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1.
The identification of pointer years is central in tree-ring studies, for example, for crossdating or describing growth responses of trees to extreme events. Various methods exist to define pointer years. To these methods, a presumably new method was recently added, i.e. the standardized growth change (SGC) method (Buras et al., 2020; 2022), which was claimed by the authors to outperform existing methods. In this Communication, we perform a concise method review and compare the SGC method with existing pointer year detection methods using simulated tree-ring data that contain diverse signal patterns. We not only provide evidence that the SGC method has already been proposed in 1994, but furthermore show that each method for pointer year detection, including SGC, has its own strengths and weaknesses. Given that each method highlights different aspects of extraordinary growth, we repeat our conclusion from Jetschke et al. (2019), namely that no method can be substituted by others completely, and thus be claimed as being ‘best’. As applies to all methods for pointer year detection, SGC is a complementary but certainly powerful method. We request the dendro-community to provide clear explanations in future studies on how the term pointer year was used to prevent unnecessary confusion and misunderstanding.  相似文献   

2.
Detecting pointer years in tree-ring data is a central aspect of dendroecology. Pointer years are usually represented by extraordinary secondary tree growth, which is often interpreted as a response to abnormal environmental conditions such as late-frosts or droughts. Objectively identifying pointer years in larger tree-ring networks and relating those to specific climatic conditions will allow for refining our understanding of how trees perform under extreme climate and consequently, under anticipated climate change. Recently, Buras et al. (2020) demonstrated that frequently used pointer-year detection methods were either too sensitive or insensitive for such large scale analyses. In their study, Buras et al. (2020) proposed a novel approach for detecting pointer years – the standardized growth change (SGC) method which outperformed other pointer-year detection methods in pseudopopulation trials. Yet, the authors concluded that SGC could be improved further to account for the inability to detect pointer years following successive growth decline. Under this framework, we here present a refined version of the SGC-method – the bias-adjusted standardized growth change method (BSGC). The methodological adjustment to the SGC approach comprises conflated probabilities derived from standardized growth changes with probabilities derived from a time-step specific global standardization of growth changes. In addition, BSGC allows for estimating the length of the deflection period, i.e. the period before extraordinary growth values have reached normal levels. Application of BSGC to simulated and measured tree-ring data indicated an improved performance in comparison to SGC which allows for the identification of pointer years following years of successive growth decline. Also, deflection period lengths were estimated well and revealed plausible results for an existing tree-ring data set. Based on these validations, BSGC can be considered a further refinement of pointer-year detection, allowing for a more accurate identification and consequently better understanding of the radial growth response of trees to extreme events.  相似文献   

3.
To study growth responses of trees to extreme events, analyses of so-called ‘pointer years’ are often performed. Thereby, the term pointer year refers to years in which a majority of trees shows extraordinary growth responses, like very narrow (or wide) ring widths. A wide variety of methods has been used to analyze pointer years, hampering comparisons between studies. The latter illustrates that there is a strong need to harmonize pointer year detection methods. This review contributes to that by describing and comparing the main methods (and variants thereof) found in the literature for the last two decades, both in a qualitative and quantitative way. We discuss methodological considerations and provide recommendations how to proceed with pointer year detection in future. Given that the individual methods for pointer year detection were found to highlight different aspects of extraordinary growth, the one method cannot completely substitute the other. Hence, we suggest to use multiple methods in a harmonized way to get the deepest insight into the nature of pointer years.  相似文献   

4.
For a long time, radial growth of pedunculate oak (Quercus robur) in relation to environmental factors has been studied in Central Europe. However, there is insufficient information on oak growth in the Baltic region. Climate–growth interactions have been mostly investigated by correlation/response analysis between ring width and climatic factors. Other wood anatomical proxies, and also pointer year analysis, which focuses on weather extremes, can be sources of additional information. Wood samples were taken from 40 sites across Latvia. Tree-ring width (TRW) and mean area of earlywood vessels (EVA) were measured. To assess differences in wood formation among sites, a PCA was performed based on pointer year series of TRW and EVA per site. The relation with climate was assessed by Pearson's correlation. Patterns of wood formation differed along west/east gradient, so that two regions (western and eastern) of Latvia were distinguished. Pointer year values were higher for TRW than for EVA and higher in the eastern than in the western region. However, the pointer year values were moderate, suggesting that there was no strict limiting factor for their formation. Pointer years of EVA were strongly related with temperature during the dormant period in both regions; June precipitation had a significant effect in the eastern region. Pointer years of TRW were correlated with spring and summer temperature in the western region and with February temperature in the eastern region; precipitation in autumn showed a negative effect. These differences can be explained by a gradient from maritime to continental climate, leading to shifts in the growth limiting factors. Extremely cold winters resulted in most of the negative pointer years of TRW and EVA.  相似文献   

