Production of antibodies and antibody fragments containing non-natural amino acids in Escherichia coli.

Therapeutic bioconjugates are emerging as an essential tool to combat human disease. Site-specific conjugation technologies are widely recognized as the optimal approach for producing homogeneous drug products. Non-natural amino acid (nnAA) incorporation allows the introduction of bioconjugation handles at genetically defined locations. Escherichia coli (E. coli) is a facile host for therapeutic nnAA protein synthesis because it can stably replicate plasmids encoding genes for product and nnAA incorporation. Here, we demonstrate that by engineering E. coli to incorporate high levels of nnAAs, it is feasible to produce nnAA-containing antibody fragments and full-length immunoglobulin Gs (IgGs) in the cytoplasm of E. coli. Using high-density fermentation, it was possible to produce both of these types of molecules with site-specifically incorporated nnAAs at titers > 1 g/L. We anticipate this strategy will help simplify the production and manufacture of promising antibody therapeutics.

Mapping the planet's critical areas for biodiversity and nature's contributions to people.

Meeting global commitments to conservation, climate, and sustainable development requires consideration of synergies and tradeoffs among targets. We evaluate the spatial congruence of ecosystems providing globally high levels of nature's contributions to people, biodiversity, and areas with high development potential across several sectors. We find that conserving approximately half of global land area through protection or sustainable management could provide 90% of the current levels of ten of nature's contributions to people and meet minimum representation targets for 26,709 terrestrial vertebrate species. This finding supports recent commitments by national governments under the Global Biodiversity Framework to conserve at least 30% of global lands and waters, and proposals to conserve half of the Earth. More than one-third of areas required for conserving nature's contributions to people and species are also highly suitable for agriculture, renewable energy, oil and gas, mining, or urban expansion. This indicates potential conflicts among conservation, climate and development goals.

Pathologist-trained machine learning classifiers developed to quantitate celiac disease features differentiate endoscopic biopsies according to modified marsh score and dietary intervention response.

BACKGROUND: Histologic evaluation of the mucosal changes associated with celiac disease is important for establishing an accurate diagnosis and monitoring the impact of investigational therapies. While the Marsh-Oberhuber classification has been used to categorize the histologic findings into discrete stages (i.e., Type 0-3c), significant variability has been documented between observers using this ordinal scoring system. Therefore, we evaluated whether pathologist-trained machine learning classifiers can be developed to objectively quantitate the pathological changes of villus blunting, intraepithelial lymphocytosis, and crypt hyperplasia in small intestine endoscopic biopsies. METHODS: A convolutional neural network (CNN) was trained and combined with a secondary algorithm to quantitate intraepithelial lymphocytes (IEL) with 5 classes on CD3 immunohistochemistry whole slide images (WSI) and used to correlate feature outputs with ground truth modified Marsh scores in a total of 116 small intestine biopsies. RESULTS: Across all samples, median %CD3 counts (positive cells/enterocytes) from villous epithelium (VE) increased with higher Marsh scores (Type 0%CD3 VE = 13.4; Type 1-3%CD3 VE = 41.9, p < 0.0001). Indicators of villus blunting and crypt hyperplasia were also observed (Type 0-2 villous epithelium/lamina propria area ratio = 0.81; Type 3a-3c villous epithelium/lamina propria area ratio = 0.29, p < 0.0001), and Type 0-1 crypt/villous epithelial area ratio = 0.59; Type 2-3 crypt/villous epithelial area ratio = 1.64, p < 0.0001). Using these individual features, a combined feature machine learning score (MLS) was created to evaluate a set of 28 matched pre- and post-intervention biopsies captured before and after dietary gluten restriction. The disposition of the continuous MLS paired biopsy result aligned with the Marsh score in 96.4% (27/28) of the cohort. CONCLUSIONS: Machine learning classifiers can be developed to objectively quantify histologic features and capture additional data not achievable with manual scoring. Such approaches should be further investigated to improve biopsy evaluation, especially for clinical trials.

Priority areas to protect mangroves and maximise ecosystem services.

Anthropogenic activities threaten global biodiversity and ecosystem services. Yet, area-based conservation efforts typically target biodiversity protection whilst minimising conflict with economic activities, failing to consider ecosystem services. Here we identify priority areas that maximise both the protection of mangrove biodiversity and their ecosystem services. We reveal that despite 13.5% of the mangrove distribution being currently strictly protected, all mangrove species are not adequately represented and many areas that provide disproportionally large ecosystem services are missed. Optimising the placement of future conservation efforts to protect 30% of global mangroves potentially safeguards an additional 16.3 billion USD of coastal property value, 6.1 million people, 1173.1 Tg C, and 50.7 million fisher days yr-1. Our findings suggest that there is a pressing need for including ecosystem services in protected area design and that strategic prioritisation and coordination of mangrove conservation could provide substantial benefits to human wellbeing.

