Identification of chilling and heat requirements of cherry trees—a statistical approach

Most trees from temperate climates require the accumulation of winter chill and subsequent heat during their dormant phase to resume growth and initiate flowering in the following spring. Global warming could reduce chill and hence hamper the cultivation of high-chill species such as cherries. Yet determining chilling and heat requirements requires large-scale controlled-forcing experiments, and estimates are thus often unavailable. Where long-term phenology datasets exist, partial least squares (PLS) regression can be used as an alternative, to determine climatic requirements statistically. Bloom dates of cherry cv. ‘Schneiders späte Knorpelkirsche’ trees in Klein-Altendorf, Germany, from 24 growing seasons were correlated with 11-day running means of daily mean temperature. Based on the output of the PLS regression, five candidate chilling periods ranging in length from 17 to 102 days, and one forcing phase of 66 days were delineated. Among three common chill models used to quantify chill, the Dynamic Model showed the lowest variation in chill, indicating that it may be more accurate than the Utah and Chilling Hours Models. Based on the longest candidate chilling phase with the earliest starting date, cv. ‘Schneiders späte Knorpelkirsche’ cherries at Bonn exhibited a chilling requirement of 68.6?±?5.7 chill portions (or 1,375?±?178 chilling hours or 1,410?±?238 Utah chill units) and a heat requirement of 3,473?±?1,236 growing degree hours. Closer investigation of the distinct chilling phases detected by PLS regression could contribute to our understanding of dormancy processes and thus help fruit and nut growers identify suitable tree cultivars for a future in which static climatic conditions can no longer be assumed. All procedures used in this study were bundled in an R package (‘chillR’) and are provided as Supplementary materials. The procedure was also applied to leaf emergence dates of walnut (cv. ‘Payne’) at Davis, California.     

中国北方主产地苹果冷热积累变化及其对始花期的影响

开展气候变化背景下苹果冷热积累变化及其对始花期的影响研究,对指导苹果种植及生产具有重要意义。本研究选取山东福山、山西万荣、甘肃西峰和新疆阿克苏代表中国北方苹果主产地,利用1996—2018年红富士苹果的始花期观测资料和逐时气温数据,采用动态模型、生长度小时模型分别计算逐日冷积累量(CP)和热积累量(GDH),并利用偏最小二乘回归法,对逐日冷、热积累量和各地苹果始花期进行相关分析,以明确各地苹果冷、热积累起止日期和积累量,以及冷、热积累期内温度变化对始花期的影响规律。结果表明: 我国北方主产地苹果冷积累时段集中于10月1日前后至2月中下旬或3月中旬,积累量为74.1～89.3 CP;热积累时段集中于1月下旬前后至始花期,积累量为4010～5770 GDH。西峰和阿克苏冷积累期内平均气温每升高1 ℃,冷积累量将分别增加3.8和5.0 CP;各地热积累期内平均气温每升高1 ℃,热积累量将增加725～967 GDH。与冷积累期内温度变化的影响效应相比,热积累期内温度变化主控我国北方主产地苹果始花期,且气候变暖总体有利于冷积累期内平均气温较低地区的苹果开花和生产。     

Differences in Heat Requirements of Flower and Leaf Buds Make Hysteranthous Trees Bloom before Leaf Unfolding

To clarify which agroclimatic requirements control the sequential occurrence of flowering and leaf unfolding in hysteranthous plants, Partial Least Squares (PLS) regression analysis was used to identify the chilling and forcing period of leaf and flower buds. The Dynamic Model and the Growing Degree Hour Model were applied to estimate the chilling and heat requirement for leaf unfolding and flowering, based on the phenological records of apricot and mountain peach and daily maximum and minimum temperature data in Beijing during 1963-1988. The results indicated that PLS regression analysis is a useful approach to calculate the chilling and heat requirements of plants when long term phenological observations are available. Leaf and flower buds were found to have similar chilling requirements but different heat requirements, which explained the earlier occurrence of flowering compared to leaf unfolding. The heat requirements of flower buds of apricot and mountain peach were 28297±8762 and 14572±5812 Growing Degree Hours, respectively, while heat requirements of vegetative buds were almost twice as high. In view of the importance and usefulness of phenological observations, species level ground observations in China should be continued and extended.     