5.
 Dendroclimatological techniques were used to assess the impact of climatic factors on radial tree growth (total ring-width and latewood-width) of stunted Scots pine trees (Pinus sylvestris L.) exposed to soil dryness and nutrient deficiency on a dolomite substrate. The response of eight scattered populations representing various habitats, yet influenced by the same regional climate was investigated. Total ring-width and latewood-width were dated, standardized and several chronology statistics, which estimate the chronology signal strength and the potential climate signal in the series, were determined. Dendroclimatic analysis comprised evaluation of event/pointer years as well as response function elements. Response function analysis indicates that at most sites wide rings are significantly associated with high precipitation in April to June and cool conditions in May of the current year, and high precipitation in August to September of the preceding year. Latewood chronologies show a lower climate signal at all sites. Limited water availability causes a homogeneous relationship of radial tree growth to climate at all habitats, though site characteristics (slope magnitude, slope aspect, soil depth, vegetation cover) differ substantially. Cluster analysis of negative event/pointer years suggests that within the study area stands react to extreme climatic events depending on susceptibility to soil dryness, which is primarily determined by site topography. Received: 5 January 1998 / Accepted: 22 April 1998  相似文献   

6.
BACKGROUND: In human pedigree data age at disease occurrence frequently is missing and is imputed using various methods. However, little is known about the performance of these methods when applied to families. In particular, there is little information about the level of agreement between imputed and actual values of temporal data and their effects on inferences. METHODS: We performed two evaluations of five imputation methods used to generate complete data for repositories to be shared by many investigators. Two of the methods are mean substitution methods, two are regression methods and one is a multiple imputation method based on one of the regression methods. To evaluate the methods, we randomly deleted the years of disease diagnosis of some men in a sample of pedigrees ascertained as part of a prostate cancer study. In the first evaluation, we used the five methods to impute the missing diagnosis years and evaluated agreement between imputed and actual values. In the second evaluation, we compared agreement between regression coefficients estimated using imputed diagnosis years with those estimated using the actual years. RESULTS/CONCLUSIONS: For both evaluations, we found optimal or near-optimal performance from a regression method that imputes a man's diagnosis year based on the year of birth and year of last observation of all affected men with complete data. The multiple imputation analogue of this method also performed well.  相似文献   

7.
Disturbances play an important role in forest dynamics across the globe. Researchers have mainly focused on the temporal context of disturbances, but have largely ignored the spatial patterns of tree recruitment they create. Geostatistical tools enable the analysis of spatial patterns and variability in tropical forest disturbance histories. Here, we examine the potential of combining dendroecological analysis and spatial statistics to reconstruct the disturbance history of a seasonal dry evergreen tropical forest plot at the Huai Kha Khaeng Wildlife Sanctuary (HKK), western Thailand. We used tree‐ring‐derived age estimates for 70 individuals of the shade‐intolerant pioneer species Melia azederach (Meliaceae) and tree locations across a 316‐ha study plot to identify the timing and spatial extent of past disturbances. Although the age distribution for Melia suggested that regeneration had been continuous over the past 60 yr, spatial analyses (mark correlation function and kriging) demonstrated the presence of three spatially discrete age cohorts. Two of these cohorts suggested a severe disturbance ~20 yr before present. A third cohort appears to have established ~50 years ago. Using historical records, we conclude that fire disturbance is the most likely disturbance factor affecting HKK. Nevertheless, we do not rule out other disturbance factors. The combined application of tree‐ring analysis and spatial statistics as applied in this study could be readily applied to reconstruct disturbance histories in other tropical regions where tree species with annual growth rings are present.  相似文献   

