Derivative-based time-adjusted analysis of diurnal and within-tree variation in the OJIP fluorescence transient of silver birch.

The JIP test, based on fast chlorophyll fluorescence (ChlF) kinetics and derived parameters, is a dependable tool for studying photosynthetic efficiency under varying environmental conditions. We extracted additional information from the whole OJIP and the normalized variable fluorescence (Vt) transient curve using first and second-order derivatives to visualize and localize points of landmark events. To account for light-induced variations in the fluorescence transient, we present a time-adjusted JIP test approach in which the derivatives of the transient curve are used to determine the exact timing of the J and I steps instead of fixed time points. We compared the traditional JIP test method with the time-adjusted method in analyzing fast ChlF measurements of silver birch (Betula pendula) in field conditions studying diurnal and within-crown variation. The time-adjusted JIP test method showed potential for studying ChlF dynamics, as it takes into account potential time shifts in the occurrence of J and I steps. The exact occurrence times of J and I steps and other landmark events coincided with the times of significant differences in fluorescence intensity. Chlorophyll fluorescence parameters were linearly related to photosynthetic photon flux density (PPFD) at different times of day, and the values obtained by the time-adjusted JIP test showed a stronger linear regression than the traditional JIP test. For fluorescence parameters having significant differences among different times of day and crown layers, the time-adjusted JIP test resulted in more clear differences than the traditional JIP test. Diurnal ChlF intensity data indicated that differences between the southern and northern provenance were only evident under low light conditions. Taken together, our results emphasize the potential relevance of considering the time domain in the analysis of the fast ChlF induction.

In vitro fertilization program in white rhinoceros.

In brief: To save endangered rhinoceros species, assisted reproductive technologies are warranted. We here report in vitro blastocyst generation of the Near-Threatened Southern white rhinoceros and, for the first time, also of the technically Extinct Northern white rhinoceros. Abstract: The Anthropocene is marked by a dramatic biodiversity decline, particularly affecting the family Rhinocerotidae. Three of five extant species are listed as Critically Endangered (Sumatran, Javan, black rhinoceros), one as Vulnerable (Indian rhinoceros), and only one white rhino (WR) subspecies, the Southern white rhinoceros (SWR), after more than a century of successful protection is currently classified as Near Threatened by the IUCN, while numbers again are declining. Conversely, in 2008, the SWR's northern counterpart and second WR subspecies, the Northern white rhinoceros (NWR), was declared extinct in the wild. Safeguarding these vanishing keystone species urgently requires new reproductive strategies. We here assess one such strategy, the novel in vitro fertilization program in SWR and - for the first-time NWR - regarding health effects, donor-related, and procedural factors. Over the past 8 years, we performed 65 procedures in 22 white rhinoceros females (20 SWR and 2 NWR) comprising hormonal ovarian stimulation, ovum pick-up (OPU), in vitro oocyte maturation, fertilization, embryo culture, and blastocyst cryopreservation, at an efficiency of 1.0 ± 1.3 blastocysts per OPU, generating 22 NWR, 19 SWR and 10 SWR/NWR hybrid blastocysts for the future generation of live offspring.

Application of decision tools to ethical analysis in biodiversity conservation.

Achieving ethically responsible decisions is crucial for the success of biodiversity conservation projects. We adapted the ethical matrix, decision tree, and Bateson's cube to assist in the ethical analysis of complex conservation scenarios by structuring these tools so that they can implement the different value dimensions (environmental, social, and animal welfare) involved in conservation ethics. We then applied them to a case study relative to the decision-making process regarding whether or not to continue collecting biomaterial on the oldest of the two remaining northern white rhinoceroses (Ceratotherium simum cottoni), a functionally extinct subspecies of the white rhinoceros. We used the ethical matrix to gather ethical pros and cons and as a starting point for a participatory approach to ethical decision-making. We used decision trees to compare the different options at stake on the basis of a set of ethical desiderata. We used Bateson's cube to establish a threshold of ethical acceptability and model the results of a simple survey. The application of these tools proved to be pivotal in structuring the decision-making process and in helping reach a shared, reasoned, and transparent decision on the best option from an ethical point of view among those available.

