Environmental determinants of genetic diversity in Caragana microphylla (Fabaceae) in northern China

Non‐rewarding orchids rely on various ruses to attract their pollinators. One of the most common is for them to resemble flowers sought by insects as food sources. This can range from generalized food deception to the mimicry of specific sympatric food plants. We investigated the basis of pollinator deception in the European food‐deceptive orchid Traunsteinera globosa, which has unusually compact flowerheads resembling those of sympatric rewarding species of Knautia and Scabiosa (Dipsacaceae), and Valeriana (Caprifoliaceae). Visual signals of T. globosa are similar in both fly and bee vision models to those of the sympatric food plants used in the choice experiments, but scent signals are divergent. Field experiments conducted in Austria and the Czech Republic showed that both naive and experienced (with respect to visitation of T. globosa) insect species approached the orchids at the same rate as food plants, but direct contact with orchid flowers was taxon specific. Flies were most easily duped into probing the orchid, and, in doing so, frequently received and deposited pollinaria, whereas most bees and butterflies avoided landing on orchid flowers. We conclude that T. globosa is a mimic of a guild of fly‐pollinated plants, but the ecological dependence of the orchid on its models remains to be fully tested. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 269–294.     

Molecular Characterization of Three Canine Models of Human Rare Bone Diseases: Caffey,van den Ende-Gupta,and Raine Syndromes

One to two percent of all children are born with a developmental disorder requiring pediatric hospital admissions. For many such syndromes, the molecular pathogenesis remains poorly characterized. Parallel developmental disorders in other species could provide complementary models for human rare diseases by uncovering new candidate genes, improving the understanding of the molecular mechanisms and opening possibilities for therapeutic trials. We performed various experiments, e.g. combined genome-wide association and next generation sequencing, to investigate the clinico-pathological features and genetic causes of three developmental syndromes in dogs, including craniomandibular osteopathy (CMO), a previously undescribed skeletal syndrome, and dental hypomineralization, for which we identified pathogenic variants in the canine SLC37A2 (truncating splicing enhancer variant), SCARF2 (truncating 2-bp deletion) and FAM20C (missense variant) genes, respectively. CMO is a clinical equivalent to an infantile cortical hyperostosis (Caffey disease), for which SLC37A2 is a new candidate gene. SLC37A2 is a poorly characterized member of a glucose-phosphate transporter family without previous disease associations. It is expressed in many tissues, including cells of the macrophage lineage, e.g. osteoclasts, and suggests a disease mechanism, in which an impaired glucose homeostasis in osteoclasts compromises their function in the developing bone, leading to hyperostosis. Mutations in SCARF2 and FAM20C have been associated with the human van den Ende-Gupta and Raine syndromes that include numerous features similar to the affected dogs. Given the growing interest in the molecular characterization and treatment of human rare diseases, our study presents three novel physiologically relevant models for further research and therapy approaches, while providing the molecular identity for the canine conditions.     