叶芽花芽需热量差异导致植物先花后叶

为探究植物先花后叶的影响因素,本研究以1963—1988年间北京地区杏和山桃展叶和始花物候资料及相应的日最高、最低温度数据为基础,利用偏最小二乘回归法确定杏和山桃叶芽及花芽的需冷期和需热期,进而利用动态模型和生长度小时模型分别估算叶芽和花芽的需冷和需热量。结果表明,依据长期物候观测资料,利用偏最小二乘回归法进行植物需冷和需热量的估算非常有效。先花后叶植物叶芽和花芽需冷量几乎相同,需热量的差异是导致植物先花后叶的主要原因。杏和山桃花芽的需热量分别为2829．7±876．2和1457．2±581．2生长度小时,而相应叶芽需热量却是花芽的两倍之多。基于物候观测的重要性及实用性．中国物种水平上的地面观测应得到进一步深入发展。     

Chilling and forcing temperatures interact to predict the onset of wood formation in Northern Hemisphere conifers

The phenology of wood formation is a critical process to consider for predicting how trees from the temperate and boreal zones may react to climate change. Compared to leaf phenology, however, the determinism of wood phenology is still poorly known. Here, we compared for the first time three alternative ecophysiological model classes (threshold models, heat‐sum models and chilling‐influenced heat‐sum models) and an empirical model in their ability to predict the starting date of xylem cell enlargement in spring, for four major Northern Hemisphere conifers (Larix decidua, Pinus sylvestris, Picea abies and Picea mariana). We fitted models with Bayesian inference to wood phenological data collected for 220 site‐years over Europe and Canada. The chilling‐influenced heat‐sum model received most support for all the four studied species, predicting validation data with a 7.7‐day error, which is within one day of the observed data resolution. We conclude that both chilling and forcing temperatures determine the onset of wood formation in Northern Hemisphere conifers. Importantly, the chilling‐influenced heat‐sum model showed virtually no spatial bias whichever the species, despite the large environmental gradients considered. This suggests that the spring onset of wood formation is far less affected by local adaptation than by environmentally driven plasticity. In a context of climate change, we therefore expect rising winter–spring temperature to exert ambivalent effects on the spring onset of wood formation, tending to hasten it through the accumulation of forcing temperature, but imposing a higher forcing temperature requirement through the lower accumulation of chilling.     

A global analysis of the comparability of winter chill models for fruit and nut trees

Many fruit and nut trees must fulfill a chilling requirement to break their winter dormancy and resume normal growth in spring. Several models exist for quantifying winter chill, and growers and researchers often tacitly assume that the choice of model is not important and estimates of species chilling requirements are valid across growing regions. To test this assumption, Safe Winter Chill (the amount of winter chill that is exceeded in 90% of years) was calculated for 5,078 weather stations around the world, using the Dynamic Model [in Chill Portions (CP)], the Chilling Hours (CH) Model and the Utah Model [Utah Chill Units (UCU)]. Distributions of the ratios between different winter chill metrics were mapped on a global scale. These ratios should be constant if the models were strictly proportional. Ratios between winter chill metrics varied substantially, with the CH/CP ratio ranging between 0 and 34, the UCU/CP ratio between −155 and +20 and the UCU/CH ratio between −10 and +5. The models are thus not proportional, and chilling requirements determined in a given location may not be valid elsewhere. The Utah Model produced negative winter chill totals in many Subtropical regions, where it does not seem to be useful. Mean annual temperature and daily temperature range influenced all winter chill ratios, but explained only between 12 and 27% of the variation. Data on chilling requirements should always be amended with information on the location and experimental conditions of the study in which they were determined, ideally including site-specific conversion factors between winter chill models. This would greatly facilitate the transfer of such information across growing regions, and help prepare growers for the impact of climate change.     

梨枣花果期耗水规律及其与茎直径变化的相关分析

设置4个水分处理,研究了4年生梨枣2010年及2011年花果期不同供水条件下土壤水分动态和耗水规律,分析了梨枣日耗水量与茎直径变化间的相关性,建立回归模型.结果表明:(1)2a内各处理梨枣耗水量随土壤供水量的增加而增大,其日耗水量最大值均出现在灌水后1周内;各处理果实膨大期日耗水强度大于开花坐果期.(2)2a内各处理茎直径日变化平均值(MTD)、茎直径日最大值(MXTD)均符合Logistic函数关系,MXTD与MTD在表征梨枣茎秆生长规律方面效果一致,各处理茎直径变化指标(MTD、MXTD)增长率因水分处理的不同而存在差异.(3)高水分(T1处理)条件下茎直径变化指标(MTD、MXTD、MDS(茎直径日最大收缩量)、DG(茎直径日生长量))在表征枣树耗水状况方面不敏感;在低水分(T4处理)条件下,日耗水量与茎直径日最大收缩量(MDS)相关系数较其他3个茎直径变化指标(MTD、MXTD、DG)高且达极显著水平,说明MDS能够更好的表征低水分处理的梨枣耗水规律.在此基础上建立耗水量与茎直径变化回归模型,为评价梨枣耗水状况提供依据.     