A metric-based framework for climate-smart conservation planning.

Climate change is already having profound effects on biodiversity, but climate change adaptation has yet to be fully incorporated into area-based management tools used to conserve biodiversity, such as protected areas. One main obstacle is the lack of consensus regarding how impacts of climate change can be included in spatial conservation plans. We propose a climate-smart framework that prioritizes the protection of climate refugia-areas of low climate exposure and high biodiversity retention-using climate metrics. We explore four aspects of climate-smart conservation planning: (1) climate model ensembles; (2) multiple emission scenarios; (3) climate metrics; and (4) approaches to identifying climate refugia. We illustrate this framework in the Western Pacific Ocean, but it is equally applicable to terrestrial systems. We found that all aspects of climate-smart conservation planning considered affected the configuration of spatial plans. The choice of climate metrics and approaches to identifying refugia have large effects in the resulting climate-smart spatial plans, whereas the choice of climate models and emission scenarios have smaller effects. As the configuration of spatial plans depended on climate metrics used, a spatial plan based on a single measure of climate change (e.g., warming) will not necessarily be robust against other measures of climate change (e.g., ocean acidification). We therefore recommend using climate metrics most relevant for the biodiversity and region considered based on a single or multiple climate drivers. To include the uncertainty associated with different climate futures, we recommend using multiple climate models (i.e., an ensemble) and emission scenarios. Finally, we show that the approaches we used to identify climate refugia feature trade-offs between: (1) the degree to which they are climate-smart, and (2) their efficiency in meeting conservation targets. Hence, the choice of approach will depend on the relative value that stakeholders place on climate adaptation. By using this framework, protected areas can be designed with improved longevity and thus safeguard biodiversity against current and future climate change. We hope that the proposed climate-smart framework helps transition conservation planning toward climate-smart approaches.

An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates.

The XpressCF+® cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50-100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+® reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.

Protected area planning to conserve biodiversity in an uncertain future.

Protected areas are a key instrument for conservation. Despite this, they are vulnerable to risks associated with weak governance, land-use intensification, and climate change. We used a novel hierarchical optimization approach to identify priority areas for expanding the global protected area system that explicitly accounted for such risks while maximizing protection of all known terrestrial vertebrate species. To incorporate risk categories, we built on the minimum set problem, where the objective is to reach species distribution protection targets while accounting for 1 constraint, such as land cost or area. We expanded this approach to include multiple objectives accounting for risk in the problem formulation by treating each risk layer as a separate objective in the problem formulation. Reducing exposure to these risks required expanding the area of the global protected area system by 1.6% while still meeting conservation targets. Incorporating risks from weak governance drove the greatest changes in spatial priorities for protection, and incorporating risks from climate change required the largest increase (2.52%) in global protected area. Conserving wide-ranging species required countries with relatively strong governance to protect more land when they bordered nations with comparatively weak governance. Our results underscore the need for cross-jurisdictional coordination and demonstrate how risk can be efficiently incorporated into conservation planning. Planeación de las áreas protegidas para conservar la biodiversidad en un futuro incierto.

Aunque las áreas protegidas son un instrumento clave para la conservación, no dejan de ser vulnerables a los riesgos asociados a una gestión pobre, la intensificación del uso de suelo y al cambio climático. Usamos una estrategia novedosa de optimización jerárquica para identificar las áreas prioritarias para la expansión del sistema global de áreas protegidas. La estrategia consideró de manera explícita los riesgos mencionados y también maximizó la protección de todas las especies conocidas de vertebrados terrestres. Para incorporar a las categorías de riesgo partimos del mínimo problema establecido, en donde el objetivo es lograr los objetivos de protección de la distribución de especies mientras se considera sólo una restricción, como el costo o área del suelo. Expandimos esta estrategia para que incluyera varios objetivos que consideraran el riesgo desde la formulación del problema mediante el manejo de cada nivel de riesgo como un objetivo aparte durante la formulación del problema. La reducción de la exposición a estos riesgos requirió que se expandiera el área total del sistema global de áreas protegidas en un 1.6% y así todavía cumplir con los objetivos de conservación. La incorporación de riesgos a partir de una gestión pobre fue el principal impulsor de cambios en las prioridades espaciales para la protección, mientras que la incorporación de riesgos a partir del cambio climático requirió el mayor incremento (2.52%) del área protegida a nivel mundial. La conservación de especies con distribución amplia requirió que los países con una gestión relativamente fuerte protegieran más suelo al tener fronteras con países con una gestión pobre en comparación son la suya. Nuestros resultados destacan la necesidad de una coordinación entre jurisdicciones y demuestran cómo puede incorporarse el riesgo de manera exitosa a la planeación de la conservación.