Que se logren decisiones éticamente responsables es crucial para el éxito de los proyectos de conservación de la biodiversidad. Adaptamos la matriz ética, el árbol de decisión y el cubo de Bateson para apoyar con el análisis ético de escenarios de conservación compleja mediante la estructuración de estas herramientas de tal manera que puedan ejecutar las diferentes dimensiones de valor (ambiental, social y bienestar animal) involucradas en la ética de la conservación. Después aplicamos las herramientas a un estudio de caso relacionado con el proceso de toma de decisiones respecto a si se debe seguir o no recolectando material biológico del rinoceronte blanco del norte (Ceratotherium simum cottoni) más viejo (una subespecie funcionalmente extinta) de los dos que existen. Usamos la matriz ética como un punto de partida para una estrategia participativa para la toma ética de decisiones y para recopilar los pros y contras éticos. Usamos el árbol de decisión para comparar las diferentes opciones en juego con base en un conjunto de deseos éticos. Usamos el cubo de Bateson para establecer un umbral de aceptación ética y modelar los resultados de una encuesta simple. La aplicación de estas herramientas demostró ser central en la estructuración del proceso de toma de decisiones y en el apoyo para lograr una decisión compartida, razonada y transparente sobre la mejor opción a partir de un punto de vista ético entre aquellos disponibles.

Contrasting evolutionary history, anthropogenic declines and genetic contact in the northern and southern white rhinoceros (Ceratotherium simum).

The white rhinoceros (Ceratotherium simum) has a discontinuous African distribution, which is limited by the extent of sub-Saharan grasslands. The southern population (SWR) declined to its lowest number around the turn of the nineteenth century, but recovered to become the world's most numerous rhinoceros. In contrast, the northern population (NWR) was common during much of the twentieth century, declining rapidly since the 1970s, and now only two post-reproductive individuals remain. Despite this species's conservation status, it lacks a genetic assessment of its demographic history. We therefore sampled 232 individuals from extant and museum sources and analysed ten microsatellite loci and the mtDNA control region. Both marker types reliably partitioned the species into SWR and NWR, with moderate nuclear genetic diversity and only three mtDNA haplotypes for the species, including historical samples. We detected ancient interglacial demographic declines in both populations. Both populations may also have been affected by recent declines associated with the colonial expansion for the SWR, and with the much earlier Bantu migrations for the NWR. Finally, we detected post-divergence secondary contact between NWR and SWR, possibly occurring as recently as the last glacial maximum. These results suggest the species was subjected to regular periods of fragmentation and low genetic diversity, which may have been replenished upon secondary contact during glacial periods. The species's current situation thus reflects prehistoric declines that were exacerbated by anthropogenic pressure associated with the rise of late Holocene technological advancement in Africa. Importantly, secondary contact suggests a potentially positive outcome for a hybrid rescue conservation strategy, although further genome-wide data are desirable to corroborate these results.

Rewinding the process of mammalian extinction.

With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. 35:280-292, 2016. © 2016 The Authors. Zoo Biology published by Wiley Periodicals, Inc.

A new perspective for European SMEs' innovative support analysis: Does non-financial support matter?

Most of the previous research focused on the effects of public support for SMEs' innovative activities dealt with the effects of financial incentives, while non-financial support was considered a kind of "black box". This research therefore aims to fill this gap by exploring the role non-financial support systems play in triggering SMEs' collaborations and innovativeness in Europe. We show that non-financial support (framework condition) significantly facilitates collaboration and innovation of European SMEs, by using partial least squares structural equation modelling (PLS-SEM) and 216 sampled datasets of the 27 EU countries from 2012 to 2020 European Innovation Scoreboard (EIS). Our expectation was confirmed about the SMEs' collaboration significant effects on their innovative activities, which subsequently translate into innovative outputs. We also found indirect significant effect of the non-financial support on SME's innovation output. This study serves several practical implications and contributes to the ongoing debate on the effects of non-financial support for cooperation and innovation activities of European SMEs.