Domesticated Animal Biobanking: Land of Opportunity

In the past decade, biobanking has fuelled great scientific advances in the human medical sector. Well-established domesticated animal biobanks and integrated networks likewise harbour immense potential for great scientific advances with broad societal impacts, which are currently not being fully realised. Political and scientific leaders as well as journals and ethics committees should help to ensure that we are well equipped to meet future demands in livestock production, animal models, and veterinary care of companion animals.In the last decade, human biobanking has emerged as an important driver of scientific activities, and biobanks are indisputably an invaluable resource for all types of research aimed at improving public health. The combination of accessible and well-characterized biological samples of various types linked with a multitude of associated data is driving scientific discoveries at unprecedented speed and making previously unthinkable lines of research a reality [1,2].Unfortunately, biobanking of animal samples is by far less well-established. In March 2015, Nature published an article, titled “Inside the first pig biobank,” describing a newly established biobank of porcine samples to be used in studying human diabetes and hailing it as a pioneering effort in animal biobanking [3]. A PubMed search confirmed that in comparison to human biobanking there appears to be negligible activity in the animal biobanking sector. Searching titles, abstracts, and keywords with the search keys “biobank,” “biobanking,” “genebank,” and “gene bank” and limiting the results to publication dates in 2015, only 9 of 498 search results referred to animal biobanks (see S1 Data). This apparent lack of activity in the animal biobanking sector is also reflected in a 2015 editorial of Biopreservation and Biobanking, the official journal of the International Society for Biological and Environmental Repositories (ISBER), which caters to biobanks of any species. The authors conclude that even though there has been increasing participation from the non-human biobanking sector in the annual ISBER meetings, there is still a pronounced lack of submissions to the journal pertaining to non-human biobanking, and human biobanking issues continue to dominate ISBER activities [4]. The roadmap of the European Council’s European Strategy Forum on Research Infrastructures (ESFRI) reveals that there are projects under way involving human (Biobanking and BioMolecular Resources Research Infrastructure [BBMRI]), marine (European Marine Biological Resource Centre [EMBRC]), microbial (Microbial Resource Research Infrastructure [MIRRI]), and mouse model (Infrafrontier) biobanks, with general animal biobanks starkly missing on that list [5].Naturally, some non-human biobanks storing animal samples, amongst others, do exist. The most active are likely the natural history collections, because they have the intrinsic task to collect, catalogue, and store specimens. The Global Genome Biodiversity Network (GGBN), established in 2011, acts as an umbrella organisation for biodiversity repositories and aims to establish standards and best practices as well as increase sample accessibility through its data portal [6]. A search of the most common domesticated animal species (cattle, sheep, goat, pig, horse, chicken, and dog) yielded only 13 records in the GGBN member repositories.However, some domesticated animal biobanks and less formalized sample collections can be found. Their hosting institutions range from veterinary hospitals, zoos, breeding and diagnostics companies, national farm animal genetic resource gene banks, to research institutes and universities. Depending on their purpose, the stored types of samples vary greatly and range from healthy tissue samples, diseased pathogenic tissue samples, DNA, and RNA to reproductive materials. An example of a well-established physical non-human biobanking infrastructure serving a university is the Swedish University of Agricultural Sciences’ (SLU) Biobank (http://www.slu.se/slubiobank). This biobank also offers a data portal for increasing the visibility and accessibility of non-human sample collections no matter where they are stored. This data portal would be redundant if all samples, together with their associated data, were stored in established biobanks that ensured the visibility of their samples through a network such as GGBN. In contrast, the European Genebank Network for Animal Genetic Resources (EUGENA), coordinated by the European Regional Focal Point on Animal Genetic Resources (http://www.rfp-europe.org), is an emerging networking activity specifically targeting only national farm animal genetic resource collections [7]. These disparate examples demonstrate that there is a lack of a unified and generalized approach to sample collections in the domesticated animal sector.Nonetheless, there are numerous examples of how different disciplines and stakeholders, and ultimately the general public, have already benefitted from the availability of biobanked domesticated animal samples.Even though the pig biobank was commended as a pioneering effort [3], there are in fact a number of biobanks that accommodate animal models for the study of human disease. The domestic dog, for example, with its unique population history, breed structure, and hundreds of spontaneous genetic conditions has proven to be an excellent model for gene mapping in simple and complex disorders [8]. Targeted and effective breeding programs over the past 150 years have created hundreds of distinct breeds that form genetic isolates with reduced genetic heterogeneity. This simplifies genetic studies because fewer susceptibility loci with higher impact contribute to complex disease and allow genetic breakthroughs with smaller study cohorts as compared to the corresponding human conditions [9].The annotation of the canine genome facilitated a rapid evolution of genomic tools and development of several canine biobanks across the continents [10]. Collectively, these biobanks house hundreds of thousands of DNA samples and tissue specimens for hundreds of conditions with medical relevance to humans. Importantly, many canine biobanks maintain active collaborative networks with the breeder community and dog fanciers as well as veterinary clinics and hospitals for patient recruitment and health updates.Besides playing an instrumental role for human health, biobanked animal samples heavily impact developments in food production and the sustainable management of the world’s finite resources. Biobanks in animal breeding, often referred to as gene banks, were initially established with the advent of new reproductive techniques, such as artificial insemination, and typically stored semen and embryos. These biobanks recently played a critical role in the swift implementation of genomic selection, which uses genome-wide SNP markers to predict the genetic merit of breeding individuals [11,12]. The efficient use of genomic selection requires large reference panels of individuals whose genetic values are known with high confidence. In cattle breeding, these are bulls with large numbers of offspring with recorded performance data, such as milk yield. Genomic selection could only be implemented so swiftly and successfully because DNA or semen samples from a large number of bulls were available from cattle breeding company biobanks, and these samples could be linked to performance records of the respective bulls’ offspring. This technology was first adopted by the dairy industry and can potentially result in a 60%–120% increase in the rate of genetic gain. Together with advanced