8.
Phosphorylation is one of the most common forms of protein modification. The most frequent targets for protein phosphorylation in eukaryotes are serine and threonine residues, although tyrosine residues also undergo phosphorylation. Many of the currently applied methods for the detection and localization of protein phosphorylation sites are mass spectrometry-based and are biased against the analysis of tyrosine-phosphorylated residues because of the stability and low reactivity of phosphotyrosines. To overcome this lack of sensitive methods for the detection of phosphotyrosine-containing peptides, we have recently developed a method that is not affected by the more predominant threonine or serine phosphorylation within cells. It is based on the specific detection of immonium ion of phosphotyrosine at 216.043 Da and does not require prior knowledge of the protein sequence. In this report, we describe the first application of this new method in a proteomic strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and one-dimensional gel electrophoresis, we have identified 10 proteins in the epidermal growth factor receptor signaling pathway, of which 8 have been shown previously to be involved in epidermal growth factor signaling. Most importantly, in addition to several known tyrosine phosphorylation sites, we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2, most of which were not predicted to be phosphorylated. Because of its sensitivity and selectivity, this approach will be useful in proteomic approaches to study tyrosine phosphorylation in a number of signal transduction pathways.  相似文献   

9.
Plant pathogenic bacteria, phytoplasmas, viruses and viroids are difficult to control, and preventive measures are essential to minimize the losses they cause each year in different crops. In this context, rapid and accurate methods for detection and diagnosis of these plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools being those based on PCR or RT-PCR now routinely applied worldwide. Nucleic acid extraction is still necessary in many cases and in practice inhibition problems are decreasing the theoretical sensitivity of molecular detection. For these reasons, integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques supported by different biological principles are advisable. Overall, molecular techniques based on different types of PCR amplification and very especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture. Other technologies, such as isothermal amplification, microarrays, etc. have great potential, but their practical development in plant pathology is still underway. Despite these advances, there are some unsolved problems concerning the detection of many plant pathogens due to their low titre in the plants, their uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research based on genomic advances and innovative detection methods as well as better knowledge of the pathogens' lifecycle, will facilitate their early and accurate detection, thus improving the sanitary status of cultivated plants in the near future.  相似文献   

10.
目的:探究Mi R-935调控胃癌SGC7901细胞的增殖和浸润与Notch1基因表达的关系。方法:分别检测40例正常人胃粘膜组织与40例胃印戒细胞癌的Notch1表达情况,并分析胃印戒细胞癌组织中Notch1表达与患者年龄、性别、组织进展程度、TNM分期、有无淋巴结转移的关系;采用Mi R-935转染体外培养的SGC7901细胞系,检测Notch1的表达情况,其后采用Mi R-935抑制剂处理,通过Transwell实验检测胃癌细胞的侵袭能力,细胞划痕实验检测胃癌细胞迁移能力。结果:正常人胃粘膜组织中Notch1表达呈阴性,而胃印戒细胞癌组织中Notch1表达呈阳性;Notch1的表达与胃印戒细胞癌的TNM分期、有无淋巴结转移存在着显著的相关性;转染Mi R-935的SGC7901细胞Notch1表达明显上调,采用Mi R-935抑制剂处理后,Notch1的表达显著下降。结论:Mi R-935可能通过调控Notch1的表达调控胃癌的扩增和浸润。  相似文献   

11.
Protozoa of the genus Leishmania are the causative agents of leishmaniosis. Although the polymerase chain reaction (PCR) has proved very effective in the detection of Leishmania DNA, a standardized method does not exist. In this study we attempt a comparative evaluation between one real time PCR (Method D), two in-house (Methods A and C), and a commercially available PCR assay (Method B) for the detection of Leishmania DNA, in order to support reliable diagnostic investigation of leishmaniosis. This evaluation was performed in regard to relative specificity and sensitivity, minimum detection limit (MDL), repeatability and reproducibility using cultured isolates and clinical samples. All the methods under study produced the expected result with the positive and negative controls. However with regard to clinical samples, Method C showed a statistically significant higher level of positivity. Relative sensitivity and specificity of Methods A, B and D in comparison to C was calculated respectively at 50.7%, 43%, 40%, and 90.8%, 93.4% and 89.5%. The MDL for Methods A-D was defined respectively at 30.7, 5, 3.7, and 5 promastigotes/ml. Repeatability and reproducibility were excellent in all cases with only the exception of Method A regarding reproducibility with a different brand of PCR reagents. The results that were recorded indicate that evaluation of PCR assays before their application for research and clinical diagnosis can provide useful evidence for their reliable application. Within this context the use of internal amplification controls and the confirmation of the specificity of the amplification product is recommended.  相似文献   