Do R&D intensity and capacity utilisation matter for SMEs' innovations within the CEE region? Testing moderating roles of different ownership structures.

Existing innovation literature has assumed that the relationship between firms' R&D intensity and innovation take place without the interplay of other organizational factors. However, the reality differs, and research to date has shown that other factors affecting firms' innovation need to be considered. This is important especially in Central and Eastern Europe (CEE) countries, which are highly dependent on both internal and external R&D and are associated with an inability to use R&D resources effectively. This study therefore responds to calls for further analysis, especially within the CEE region, and focuses on the role of two factors affecting SMEs' innovativeness and their effects, which have been mixed so far. First, we investigate the effects of SMEs' R&D intensity and capacity utilisation on product innovation. Second, we reveal the moderating role of SMEs' different ownership structures (ownership concentration; private/public ownership; family/non-family ownership) in the relationship between R&D intensity and product innovation. We confirm that CEE SMEs' ownership concentration and private ownership moderate the relationship between R&D intensity and product innovation. In contrast, we reject our hypothesis expecting that family ownership of SMEs can significantly moderate the relationship between R&D intensity and product innovation. Interestingly, we also show that the relationship between capacity utilisation and innovation is non-linear (inverted U-shaped). This study makes a significant contribution in the form of analysis within the CEE region, whose innovation systems are seen to be weak, and it is therefore necessary to bring new knowledge and recommendations to managers and public policymakers.

Making the Genotypic Variation Visible: Hyperspectral Phenotyping in Scots Pine Seedlings.

Hyperspectral reflectance contains valuable information about leaf functional traits, which can indicate a plant's physiological status. Therefore, using hyperspectral reflectance for high-throughput phenotyping of foliar traits could be a powerful tool for tree breeders and nursery practitioners to distinguish and select seedlings with desired adaptation potential to local environments. We evaluated the use of 2 nondestructive methods (i.e., leaf and proximal/canopy) measuring hyperspectral reflectance in the 350- to 2,500-nm range for phenotyping on 1,788 individual Scots pine seedlings belonging to lowland and upland ecotypes of 3 different local populations from the Czech Republic. Leaf-level measurements were collected using a spectroradiometer and a contact probe with an internal light source to measure the biconical reflectance factor of a sample of needles placed on a black background in the contact probe field of view. The proximal canopy measurements were collected under natural solar light, using the same spectroradiometer with fiber optical cable to collect data on individual seedlings' hemispherical conical reflectance factor. The latter method was highly susceptible to changes in incoming radiation. Both spectral datasets showed statistically significant differences among Scots pine populations in the whole spectral range. Moreover, using random forest and support vector machine learning algorithms, the proximal data obtained from the top of the seedlings offered up to 83% accuracy in predicting 3 different Scots pine populations. We conclude that both approaches are viable for hyperspectral phenotyping to disentangle the phenotypic and the underlying genetic variation within Scots pine seedlings.

Revealing the Complex Relationship Among Hyperspectral Reflectance, Photosynthetic Pigments, and Growth in Norway Spruce Ecotypes.

Norway spruce has a wide natural distribution range, harboring substantial physiological and genetic variation. There are three altitudinal ecotypes described in this species. Each ecotype has been shaped by natural selection and retains morphological and physiological characteristics. Foliar spectral reflectance is readily used in evaluating the physiological status of crops and forest ecosystems. However, underlying genetics of foliar spectral reflectance and pigment content in forest trees has rarely been investigated. We assessed the reflectance in a clonal bank comprising three ecotypes in two dates covering different vegetation season conditions. Significant seasonal differences in spectral reflectance among Norway spruce ecotypes were manifested in a wide-ranging reflectance spectrum. We estimated significant heritable variation and uncovered phenotypic and genetic correlations among growth and physiological traits through bivariate linear models utilizing spatial corrections. We confirmed the relative importance of the red edge within the context of the study site's ecotypic variation. When interpreting these findings, growth traits such as height, diameter, crown length, and crown height allowed us to estimate variable correlations across the reflectance spectrum, peaking in most cases in wavelengths connected to water content in plant tissues. Finally, significant differences among ecotypes in reflectance and other correlated traits were detected.

