Project description:BackgroundA major challenge for ancient DNA (aDNA) studies on insect remains is that sampling procedures involve at least partial destruction of the specimens. A recent extraction protocol reveals the possibility of obtaining DNA from past insect remains without causing visual morphological damage. We test the applicability of this protocol on historic museum beetle specimens dating back to AD 1820 and on ancient beetle chitin remains from permafrost (permanently frozen soil) dating back more than 47,000 years. Finally, we test the possibility of obtaining ancient insect DNA directly from non-frozen sediments deposited 3280-1800 years ago -- an alternative approach that also does not involve destruction of valuable material.Methodology/principal findingsThe success of the methodological approaches are tested by PCR and sequencing of COI and 16S mitochondrial DNA (mtDNA) fragments of 77-204 base pairs (-bp) in size using species-specific and general insect primers.Conclusion/significanceThe applied non-destructive DNA extraction method shows promising potential on insect museum specimens of historical age as far back as AD 1820, but less so on the ancient permafrost-preserved insect fossil remains tested, where DNA was obtained from samples up to ca. 26,000 years old. The non-frozen sediment DNA approach appears to have great potential for recording the former presence of insect taxa not normally preserved as macrofossils and opens new frontiers in research on ancient biodiversity.
Project description:Herbarium specimens are an important source of DNA for plant research but current sampling methods require the removal of material for DNA extraction. This is undesirable for irreplaceable specimens such as rare species or type material. Here I present the first non-destructive sampling method for extracting DNA from herbarium specimens. DNA was successfully retrieved from robust leaves and/or stems of herbarium specimens up to 73 years old.
Project description:Tein-Shun Tsai and Jean-Jay Mao (2017) Shed snake skins have many applications for humans and other animals, and can provide much useful information to a field survey. When properly prepared and identified, a shed snake skin can be used as an important voucher; the morphological descriptions of the shed skins may be critical for taxonomic research, as well as studies of snake ecology and conservation. However, few convenient/ expeditious methods or techniques to identify shed snake skins in specific areas have been developed. In this study, we collected and examined a total of 1,260 shed skin samples - including 322 samples from neonates/ juveniles and 938 from subadults/adults - from 53 snake species in Taiwan and adjacent islands, and developed the first guide to identify them. To the naked eye or from scanned images, the sheds of almost all species could be identified if most of the shed was collected. The key features that aided in identification included the patterns on the sheds and scale morphology. Ontogenetic differences and intraspecific variation in the patterns of sheds were evident in some snake species, and the proportion of young snakes with patterned shed skins was larger than that of adults. The retention of markings on the ventral side of the body (especially the ventral head) during sloughing was much lower than that on the dorsal side. We hope that this pioneering work will not only encourage other researchers to develop similar keys for their country, but also promote local schools, organizations, and citizen scientists to conduct snake inventories.
Project description:BackgroundPhysical dormancy (hard seed) occurs in most species of Leguminosae family and has great consequences not only for ecological adaptation but also for agricultural practice of these species. A rapid, nondestructive and on-site screening method to detect hard seed within species is fundamental important for maintaining seed vigor and germplasm storage as well as understanding seed adaptation to various environment. In this study, the potential of multispectral imaging with object-wise multivariate image analysis was evaluated as a way to identify hard and soft seeds in Acacia seyal, Galega orientulis, Glycyrrhiza glabra, Medicago sativa, Melilotus officinalis, and Thermopsis lanceolata. Principal component analysis (PCA), linear discrimination analysis (LDA), and support vector machines (SVM) methods were applied to classify hard and soft seeds according to their morphological features and spectral traits.ResultsThe performance of discrimination model via multispectral imaging analysis was varied with species. For M. officinalis, M. sativa, and G. orientulis, an excellent classification could be achieved in an independent validation data set. LDA model had the best calibration and validation abilities with the accuracy up to 90% for M. sativa. SVM got excellent seed discrimination results with classification accuracy of 91.67% and 87.5% for M. officinalis and G. orientulis, respectively. However, both LDA and SVM model failed to discriminate hard and soft seeds in A. seyal, G. glabra, and T. lanceolate.ConclusionsMultispectral imaging together with multivariate analysis could be a promising technique to identify single hard seed in some legume species with high efficiency. More legume species with physical dormancy need to be studied in future research to extend the use of multispectral imaging techniques.