genotyping and reproductive technologies, genomic selection has the potential to increase genetic improvement both in often neglected traits, such as feed efficiency and fertility, and in traits that only recently have become of interest, such as methane output in ruminants or adaptation to climate change [12]. Improvements in these traits are of great interest for ensuring global food security and sustainable management of our limited resources. Without the availability of the gene bank samples, as well as associated performance data records, this transformation would have taken decades, if it had happened at all.Biobanks also play an integral part in worldwide conservation efforts to counteract the well-documented loss of genetic diversity in production animals [13,14]. Slowly, the general perception that these repositories are only to be used in emergencies and as a last resort is changing. In 2012, the USDA National Animal Germplasm Program, for example, harboured more than 700,000 gamete and tissue samples from over 18,000 animals representing more than 130 breeds. From this repository, samples from more than 3,300 animals had been requested and distributed for use in research and industry by 2012. The applications included quantitative trait locus (QTL) studies, assessment of genetic distances, cryobiology research, generation of an experimental research line, reduction of inbreeding, and re-introduction of genotypic combinations lost in current production populations [14]. Samples from rare and endangered breeds are also finding use in research and development of the leading breeding companies. For example, in the Netherlands, a consortium of university and dairy industry partners genotyped samples from rare local cattle breeds to gain insight into the genetic background of milk fatty acid composition. Genomic-assisted introgression could ultimately be used to introduce favourable alleles found in the rare breeds into more widely used breeds.Biobanked samples also played an important role in fighting a viral infection, infectious pancreatic necrosis (IPN), which is common in farmed fish. This virus can lead to rates of >90% mortality in farmed Atlantic salmon, which, therefore, poses a threat to animal welfare and aquaculture industries. In 2008, a major QTL for IPN-resistance was detected in Atlantic salmon. Already, a year later, AquaGen, which supplies about 55% of Atlantic salmon eggs used commercially in Norway, was employing marker-assisted selection to produce IPN-resistant fish. This swift implementation of the QTL in marker-assisted selection was only possible due to the availability of biobanked samples collected in a challenge test in 2005 [15].In addition to combatting disease in animals, biobanked domestic animal samples also play a crucial role in fighting emerging infectious diseases that are often zoonotic, meaning that they can be transmitted between vertebrate animals and humans. Having access to samples of species that act as reservoirs of a disease greatly facilitates the work of public health responders during infectious disease outbreaks [16]. In this context, the collection and traceable link of associated samples, such as parasites, pathogens, and other microbiota, to their parent sample becomes especially important.We are convinced that these examples leave no doubt that biobanked animal samples hold great potential both for advancing human and animal health and welfare as well as securing future food production. Furthermore, the recent advent of cost-efficient gene modification technologies [17] envisages many production, performance, and health applications in livestock and companion animals and further adds interest in animal biobanks.When examining the causes for the low levels of activity in large-scale domesticated animal biobanking, both in regard to the establishment or use of existing physical biobanking infrastructures as well as overarching data portals, a number of hypotheses come to mind. The industries connected to domesticated animal biobanking, such as livestock and companion animal production and veterinary care, are dwarfed by the healthcare industry, so monetary incentives would presumably play a much smaller role. Legislation may have acted as a driver in the formalization and shaping of biobanks and differential legislation regarding the handling, storage, and sharing of human versus animal biosamples, and associated data may thus have led to disparate developments. It is moreover conceivable that the community around domesticated animal biobanking is more fragmented and consists of more diverse stakeholders (academic, non-profit, industrial) than the human biobanking community, which could explain the absence of large-scale cooperative umbrella projects. Moreover, there may be greater difficulties in drafting material transfer agreements for reproductive materials than for other types of samples.We will only be able to exploit the full potential if we, in parallel with human and biodiversity biobanking, tackle the challenges of standardized sampling, processing, and storage, sample visibility and accessibility, standardized codes for diagnoses, collection and storage of associated data with the possibility for updates, as well as ethical and regulatory issues. Here, it is advisable that the domesticated animal sector ensures full compatibility with and relies on existing initiatives wherever feasible. Especially important in this context is to ensure a link between samples and associated phenomic and genomic data, such as derived sequence data. To achieve agreement on standards, both in terms of sample processing and storage and sample visibility and accessibility, actors from veterinary hospitals, zoos, breeding and diagnostics companies, national farm animal genetic resource gene banks, research institutes, universities, and policymakers need to join forces. This is where we momentarily see a lack of coordinated efforts.To respond to these challenges and to ensure that we are well equipped to meet future demands in livestock production, animal models, and veterinary care of companion animals, we propose that scientific and political leaders need to (i) acknowledge the inadequacy of the current situation, (ii) create opportunity and support for the establishment of an international research infrastructure for animal biobanking, and (iii) motivate academic and industrial stakeholders to develop and coordinate biobanks based on lessons learned from human and biodiversity biobanking.In Europe, the European Council’s ESFRI could play a leading role in the establishment of a domesticated animal biobanking network, including best practices, direly needed standards, and a common ontology. In a landscape analysis of European research infrastructures, the 2016 ESFRI roadmap acknowledges a gap in the agricultural and bio-economy sector and explicitly lists livestock facilities including gene banks [5]. While an increase in activities regarding biobanking of farm animal genetic resources is certainly relevant, we consider this not to be far-reaching enough. A step in the right direction would be to begin with compiling information on all existing animal biobanks, analogous to BBMRI’s catalogue for European human biobanks [18], which currently contains information on 340 biobanks (http://www.bbmriportal.eu/).Moreover, ethics committees should require the storage of samples and associated data in formalized biobanks for the approval of scientific experiments. Similarly, journals should apply the same standard to samples and associated data, as they currently apply to molecular data, in terms of storage in formalized repositories prior to publication.     