Robust induction of primordial germ cells of white rhinoceros on the brink of extinction.

In vitro gametogenesis, the process of generating gametes from pluripotent cells in culture, is a powerful tool for improving our understanding of germ cell development and an alternative source of gametes. Here, we induced primordial germ cell-like cells (PGCLCs) from pluripotent stem cells of the northern white rhinoceros (NWR), a species for which only two females remain, and southern white rhinoceros (SWR), the closest species to the NWR. PGCLC differentiation from SWR embryonic stem cells is highly reliant on bone morphogenetic protein and WNT signals. Genetic analysis revealed that SRY-box transcription factor 17 (SOX17) is essential for SWR-PGCLC induction. Under the defined condition, NWR induced pluripotent stem cells differentiated into PGCLCs. We also identified cell surface markers, CD9 and Integrin subunit alpha 6 (ITGA6), that enabled us to isolate PGCLCs without genetic alteration in pluripotent stem cells. This study provides a first step toward the production of NWR gametes in culture and understanding of the basic mechanism of primordial germ cell specification in a large animal.

Naïve-like pluripotency to pave the way for saving the northern white rhinoceros from extinction.

The northern white rhinoceros (NWR) is probably the earth's most endangered mammal. To rescue the functionally extinct species, we aim to employ induced pluripotent stem cells (iPSCs) to generate gametes and subsequently embryos in vitro. To elucidate the regulation of pluripotency and differentiation of NWR PSCs, we generated iPSCs from a deceased NWR female using episomal reprogramming, and observed surprising similarities to human PSCs. NWR iPSCs exhibit a broad differentiation potency into the three germ layers and trophoblast, and acquire a naïve-like state of pluripotency, which is pivotal to differentiate PSCs into primordial germ cells (PGCs). Naïve culturing conditions induced a similar expression profile of pluripotency related genes in NWR iPSCs and human ESCs. Furthermore, naïve-like NWR iPSCs displayed increased expression of naïve and PGC marker genes, and a higher integration propensity into developing mouse embryos. As the conversion process was aided by ectopic BCL2 expression, and we observed integration of reprogramming factors, the NWR iPSCs presented here are unsuitable for gamete production. However, the gained insights into the developmental potential of both primed and naïve-like NWR iPSCs are fundamental for in future PGC-specification in order to rescue the species from extinction using cryopreserved somatic cells.

Ethical Analysis of the Application of Assisted Reproduction Technologies in Biodiversity Conservation and the Case of White Rhinoceros (Ceratotherium simum) Ovum Pick-Up Procedures.

Originally applied on domestic and lab animals, assisted reproduction technologies (ARTs) have also found application in conservation breeding programs, where they can make the genetic management of populations more efficient, and increase the number of individuals per generation. However, their application in wildlife conservation opens up new ethical scenarios that have not yet been fully explored. This study presents a frame for the ethical analysis of the application of ART procedures in conservation based on the Ethical Matrix (EM), and discusses a specific case study-ovum pick-up (OPU) procedures performed in the current conservation efforts for the northern white rhinoceros (Ceratotherium simum cottoni)-providing a template for the assessment of ART procedures in projects involving other endangered species.

The role of foreign technologies and R&D in innovation processes within catching-up CEE countries.

