention of Pollution from Ships regulations.

Receding ice drove parallel expansions in Southern Ocean penguins.

Climate shifts are key drivers of ecosystem change. Despite the critical importance of Antarctica and the Southern Ocean for global climate, the extent of climate-driven ecological change in this region remains controversial. In particular, the biological effects of changing sea ice conditions are poorly understood. We hypothesize that rapid postglacial reductions in sea ice drove biological shifts across multiple widespread Southern Ocean species. We test for demographic shifts driven by climate events over recent millennia by analyzing population genomic datasets spanning 3 penguin genera (Eudyptes, Pygoscelis, and Aptenodytes). Demographic analyses for multiple species (macaroni/royal, eastern rockhopper, Adélie, gentoo, king, and emperor) currently inhabiting southern coastlines affected by heavy sea ice conditions during the Last Glacial Maximum (LGM) yielded genetic signatures of near-simultaneous population expansions associated with postglacial warming. Populations of the ice-adapted emperor penguin are inferred to have expanded slightly earlier than those of species requiring ice-free terrain. These concerted high-latitude expansion events contrast with relatively stable or declining demographic histories inferred for 4 penguin species (northern rockhopper, western rockhopper, Fiordland crested, and Snares crested) that apparently persisted throughout the LGM in ice-free habitats. Limited genetic structure detected in all ice-affected species across the vast Southern Ocean may reflect both rapid postglacial colonization of subantarctic and Antarctic shores, in addition to recent genetic exchange among populations. Together, these analyses highlight dramatic, ecosystem-wide responses to past Southern Ocean climate change and suggest potential for further shifts as warming continues.

Additive Traits Lead to Feeding Advantage and Reproductive Isolation, Promoting Homoploid Hybrid Speciation.

Speciation through homoploid hybridization (HHS) is considered extremely rare in animals. This is mainly because the establishment of reproductive isolation as a product of hybridization is uncommon. Additionally, many traits are underpinned by polygeny and/or incomplete dominance, where the hybrid phenotype is an additive blend of parental characteristics. Phenotypically intermediate hybrids are usually at a fitness disadvantage compared with parental species and tend to vanish through backcrossing with parental population(s). It is therefore unknown whether the additive nature of hybrid traits in itself could lead successfully to HHS. Using a multi-marker genetic data set and a meta-analysis of diet and morphology, we investigated a potential case of HHS in the prions (Pachyptila spp.), seabirds distinguished by their bills, prey choice, and timing of breeding. Using approximate Bayesian computation, we show that the medium-billed Salvin's prion (Pachyptila salvini) could be a hybrid between the narrow-billed Antarctic prion (Pachyptila desolata) and broad-billed prion (Pachyptila vittata). Remarkably, P. salvini's intermediate bill width has given it a feeding advantage with respect to the other Pachyptila species, allowing it to consume a broader range of prey, potentially increasing its fitness. Available metadata showed that P. salvini is also intermediate in breeding phenology and, with no overlap in breeding times, it is effectively reproductively isolated from either parental species through allochrony. These results provide evidence for a case of HHS in nature, and show for the first time that additivity of divergent parental traits alone can lead directly to increased hybrid fitness and reproductive isolation.

Mitogenomes Uncover Extinct Penguin Taxa and Reveal Island Formation as a Key Driver of Speciation.

The emergence of islands has been linked to spectacular radiations of diverse organisms. Although penguins spend much of their lives at sea, they rely on land for nesting, and a high proportion of extant species are endemic to geologically young islands. Islands may thus have been crucial to the evolutionary diversification of penguins. We test this hypothesis using a fossil-calibrated phylogeny of mitochondrial genomes (mitogenomes) from all extant and recently extinct penguin taxa. Our temporal analysis demonstrates that numerous recent island-endemic penguin taxa diverged following the formation of their islands during the Plio-Pleistocene, including the Galápagos (Galápagos Islands), northern rockhopper (Gough Island), erect-crested (Antipodes Islands), Snares crested (Snares) and royal (Macquarie Island) penguins. Our analysis also reveals two new recently extinct island-endemic penguin taxa from New Zealand's Chatham Islands: Eudyptes warhami sp. nov. and a dwarf subspecies of the yellow-eyed penguin, Megadyptes antipodes richdalei ssp. nov. Eudyptes warhami diverged from the Antipodes Islands erect-crested penguin between 1.1 and 2.5 Ma, shortly after the emergence of the Chatham Islands (∼3 Ma). This new finding of recently evolved taxa on this young archipelago provides further evidence that the radiation of penguins over the last 5 Ma has been linked to island emergence. Mitogenomic analyses of all penguin species, and the discovery of two new extinct penguin taxa, highlight the importance of island formation in the diversification of penguins, as well as the extent to which anthropogenic extinctions have affected island-endemic taxa across the Southern Hemisphere's isolated archipelagos.