Molecular prey identification in Central European piscivores

Diet analysis is an important aspect when investigating the ecology of fish‐eating animals and essential for assessing their functional role in food webs across aquatic and terrestrial ecosystems. The identification of fish remains in dietary samples, however, can be time‐consuming and unsatisfying using conventional morphological analysis of prey remains. Here, we present a two‐step multiplex PCR system, comprised of six assays, allowing for rapid, sensitive and specific detection of fish DNA in dietary samples. This approach encompasses 78 fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. All targeted taxa were successfully amplified from 25 template molecules, and each assay was specific when tested against a wide range of invertebrates and vertebrates inhabiting aquatic environments. The applicability of the multiplex PCR system was evaluated in a feeding trial, wherein it outperformed morphological prey analysis regarding species‐specific prey identification in faeces of Eurasian otters. Additionally, a wide spectrum of fish species was detected in field‐collected faecal samples and regurgitated pellets of Common Kingfishers and Great Cormorants, demonstrating the broad applicability of the approach. In conclusion, this multiplex PCR system provides an efficient, easy to use and cost‐effective tool for assessing the trophic ecology of piscivores in Central Europe. Furthermore, the multiplex PCRs and the primers described therein will be applicable wherever DNA of the targeted fish species needs to be detected at high sensitivity and specificity.     