Project description:Tapa (barkcloth) is a non-woven textile made from the inner bark of some plant species. Tapa manufacture was once widespread throughout the Pacific and tapa from the eighteenth and nineteenth century form part of Pacific collections in many museums. Here we examined the feasibility of DNA identification of the plants used to make tapa artefacts by developing and testing a DNA reference database of chloroplast trnL intron P6 loop sequences from many of the plant species used to make tapa, as well as other New Zealand textile plants. This database enabled identification to genus for most species but many species shared identical sequences. Despite the lack of species-level resolution, this technique will still aid with identifying the origins of tapa artefacts made from plants with restricted distributions, such as endemic New Zealand and Hawaiian Islands plants. A second aim was to test a number of DNA extraction methods, including non-destructive methods of interest to the heritage sector, on tapa samples. Only one of the non-destructive sampling methods produced amplifiable DNA. However, we did find variation in the success of the destructive methods tested, with the Qiagen DNeasy Plant Mini Kit having the highest success rate.
Project description:Limiting harm to organisms caused by genetic sampling is an important consideration for rare species, and a number of non-destructive sampling techniques have been developed to address this issue in freshwater mussels. Two methods, visceral swabbing and tissue biopsies, have proven to be effective for DNA sampling, though it is unclear as to which method is preferable for genotyping-by-sequencing (GBS). Tissue biopsies may cause undue stress and damage to organisms, while visceral swabbing potentially reduces the chance of such harm. Our study compared the efficacy of these two DNA sampling methods for generating GBS data for the unionid freshwater mussel, the Texas pigtoe (Fusconaia askewi). Our results find both methods generate quality sequence data, though some considerations are in order. Tissue biopsies produced significantly higher DNA concentrations and larger numbers of reads when compared with swabs, though there was no significant association between starting DNA concentration and number of reads generated. Swabbing produced greater sequence depth (more reads per sequence), while tissue biopsies revealed greater coverage across the genome (at lower sequence depth). Patterns of genomic variation as characterized in principal component analyses were similar regardless of the sampling method, suggesting that the less invasive swabbing is a viable option for producing quality GBS data in these organisms.
Project description:The recent expansion of oil palm (OP, Elaeis guineensis) plantations into tropical forest peatlands has resulted in ecosystem carbon emissions. However, estimates of net carbon flux from biomass changes require accurate estimates of the above ground biomass (AGB) accumulation rate of OP on peat. We quantify the AGB stocks of an OP plantation on drained peat in Malaysia from 3 to 12 years after planting using destructive harvests supported by non-destructive surveys of a further 902 palms. Peat specific allometric equations for palm (R2 = 0.92) and frond biomass are developed and contrasted to existing allometries for OP on mineral soils. Allometries are used to upscale AGB estimates to the plantation block-level. Aboveground biomass stocks on peat accumulated at ~6.39 ± 1.12 Mg ha-1 per year in the first 12 years after planting, increasing to ~7.99 ± 0.95 Mg ha-1 yr-1 when a 'perfect' plantation was modelled. High inter-palm and inter-block AGB variability was observed in mature classes as a result of variations in palm leaning and mortality. Validation of the allometries defined and expansion of non-destructive inventories across alternative plantations and age classes on peat would further strengthen our understanding of peat OP AGB accumulation rates.