Discovery of STRO-002, a Novel Homogeneous ADC Targeting Folate Receptor Alpha, for the Treatment of Ovarian and Endometrial Cancers.

STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody-drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug-antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non-small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).

Molecular ecology meets systematic conservation planning.

Integrative and proactive conservation approaches are critical to the long-term persistence of biodiversity. Molecular data can provide important information on evolutionary processes necessary for conserving multiple levels of biodiversity (genes, populations, species, and ecosystems). However, molecular data are rarely used to guide spatial conservation decision-making. Here, we bridge the fields of molecular ecology (ME) and systematic conservation planning (SCP) (the 'why') to build a foundation for the inclusion of molecular data into spatial conservation planning tools (the 'how'), and provide a practical guide for implementing this integrative approach for both conservation planners and molecular ecologists. The proposed framework enhances interdisciplinary capacity, which is crucial to achieving the ambitious global conservation goals envisioned for the next decade.

Suffering, authenticity, and meaning in life: Toward an integrated conceptualization of well-being.

Most conceptions of well-being either ignore suffering or assume an ideal version of human life in which suffering would be eliminated. This trend is especially emblematic of positive psychology. Recent research on well-being indicates a mediating function of meaning in life between suffering and well-being demonstrating that making sense of past experiences is significantly correlated with high presence of meaning in life. Hence, meaning-making serves the role of an active coping mechanism that alleviates suffering. This and related strategies of defining, measuring, and augmenting well-being however overlook a form of suffering that is ineliminable and in fact essential to personal growth. In this paper the insights of the existentialist philosopher Søren Kierkegaard are developed to formulate an integrated conceptualization of well-being that regards "negative" affects as crucial for a rich and complete life. The complexity of the relationship between meaning in life, suffering, and authenticity concerning well-being are discussed. A synthetic perspective on the subjective dimension of the experience of suffering and on the objective nature of human limitations that often cause suffering is discussed in relation to the notions of meaning in life and authenticity. Finally, an integrated conceptualization of well-being is posited. It entails suffering as constitutive of meaning in life and authenticity, which are key components of a well-lived life.

Development of an E. coli strain for cell-free ADC manufacturing.

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody-drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.

Areas of global importance for conserving terrestrial biodiversity, carbon and water.

To meet the ambitious objectives of biodiversity and climate conventions, the international community requires clarity on how these objectives can be operationalized spatially and how multiple targets can be pursued concurrently. To support goal setting and the implementation of international strategies and action plans, spatial guidance is needed to identify which land areas have the potential to generate the greatest synergies between conserving biodiversity and nature's contributions to people. Here we present results from a joint optimization that minimizes the number of threatened species, maximizes carbon retention and water quality regulation, and ranks terrestrial conservation priorities globally. We found that selecting the top-ranked 30% and 50% of terrestrial land area would conserve respectively 60.7% and 85.3% of the estimated total carbon stock and 66% and 89.8% of all clean water, in addition to meeting conservation targets for 57.9% and 79% of all species considered. Our data and prioritization further suggest that adequately conserving all species considered (vertebrates and plants) would require giving conservation attention to ~70% of the terrestrial land surface. If priority was given to biodiversity only, managing 30% of optimally located land area for conservation may be sufficient to meet conservation targets for 81.3% of the terrestrial plant and vertebrate species considered. Our results provide a global assessment of where land could be optimally managed for conservation. We discuss how such a spatial prioritization framework can support the implementation of the biodiversity and climate conventions.

Evaluating surrogates of genetic diversity for conservation planning.