12.
This paper presents the results of a dendrochronological and dendroclimatological study of birch growth in northern Iceland. Two cores from 36 trees were extracted at two locations and a 110-year-long chronology was built, which is near the maximum possible chronology length for living trees in the study area, considering the growing conditions in Iceland and the history of forest management. Birch has a very good dendroclimatological potential. Pointer year analysis indicates that above-average summer temperature and above-average snowpack result in a positive pointer year (wide ring), while below-average summer temperature and a dry winter result in a negative pointer year (narrow ring). Bootstrapped response functions confirm that June and July above-average temperature positively influences tree-ring width. This climate/growth relationship is stable throughout the 1930–2002 period. Yet the influence of above-average temperature is slightly more important in June than in July. It turned out that birch tree rings are challenging to work with in dendrochronology, considering that their boundaries are difficult to distinguish. Moreover, frequent early summer frosts, insect attacks, and sheep grazing affect birch wood formation and development of tree rings in Iceland, making dendrochronological work even harder.  相似文献   

13.
Questions: (1) How do extreme climatic events and climate variability influence radial growth of conifers (silver fir, Norway spruce, Scots pine)? (2) How do elevation and soil water capacity (SWC) modulate sensitivity to climate? Location: The sampled conifer stands are in France, in western lowland and mountain forests, at elevations from 400 to 1700 m, and an SWC from 50 to 190 mm. Methods: We established stand chronologies for total ring width, earlywood and latewood width for the 33 studied stands (985 trees in total). Responses to climate were analysed using pointer years and bootstrapped response functions. Principal component analysis was applied to pointer years and response function coefficients in order to elucidate the ecological structure of the studied stands. Results: Extreme winter frosts are responsible for greater growth reductions in silver fir than in Norway spruce, especially at the upper elevation, while Scots pine was the least sensitive species. Exceptional spring droughts caused a notable growth decrease, especially when local conditions were dry (altitude<1000 m and SWC<100 mm for silver fir, western lowlands for Scots pine). Earlywood of silver fir depended on previous September and November and current‐year February temperature, after which current June and July water supply influenced latewood. Earlywood of Norway spruce was influenced by previous September temperature, after which current spring and summer droughts influenced both ring components. In Scots pine, earlywood and latewood depended on the current summer water balance. Local conditions mainly modulated latewood formation. Conclusions: If the climate becomes drier, low‐elevation dry stands or trees growing in western lowlands may face problems, as their growth is highly dependent on soil moisture availability.  相似文献   

14.
目的:研究胃癌耐药细胞及其亲本细胞中长链非编码RNA UCA1的表达差异,探讨UCA1在胃癌多药耐药中的作用。方法:通过实时荧光定量PCR(q RT-PCR)检测胃癌耐药细胞SGC7901/ADR、SGC7901/VCR及其亲本细胞SGC7901中UCA1的表达差异;通过si RNA转染降低SGC7901/ADR中UCA1表达,MTT法检测细胞半数抑制浓度(IC50)的变化,流式细胞仪检测细胞凋亡变化。结果:QRT-PCR结果显示,UCA1在SGC7901/ADR和SGC7901/VCR胃癌耐药细胞表达显著高于SGC7901胃癌亲本细胞;MTT实验表明,干扰UCA1的SGC7901/ADR相对于阴性对照(NC)组的IC50显著降低;凋亡检测结果显示,在相同剂量化疗药物作用下,干扰UCA1后SGC7901/ADR凋亡率显著高于NC组;Western blot证实,干扰UCA1表达可显著降低BCL-2蛋白表达。结论:长链非编码RNA UCA1在胃癌耐药细胞表达显著升高,干扰UCA1表达可明显逆转胃癌耐药,UCA1可作为治疗胃癌耐药的重要分子靶标。  相似文献   