Prior research showed that there is a growing consensus among researchers, which point out a key role of external knowledge sources such as external R&D and technologies in enhancing firms´ innovation. However, firms´ from catching-up Central and Eastern European (CEE) countries have already shown in the past that their innovation models differ from those applied, for example, in Western Europe. This study therefore introduces a novel two-staged model combining artificial neural networks and random forests to reveal the importance of internal and external factors influencing firms´ innovation performance in the case of 3,361 firms from six catching-up CEE countries (Czech Republic, Slovakia, Poland, Estonia, Latvia and Lithuania), by using the World Banks´ Enterprise Survey data from 2019. We confirm the hypothesis that innovators in the catching-up CEE countries depend more on internal knowledge sources and, moreover, that participation in the firms groups represents an important factor of firms´ innovation. Surprisingly, we reject the hypothesis that foreign technologies are a crucial source of external knowledge. This study contributes to the theories of open innovation and absorptive capacity in the context of selected CEE countries and provides several practical implications for firms.

Accelerating Adaptation of Forest Trees to Climate Change Using Individual Tree Response Functions.

In forest tree breeding, assisted migration has been proposed to accelerate the adaptive response to climate change. Response functions are currently fitted across multiple populations and environments, enabling selections of the most appropriate seed sources for a specific reforestation site. So far, the approach has been limited to capturing adaptive variation among populations, neglecting tree-to-tree variation residing within a population. Here, we combined the response function methodology with the in-situ breeding approach, utilizing progeny trials of European larch (Larix decidua) across 21 test sites in Austria ranging from Alpine to lowland regions. We quantified intra-population genetic variance and predicted individual genetic performance along a climatic gradient. This approach can be adopted in most breeding and conservation programs, boosting the speed of adaptation under climate change.

Genetic diversity of Norway spruce ecotypes assessed by GBS-derived SNPs.

We investigated the genetic structure of three phenotypically distinct ecotypic groups of Norway spruce (Picea abies) belonging to three elevational classes; namely, low- (acuminata), medium- (europaea), and high-elevation (obovata) form, each represented by 150 trees. After rigorous filtering, we used 1916 Genotyping-by-Sequencing generated SNPs for analysis. Outputs from three multivariate analysis methods (Bayesian clustering algorithm implemented in STRUCTURE, Principal Component Analysis, and the Discriminant Analysis of Principal Components) indicated the presence of a distinct genetic cluster representing the high-elevation ecotypic group. Our findings bring a vital message to forestry practice affirming that artificial transfer of forest reproductive material, especially for stands under harsh climate conditions, should be considered with caution.

An Ethical Assessment Tool (ETHAS) to Evaluate the Application of Assisted Reproductive Technologies in Mammals' Conservation: The Case of the Northern White Rhinoceros (Ceratotherium simum cottoni).

Assisted reproductive technologies (ARTs) can make a difference in biodiversity conservation. Their application, however, can create risks and raise ethical issues that need addressing. Unfortunately, there is a lack of attention to the topic in the scientific literature and, to our knowledge, there is no tool for the ethical assessment of ARTs in the context of conservation that has been described. This paper reports the first applications of the Ethical Assessment Tool (ETHAS) to trans-rectal ovum pick-up (OPU) and in vitro fertilization (IVF) procedures used in a northern white rhinoceros (Ceratotherium simum cottoni) conservation project. The ETHAS consists of two checklists, the Ethical Evaluation Sheet and the Ethical Risk Assessment, and is specifically customized for each ART procedure. It provides an integrated, multilevel and standardized self-assessment of the procedure under scrutiny, generating an ethical acceptability ranking (totally, partially, not acceptable) and a risk rank (low, medium, high), and, hence, allows for implementing measures to address or manage issues beforehand. The application of the ETHAS to the procedures performed on the northern white rhinoceros was effective in ensuring a high standard of procedures, contributing to the acceptability and improved communication among the project's partners. In turn, the tool itself was also refined through an iterative consultation process between experts and stakeholders.

The ART of bringing extinction to a freeze - History and future of species conservation, exemplified by rhinos.