Protected-area systems should conserve intraspecific genetic diversity. Because genetic data require resources to obtain, several approaches have been proposed for generating plans for protected-area systems (prioritizations) when genetic data are not available. Yet such surrogate-based approaches remain poorly tested. We evaluated the effectiveness of potential surrogate-based approaches based on microsatellite genetic data collected across the Iberian Peninsula for 7 amphibian and 3 reptilian species. Long-term environmental suitability did not effectively represent sites containing high genetic diversity (allelic richness). Prioritizations based on long-term environmental suitability had similar performance to random prioritizations. Geographic distances and resistance distances based on contemporary environmental suitability were not always effective surrogates for identification of combinations of sites that contain individuals with different genetic compositions. Our results demonstrate that population genetic data based on commonly used neutral markers can inform prioritizations, and we could not find an adequate substitute. Conservation planners need to weigh the potential benefits of genetic data against their acquisition costs.

Evaluación de los Sustitutos de la Diversidad Genética para la Planeación de la Conservación Resumen Los sistemas de áreas protegidas deberían conservar la diversidad genética intraespecífica. Ya que para obtener datos genéticos se requieren recursos, se han propuesto distintas estrategias para generar los planes para los sistemas de áreas protegidas (priorizaciones) cuando los datos genéticos no están disponibles. A pesar de lo anterior, dichas estrategias basadas en sustitutos han sido poco evaluadas. Evaluamos la efectividad del potencial de las estrategias basadas en sustitutos cuya base son los datos genéticos de microsatélites obtenidos en toda la Península Ibérica y correspondientes a siete especies de anfibios y a tres de reptiles. La idoneidad ambiental a largo plazo no representó efectivamente los sitios que contienen una diversidad genética alta (riqueza de alelos). Las priorizaciones basadas en la idoneidad ambiental a largo plazo tuvieron un desempeño similar a las priorizaciones aleatorias. Las distancias geográficas y las distancias de resistencia basadas en la idoneidad ambiental contemporánea no siempre fueron sustitutos efectivos para la identificación de las combinaciones de sitios que contienen individuos con composiciones genéticas diferentes. Nuestros resultados demuestran que los datos genéticos de una población basados en marcadores neutrales de uso común pueden informar a las priorizaciones y que no pudimos encontrar un sustituto adecuado. Los planificadores de la conservación necesitan sopesar los beneficios potenciales de los datos genéticos contra sus costos de adquisición.

Comparative assessment of range-wide patterns of genetic diversity and structure with SNPs and microsatellites: A case study with Iberian amphibians.

Reduced representation genome sequencing has popularized the application of single nucleotide polymorphisms (SNPs) to address evolutionary and conservation questions in nonmodel organisms. Patterns of genetic structure and diversity based on SNPs often diverge from those obtained with microsatellites to different degrees, but few studies have explicitly compared their performance under similar sampling regimes in a shared analytical framework. We compared range-wide patterns of genetic structure and diversity in two amphibians endemic to the Iberian Peninsula: Hyla molleri and Pelobates cultripes, based on microsatellite (18 and 14 loci) and SNP (15,412 and 33,140 loci) datasets of comparable sample size and spatial extent. Model-based clustering analyses with STRUCTURE revealed minor differences in genetic structure between marker types, but inconsistent values of the optimal number of populations (K) inferred. SNPs yielded more repeatable and less admixed ancestries with increasing K compared to microsatellites. Genetic diversity was weakly correlated between marker types, with SNPs providing a better representation of southern refugia and of gradients of genetic diversity congruent with the demographic history of both species. Our results suggest that the larger number of loci in a SNP dataset can provide more reliable inferences of patterns of genetic structure and diversity than a typical microsatellite dataset, at least at the spatial and temporal scales investigated.

Spiritually Motivated Self-Forgiveness and Divine Forgiveness, and Subsequent Health and Well-Being Among Middle-Aged Female Nurses: An Outcome-Wide Longitudinal Approach.

Background: Interest in the relationship between forgiveness and health is steadily growing across disciplines within the research community. While there are multiple forms of forgiveness, past research has focused principally on studying forgiveness of others, whereas longitudinal evidence on the associations between other forms of forgiveness and health remains scarce. Methods: Using longitudinal data from the Nurses' Health Study II (from the 2008 Trauma Exposure and Post-traumatic Stress Supplementary Survey to 2015 questionnaire wave), this study employed an outcome-wide analytic approach to prospectively examine the association between two forms of religiously or spiritually motivated forgiveness, namely, self-forgiveness and divine forgiveness, and a wide array of subsequent psychosocial well-being, mental health, health behavior, and physical health outcomes among middle-aged female nurses (N = 54,703 for self-forgiveness; N = 51,661 for divine forgiveness). All models controlled for sociodemographic factors, prior religious service attendance, and prior values of all outcome variables wherever data were available. Bonferroni correction was used to account for multiple testing. Results: Self-forgiveness was strongly associated with greater psychosocial well-being (e.g., for top vs. bottom level of self-forgiveness, ß = 0.23, 95% CI: 0.20, 0.25 for positive affect) and lower psychological distress (e.g., ß = -0.21, 95% CI: -0.23, -0.18 for depressive symptoms). To a lesser extent, divine forgiveness was also associated with higher levels of psychological well-being and lower psychological distress. For both forgiveness types, there was little evidence of association with physical health or health behavior outcomes, though possible marginal evidence for an association of self-forgiveness with increased mortality. Discussion: This study provides novel evidence that religiously or spiritually motivated self-forgiveness and divine forgiveness are both positively related to several indicators of psychosocial well-being and inversely associated with psychological distress outcomes, whereas the associations with physical health and health behaviors are less clear. Further longitudinal investigation of the dynamics between these types of forgiveness and health and well-being is warranted.