15.
A fundamental decision in biodiversity assessment is the selection of one or more study taxa, a choice that is often made using qualitative criteria such as historical precedent, ease of detection, or available technical or taxonomic expertise. A more robust approach would involve selecting taxa based on the a priori expectation that they will provide the best possible information on unmeasured groups, but data to inform such hypotheses are often lacking. Using a global meta‐analysis, we quantified the proportion of variability that each of 12 taxonomic groups (at the Order level or above) explained in the richness or composition of other taxa. We then applied optimization to matrices of pairwise congruency to identify the best set of complementary surrogate groups. We found that no single taxon was an optimal surrogate for both the richness and composition of unmeasured taxa if we used simple methods to aggregate congruence data between studies. In contrast, statistical methods that accounted for well‐known drivers of cross‐taxon congruence (spatial extent, grain size, and latitude) lead to the prioritization of similar surrogates for both species richness and composition. Advanced statistical methods were also more effective at describing known ecological relationships between taxa than simple methods, and show that congruence is typically highest between taxonomically and functionally dissimilar taxa. Birds and vascular plants were most frequently selected by our algorithm as surrogates for other taxonomic groups, but the extent to which any one taxon was the ‘optimal’ choice of surrogate for other biodiversity was highly context‐dependent. In the absence of other information – such as in data‐poor areas of the globe, and under limited budgets for monitoring or assessment – ecologists can use our results to assess which taxa are most likely to reflect the distribution of the richness or composition of ‘total’ biodiversity.  相似文献   

16.
Age is a fundamental aspect of animal ecology, but is difficult to determine in many species. Humpback whales exemplify this as they have a lifespan comparable to humans, mature sexually as early as 4 years and have no reliable visual age indicators after their first year. Current methods for estimating humpback age cannot be applied to all individuals and populations. Assays for human age have recently been developed based on age‐induced changes in DNA methylation of specific genes. We used information on age‐associated DNA methylation in human and mouse genes to identify homologous gene regions in humpbacks. Humpback skin samples were obtained from individuals with a known year of birth and employed to calibrate relationships between cytosine methylation and age. Seven of 37 cytosines assayed for methylation level in humpback skin had significant age‐related profiles. The three most age‐informative cytosine markers were selected for a humpback epigenetic age assay. The assay has an R2 of 0.787 (P = 3.04e?16) and predicts age from skin samples with a standard deviation of 2.991 years. The epigenetic method correctly determined which of parent–offspring pairs is the parent in more than 93% of cases. To demonstrate the potential of this technique, we constructed the first modern age profile of humpback whales off eastern Australia and compared the results to population structure 5 decades earlier. This is the first epigenetic age estimation method for a wild animal species and the approach we took for developing it can be applied to many other nonmodel organisms.  相似文献   

17.
Methods that predict membrane helices have become increasingly useful in the context of analyzing entire proteomes, as well as in everyday sequence analysis. Here, we analyzed 27 advanced and simple methods in detail. To resolve contradictions in previous works and to reevaluate transmembrane helix prediction algorithms, we introduced an analysis that distinguished between performance on redundancy-reduced high- and low-resolution data sets, established thresholds for significant differences in performance, and implemented both per-segment and per-residue analysis of membrane helix predictions. Although some of the advanced methods performed better than others, we showed in a thorough bootstrapping experiment based on various measures of accuracy that no method performed consistently best. In contrast, most simple hydrophobicity scale-based methods were significantly less accurate than any advanced method as they overpredicted membrane helices and confused membrane helices with hydrophobic regions outside of membranes. In contrast, the advanced methods usually distinguished correctly between membrane-helical and other proteins. Nonetheless, few methods reliably distinguished between signal peptides and membrane helices. We could not verify a significant difference in performance between eukaryotic and prokaryotic proteins. Surprisingly, we found that proteins with more than five helices were predicted at a significantly lower accuracy than proteins with five or fewer. The important implication is that structurally unsolved multispanning membrane proteins, which are often important drug targets, will remain problematic for transmembrane helix prediction algorithms. Overall, by establishing a standardized methodology for transmembrane helix prediction evaluation, we have resolved differences among previous works and presented novel trends that may impact the analysis of entire proteomes.  相似文献   