The ongoing mass extinction of animal species at an unprecedented rate is largely caused by human activities. Progressive habitat destruction and fragmentation is resulting in accelerated loss of biodiversity on a global scale. Over decades, captive breeding programs of non-domestic species were characterized by efforts to optimize species-specific husbandry, to increase studbook-based animal exchange, and to improve enclosure designs. To counter the ongoing dramatic loss of biodiversity, new approaches are warranted. Recently, new ideas, particularly the application of assisted reproduction technologies (ART), have been incorporated into classical zoo breeding programs. These technologies include semen and oocyte collection, artificial insemination, and in-vitro embryo generation. More futuristic ideas of advanced ART (aART) implement recent advances in biotechnology and stem-cell related approaches such as cloning, inner cell mass transfer (ICM), and the stem-cell-associated techniques (SCAT) for the generation of gametes and ultimately embryos of highly endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni) of which only two female individuals are left. Both, ART and aART greatly depend on and benefit from the rapidly evolving cryopreservation techniques and biobanking not only of genetic, but also of viable cellular materials suitable for the generation of induced pluripotent stem cells (iPSC). The availability of cryopreserved materials bridges gaps in time and space, thereby optimizing the available genetic variability and enhancing the chance to restore viable populations.

The dehydrins gene expression differs across ecotypes in Norway spruce and relates to weather fluctuations.

Norway spruce has a broad natural distribution range, which results in a substantial variety of its physiological and genetic variation. There are three distinct altitudinal ecotypes described in this tree species. The physiological optimum of each ecotype may be shifted due to ongoing climate change, especially in traits associated with water demand that might be crucial for adaptation. Dehydrins are proteins that help to mitigate the adverse effects of dehydration. Dehydrin gene expression patterns appeared to be a suitable marker for plant stress assessment. Genetically determined differences in response between individuals and populations were formerly studied, however, mainly in controlled conditions. We evaluated ecotypic variation in dehydrin gene expression in a clonal bank comprised of all three ecotypes. A genetic relationship among targeted trees was uncovered utilizing GBS (Genotyping by Sequencing) platform. We sampled 4-6 trees of each ecotype throughout 15 months period. Subsequently, we assessed the RNA expression of dehydrin genes by qRT-PCR. For this study, we deliberately selected dehydrins from different categories. Our findings detected significant differences among ecotypes in dehydrin expression. The association of recorded climatic variables and individual gene expression across the study period was evaluated and revealed, for certain genes, a correlation between dehydrin gene expression and precipitation, temperature, and day-length.

In Situ Genetic Evaluation of European Larch Across Climatic Regions Using Marker-Based Pedigree Reconstruction.

Sustainable and efficient forestry in a rapidly changing climate is a daunting task. The sessile nature of trees makes adaptation to climate change challenging; thereby, ecological services and economic potential are under risk. Current long-term and costly gene resources management practices have been primarily directed at a few economically important species and are confined to defined ecological boundaries. Here, we present a novel in situ gene-resource management approach that conserves forest biodiversity and improves productivity and adaptation through utilizing basic forest regeneration installations located across a wide range of environments without reliance on structured tree breeding/conservation methods. We utilized 4,267 25- to 35-year-old European larch trees growing in 21 reforestation installations across four distinct climatic regions in Austria. With the aid of marker-based pedigree reconstruction, we applied multi-trait, multi-site quantitative genetic analyses that enabled the identification of broadly adapted and productive individuals. Height and wood density, proxies to fitness and productivity, yielded in situ heritability estimates of 0.23 ± 0.07 and 0.30 ± 0.07, values similar to those from traditional "structured" pedigrees methods. In addition, individual trees selected with this approach are expected to yield genetic response of 1.1 and 0.7 standard deviations for fitness and productivity attributes, respectively, and be broadly adapted to a range of climatic conditions. Genetic evaluation across broad climatic gradients permitted the delineation of suitable reforestation areas under current and future climates. This simple and resource-efficient management of gene resources is applicable to most tree species.