Anatomical Grading for Metabolic Activity of Brown Adipose Tissue

Background

Recent advances in obesity research suggest that BAT activity, or absence thereof, may be an important factor in the growing epidemic of obesity and its manifold complications. It is thus important to assess larger populations for BAT-activating and deactivating factors. ¹⁸FDG-PET/CT is the standard method to detect and quantify metabolic BAT activity, however, the manual measurement is not suitable for large studies due to its time-consuming nature and poor reproducibility across different software and devices.

Methodology/Main Findings

In a retrospective study, 1060 consecutive scans of 1031 patients receiving a diagnostic ¹⁸FDG-PET/CT were examined for the presence of active BAT. Patients were classified according to a 3-tier system (supraclavicular, mediastinal, infradiaphragmatic) depending on the anatomical location of their active BAT depots, with the most caudal location being the decisive factor. The metabolic parameters (maximum activity, total volume and total glycolysis) were measured on a standard PET/CT workstation. Mean age of the population was 60±14.6y. 41.61% of patients were female. Metabolically active BAT was found in 53 patients (5.1%). Female, younger and leaner patients tended to have more active BAT, higher metabolic activity and more caudally active BAT. In total, 15 patients showed only supraclavicular, 27 additional mediastinal, and 11 infradiaphragmal activity. Interestingly, the activation of BAT always followed a cranio-caudal gradient. This anatomical pattern correlated with age and BMI as well as with all metabolic parameters, including maximum and total glycolysis (p<0.001).

Conclusion

Based on our data we propose a simple method to grade or quantify the degree of BAT amount/activity in patients based on the most caudally activated depot. As new modalities for BAT visualization may arise in the future, this system would allow direct comparability with other modalities, in contrary to the PET-metrics, which are restricted to ¹⁸FDG-PET/CT.     

Die rolle der charakterarten bei der beurteilung fichtenreicher wälder der alpen

Zusammenfassung Die Vaccinio-Piceion-Charakterarten treten im subalpinen und montanen Fichtenwald sowie im Fichten-Tannenwald so ungleich auf, daß infolge der schwierigen Bewertung an eine erneute Krise der Charakterartenlehre gedacht werden könnte, auch wenn man den räumlich begrenzten Treuewert der Charakterarten berücksichtigt. Unmittelbare Vergleichbarkeit der Charakterarten besteht nur innerhalb geographisch einheitlicher Gebiet und analoger Gesellschaftsgruppen mit ähnlicher Synökologie und Syndynamik. Luzula-Silikat- und Adenostyles glabra- Kalk- Gesellschafts-gruppen haben selbst bei gleicher Menge und Stetigkeit der Charakterarten keine analoge soziologisch-systematische Wertigkeit. Eine rein statische Bewertung der Charakterarten für soziologisch-systematische Zwecke nach gegenwärtiger Artengarnitur, Stetigkeit und Menge ist unzureichend. Erst die Berücksichtigung von speziellem Gesellschaftsanschluß, Umwelt im engeren (Geologie, Klima) und weiteren (Konkurrenzverhältnisse, Kontaktgesellschaft, Areal) Sinne sowie die chorologische Situation ermöglicht eine eingehendere Beurteilung. Auch die nacheiszeitliche Entwicklungsgeschichte der Assoziation darf bei einer kritischen Betrachtung der heutigen soziologisch-ökologischen Zusammenhänge nicht vernachlässigt werden. Nur durch eine derartige umfassende Analyse vermögen die Charakterarten die erhofften Dienste bei der Beurteilung fichtenreicher Wälder leisten. Schwierigkeiten sind teilweise dadurch bedingt, daß die Gesamtzusammenhänge heute noch zu wenig durchschaubar sind, z.B.: Frage des Auftretens von systematisch nicht abgrenzbaren Ökotypen bei den Charakterarten; Probleme der Verbreitungsdimensionen der Assoziation: charakterartenreiche alpine Fichtenwälder mit 1000 m Höhenerstreckung und analoge charakterartenarme bis-freie boreale Fichtenwälder Skandinaviens mit über 1000 km Erstreckung.