18.
Recent new data on the important role played by lipopolysaccharides (endotoxin) of Gram negative bacteria in physiology and pathogenesis of the most important human infectious and noninfectious diseases testify to the necessity of wide clinical trials of different methods for LPS detection in blood and other physiological fluids. Among presently available diagnostic methods for endotoxinemia detection, the highly sensitive LAL (Limulus Amebocyte Lysate) test in various modifications is most widely used. The LAL test is known to be non-specific, however many drawbacks of this test have been successfully overcome. The results of clinical studies on the determination of the LPS activity in the systemic blood stream and antibody titers to its most common determinants, as well as the reserves of endotoxin binding with granulocytes give grounds for optimistic evaluation of the future studies on the role of LPS in human physiology and pathology. In clinical practice both positive sides and drawbacks of the presently known methods for LPS detection, including the LAL test, must be borne in mind for the complex evaluation of endotoxinemia levels.  相似文献   

19.
We examined the suitability of two deciduous arctic shrubs (Salix glauca L. and Betula nana L., hereafter Salix and Betula, respectively) for dendroclimatological analysis at two sites in West Greenland. Chronologies were successfully cross-dated, and the oldest covered the period 1954–2010 (Expressed Population Signal [EPS] > 0.85, 1977–2010). Distinctive pointer years, also called micro-rings, including those from a known outbreak of the irruptive moth Eurois occulta L. (Lepidoptera: Noctuidae) that peaked in 2005, assisted in the dating process. Climate-growth analyses were performed in two ways: first, using correlation analysis between residual site-level chronologies and monthly and seasonal climate data, and second, using linear mixed effects models (LMM) with seasonal climate data and standardized chronologies for each individual. We used climate data for the current and previous years for a given growth ring for both analyses. Both analyses revealed differences in climate-growth response among species and among sites of contrasting topography. Salix ring widths from south facing slopes correlated positively with current year’s summer temperatures, while those on gentle slopes associated negatively with current year’s spring precipitation. Betula was only sampled at one site (flat), and displayed associations with temperature and precipitation in spring. Results from the LMM largely corroborated the correlations for Betula and Salix on south facing slopes. Salix at the flat site displayed significant associations with a large number of climate variables, most strongly previous year’s summer and autumn temperatures, though precipitation in multiple seasons of the current and prior year did a better job of accounting for the variation in the data. Many dendrochronological studies in the Arctic illustrate clear summer temperature responses, but the majority were conducted on a single topographic position. Due to the heterogeneity of West Greenland’s landscapes, it is important to examine individuals from varying topographies. We found that samples collected from south facing slopes do appear to respond positively to summer temperatures, while those on shallow slopes respond to a wider array of seasonal temperature and precipitation parameters. Accounting for these species and topographic differences, when sampling, is imperative for improving our understanding of how plant communities in the Arctic will respond to ongoing and expected warming.  相似文献   

20.
Abstract: The genus Nothofagus in New Zealand and Australia exhibits strong mast seeding (i.e. highly variable seed crops between years). Seed crop variation is synchronized within and between species over large spatial scales, and results in greatly increased wind pollination efficiency which could provide a selective benefit favouring the maintenance of mast seeding. However, the null hypothesis (that plants simply match their reproductive effort to the variable resources available each year) has not been tested in Nothofagus. Here we use a 33‐year dataset on seedfall and wood ring increments for 19 individual Nothofagus truncata trees at Orongorongo, New Zealand, to test for the presence of switching (exaggeration of seedfall variability by diverting resources into, then out of, reproduction). A generalized least squares model explained 40.7% of the variance in standardized ring widths, using six weather variables (absolute minimum temperatures in March (lag 0) April (lag 0 and lag 1), May (lag 0) and rainfall in November and February (lag 0) ) and seedfall. Seedfall had a negative relationship with the current year's ring widths even after controlling for all significant weather variables. This shows that switching is occurring in N. truncata within individuals among years, and therefore that masting in this species is the result of selective forces such as increased wind pollination efficiency. As this result has been demonstrated for very few masting species, we call for this test to be applied more widely.  相似文献   

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