Project description:Terrestrial lichen biomass is an important indicator of forage availability for caribou in northern regions, and can indicate vegetation shifts due to climate change, air pollution or changes in vascular plant community structure. Techniques for estimating lichen biomass have traditionally required destructive harvesting that is painstaking and impractical, so we developed models to estimate biomass from relatively simple cover and height measurements. We measured cover and height of forage lichens (including single-taxon and multi-taxa "community" samples, n = 144) at 73 sites on the Seward Peninsula of northwestern Alaska, and harvested lichen biomass from the same plots. We assessed biomass-to-volume relationships using zero-intercept regressions, and compared differences among two non-destructive cover estimation methods (ocular vs. point count), among four landcover types in two ecoregions, and among single-taxon vs. multi-taxa samples. Additionally, we explored the feasibility of using lichen height (instead of volume) as a predictor of stand-level biomass. Although lichen taxa exhibited unique biomass and bulk density responses that varied significantly by growth form, we found that single-taxon sampling consistently under-estimated true biomass and was constrained by the need for taxonomic experts. We also found that the point count method provided little to no improvement over ocular methods, despite increased effort. Estimated biomass of lichen-dominated communities (mean lichen cover: 84.9±1.4%) using multi-taxa, ocular methods differed only nominally among landcover types within ecoregions (range: 822 to 1418 g m-2). Height alone was a poor predictor of lichen biomass and should always be weighted by cover abundance. We conclude that the multi-taxa (whole-community) approach, when paired with ocular estimates, is the most reasonable and practical method for estimating lichen biomass at landscape scales in northwest Alaska.
Project description:Abstract Chironomidae (Diptera) pupal exuviae samples are commonly used for biological monitoring of aquatic habitats. DNA barcoding has proved useful for species identification of chironomid life stages containing cellular tissue, but the barcoding success of chironomid pupal exuviae is unknown. We assessed whether standard DNA barcoding could be efficiently used for species identification of chironomid pupal exuviae when compared with morphological techniques and if there were differences in performance between temperate and tropical ecosystems, subfamilies, and tribes. PCR, sequence, and identification success differed significantly between geographic regions and taxonomic groups. For Norway, 27 out of 190 (14.2%) of pupal exuviae resulted in high-quality chironomid sequences that match species. For Costa Rica, 69 out of 190 (36.3%) Costa Rican pupal exuviae resulted in high-quality sequences, but none matched known species. Standard DNA barcoding of chironomid pupal exuviae had limited success in species identification of unknown specimens due to contaminations and lack of matching references in available barcode libraries, especially from Costa Rica. Therefore, we recommend future biodiversity studies that focus their efforts on understudied regions, to simultaneously use morphological and molecular identification techniques to identify all life stages of chironomids and populate the barcode reference library with identified sequences.
Project description:The visible and near-infrared (Vis-NIR) reflectance spectroscopy was utilized for the rapid and nondestructive discrimination of edible oil adulteration. In total, 110 samples of sesame oil and rapeseed oil adulterated with soybean oil in different levels were produced to obtain the reflectance spectra of 350-2500 nm. A set of multivariant methods was applied to identify adulteration types and adulteration rates. In the qualitative analysis of adulteration type, the support vector machine (SVM) method yielded high overall accuracy with multiple spectra pretreatments. In the quantitative analysis of adulteration rate, the random forest (RF) combined with multivariate scattering correction (MSC) achieved the highest identification accuracy of adulteration rate with the full wavelengths of Vis-NIR spectra. The effective wavelengths of the Vis-NIR spectra were screened to improve the robustness of the multivariant methods. The analysis results suggested that the competitive adaptive reweighted sampling (CARS) was helpful for removing the redundant information from the spectral data and improving the prediction accuracy. The PLSR + MSC + CARS model achieved the best prediction performance in the two adulteration cases of sesame oil and rapeseed oil. The coefficient of determination (RPcv2) and the root mean square error (RMSEPcv) of the prediction set were 0.99656 and 0.01832 in sesame oil adulterated with soybean oil, and the RPcv2 and RMSEPcv were 0.99675 and 0.01685 in rapeseed oil adulterated with soybean oil, respectively. The Vis-NIR reflectance spectroscopy with the assistance of multivariant analysis can effectively discriminate the different adulteration rates of edible oils.