Exact integer linear programming solvers outperform simulated annealing for solving conservation planning problems.

The resources available for conserving biodiversity are limited, and so protected areas need to be established in places that will achieve objectives for minimal cost. Two of the main algorithms for solving systematic conservation planning problems are Simulated Annealing (SA) and exact integer linear programing (EILP) solvers. Using a case study in BC, Canada, we compare the cost-effectiveness and processing times of SA used in Marxan versus EILP using both commercial and open-source algorithms. Plans for expanding protected area systems based on EILP algorithms were 12-30% cheaper than plans using SA, due to EILP's ability to find optimal solutions as opposed to approximations. The best EILP solver we examined was on average 1,071 times faster than the SA algorithm tested. The performance advantages of EILP solvers were also observed when we aimed for spatially compact solutions by including a boundary penalty. One practical advantage of using EILP over SA is that the analysis does not require calibration, saving even more time. Given the performance of EILP solvers, they can be used to generate conservation plans in real-time during stakeholder meetings and can facilitate rapid sensitivity analysis, and contribute to a more transparent, inclusive, and defensible decision-making process.

Global conservation of species' niches.

Environmental change is rapidly accelerating, and many species will need to adapt to survive1. Ensuring that protected areas cover populations across a broad range of environmental conditions could safeguard the processes that lead to such adaptations1-3. However, international conservation policies have largely neglected these considerations when setting targets for the expansion of protected areas4. Here we show that-of 19,937 vertebrate species globally5-8-the representation of environmental conditions across their habitats in protected areas (hereafter, niche representation) is inadequate for 4,836 (93.1%) amphibian, 8,653 (89.5%) bird and 4,608 (90.9%) terrestrial mammal species. Expanding existing protected areas to cover these gaps would encompass 33.8% of the total land surface-exceeding the current target of 17% that has been adopted by governments. Priority locations for expanding the system of protected areas to improve niche representation occur in global biodiversity hotspots9, including Colombia, Papua New Guinea, South Africa and southwest China, as well as across most of the major land masses of the Earth. Conversely, we also show that planning for the expansion of protected areas without explicitly considering environmental conditions would marginally reduce the land area required to 30.7%, but that this would lead to inadequate niche representation for 7,798 (39.1%) species. As the governments of the world prepare to renegotiate global conservation targets, policymakers have the opportunity to help to maintain the adaptive potential of species by considering niche representation within protected areas1,2.

Population genetics of Anopheles koliensis through Papua New Guinea: New cryptic species and landscape topography effects on genetic connectivity.

New Guinea is a topographically and biogeographically complex region that supports unique endemic fauna. Studies describing the population connectivity of species through this region are scarce. We present a population and landscape genetic study on the endemic malaria-transmitting mosquito, Anopheles koliensis (Owen). Using mitochondrial and nuclear sequence data, as well as microsatellites, we show the evidence of geographically discrete population structure within Papua New Guinea (PNG). We also confirm the existence of three rDNA ITS2 genotypes within this mosquito and assess reproductive isolation between individuals carrying different genotypes. Microsatellites reveal the clearest population structure and show four clear population units. Microsatellite markers also reveal probable reproductive isolation between sympatric populations in northern PNG with different ITS2 genotypes, suggesting that these populations may represent distinct cryptic species. Excluding individuals belonging to the newly identified putative cryptic species (ITS2 genotype 3), we modeled the genetic differences between A. koliensis populations through PNG as a function of terrain and find that dispersal is most likely along routes with low topographic relief. Overall, these results show that A. koliensis is made up of geographically and genetically discrete populations in Papua New Guinea with landscape topography being important in restricting dispersal.