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Summary The character species of Vaccinio-Piceion occur so unequally in the subalpine and montane spruce-forest as well as in the spruce-firforest that, regarding the difficulties of assessment, we could think of a renewed crisis of the Character species theory, even when taking into account the spatial limitations of the character species' degree of fidelity. An immediate comparability of character species is given only within geographically homogeneous areas and analogous groups of associations with a similar synecological and syndynamical behaviour. Association groups with Luzula on silica and with Adenostyles glabra on limestone, even in the case of equal quantities and constancies of character species, have no analogous value in the sociological system. A purely statical evaluation of character species for the purposes of sociological systematics according to present species combination, constancy and quantity is insufficient. It is but the consideration of the special position of associations, of environment in the narrower sense (geology, climate) and in the wider sense (competition conditions, contact association, area) as well as the chorological situation that will enable a detailed assessment. Also the postglacial history of development of the association must by no means be disregarded in a critical consideration of the present sociological and ecological relationships. It is but through such a comprehensive analysis that character species will be able to render the services expected in the assessment of forests rich with spruce. Partially, difficulties arise by the fact that the overall relationships are still too little transparent, e.g. the question of ecotypes without taxonomical limits among character species; problems of association area dimensions: alpine sprucewoods rich with character species over a 1000 m altitudinal range, and analogous boreal sprucewoods poor in, or even free of, character species in Scandinavia, with a horizontal range of more than 1000 km.

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Die Arbeiten wurden dankenswert durch die Deutsche Forschungsgemeinschaft unterstützt.

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(Translated by Dr. Max Onno)     

Short-term effects of boron, germanium and high light intensity on membrane permeability in boron deficient leaves of sunflower

With the ultimate purpose of clarifying the mechanism for aluminium (Al) toxicity and for Al tolerance, we tried to isolate cDNAs whose expression is induced by Al treatment and phosphate (P_i) starvation. We performed P_i starvation and Al treatment (two-step treatment) on suspension-cultured cells of Nicotiana tabacum L. cv. Samsun and then constructed a cDNA library using poly(A)⁺-RNA derived from the treated cells. Four independent cDNA clones (pAL 111, 139, 141 and 142) were isolated from the library by differential screening. Northern blot hybridization analysis indicated that the expression of these clones was induced by P_i starvation. Furthermore, we found that pAL 111 and pAL 142 are also induced by Al treatment. The complete cDNA sequencing of these 4 clones was determined. The results indicated that pAL111 is identical to the parA gene of N. tabacum, which is described as an auxin-regulated gene and that pAL142 is highly homologous to the parB gene of N. tabacum whose product has glutathione S-transferase (GST, EC 2.5.1.18) activity. Furthermore, we found a cysteine-rich domain in the amino acid sequence of pAL139. No DNA and deduced amino acid sequences homologous to the pAL141 were found.     

Monitoring of root growth and redox conditions in paddy soil rhizotrons by redox electrodes and image analysis

The root-zone of wetland rice was monitored in a paddy soil throughout a vegetation period with the aid of a rhizotron experiment. For this purpose (a) digital images of the root-zone were taken daily, and (b) the redox potential was measured in situ every day. The images were processed by image analysis in order to display areas of oxidation and reduction in the soil. Therefore, thresholds were set to simplify the localization and quantification of discrete areas which were colourized due to the redox potential. Both, images and measured redox potentials, provide the basis for the visualization of the root and redox dynamics in the root-zone. The anaerobic root-zone of flooded paddy soils is significantly influenced by the aerenchymal transport of oxygen to rice roots. The release of oxygen into the rhizosphere, which causes different patterns of oxidized and reduced areas in the course of the vegetation period, also affects microbial communities such as methane producing archaea or methane oxidizing bacteria. The visualization of redox dynamics may, therefore, be useful to localize potential hotspots for the microorganisms in the root-zone of paddy soils. The reduced and oxidized conditions changed spatiotemporally. Oxidized areas were mostly found in the surrounding of active roots and in a distinct layer next to the soil surface. Reduced areas shifted from beneath the oxidized surface layer into sparsely-rooted soil. The ratio of the analyzed oxidized and reduced areas was oscillating with increasing intensity throughout the monitored vegetation period.