Climatic Changes Lead to Declining Winter Chill for Fruit and Nut Trees in California during 1950–2099

Background

Winter chill is one of the defining characteristics of a location''s suitability for the production of many tree crops. We mapped and investigated observed historic and projected future changes in winter chill in California, quantified with two different chilling models (Chilling Hours, Dynamic Model).

Methodology/Principal Findings

Based on hourly and daily temperature records, winter chill was modeled for two past temperature scenarios (1950 and 2000), and 18 future scenarios (average conditions during 2041–2060 and 2080–2099 under each of the B1, A1B and A2 IPCC greenhouse gas emissions scenarios, for the CSIRO-MK3, HadCM3 and MIROC climate models). For each scenario, 100 replications of the yearly temperature record were produced, using a stochastic weather generator. We then introduced and mapped a novel climatic statistic, “safe winter chill”, the 10% quantile of the resulting chilling distributions. This metric can be interpreted as the amount of chilling that growers can safely expect under each scenario. Winter chill declined substantially for all emissions scenarios, with the area of safe winter chill for many tree species or cultivars decreasing 50–75% by mid-21^st century, and 90–100% by late century.

Conclusions/Significance

Both chilling models consistently projected climatic conditions by the middle to end of the 21^st century that will no longer support some of the main tree crops currently grown in California, with the Chilling Hours Model projecting greater changes than the Dynamic Model. The tree crop industry in California will likely need to develop agricultural adaptation measures (e.g. low-chill varieties and dormancy-breaking chemicals) to cope with these projected changes. For some crops, production might no longer be possible.     

近五十年北京板栗始花物候对光合有效辐射变化的响应

与全球范围内气候变暖对植物物候影响研究相比,其他气候因素（如光合有效辐射PAR等）对物候影响报道较少,果树花期物候对光合有效辐射变化响应的研究更是未见报道。本研究以1963—2008年间北京板栗始花物候资料及相应的日光合有效辐射数据为基础,利用偏最小二乘回归法确定了PAR影响板栗始花物候的两个关键阶段,进而分析了两阶段内PAR、温度及相对湿度变化对板栗花期的具体影响。结果表明,北京过去50年两相关阶段内PAR呈显著下降趋势,其中9月24日至次年2月5日问PAR下降对板栗花期提前具有促进作用,可解释12％的花期提前趋势;2月6日至次年5月31日间PAR下降促使花期延迟,但未达显著水平（P〉0．1）。板栗花期提前主要与2月6日至次年5月31日间温度升高有关,其间温度变化可解释41％的花期提前趋势;其次是相对湿度,PAR变化对花期影响较小。鉴于PAR、温度及相对湿度间的互作效应,PAR和相对湿度对花期物候的影响可由温度效应加以解释。     

Response of Chestnut Flowering in Beijing to Photosynthetically Active Radiation Variation and Change in Recent Fifty Years

Climate warming has affected plant phenology throughout the world, but few studies have evaluated plant phenology response to other climate factors (eg. photosynthetically active radiation PAR). In particular, the response of fruit flowering to PAR variation has not been explored yet. Long term (1963-2008) of chestnut (Castanea mollissima Blume) first flowering dates from Beijing, China were related with daily PAR for the 12 months, using Partial Least Squares (PLS) regression analysis. Two relevant phases were identified, during which mean PAR, temperature, and relative humidity (RH) were correlated with flowering dates, respectively. PAR during the both relevant periods decreased significantly in Beijing over the past 50 years. Reduced PAR during 24 September 5 February showed an advance impact on chestnut flowering, and could explain 12% of advance trend in flowering timing. Deceased PAR during 6 February 31 May had a delayed effect on tree flowering, but it was not significant enough to reject the null hypothesis of no impact over time. Advanced flowering of chestnut was mainly determined by increasing temperature between 6 February and 31 May which could explain 41% of flowering trend. Relative humidity variation during this period played secondly important role on tree flowering. Considering the interaction among these three climate factors, the impacts of PAR and RH on flowering timing could partially be attributed to the effects of temperature variation.     

Shifts in the temperature‐sensitive periods for spring phenology in European beech and pedunculate oak clones across latitudes and over recent decades

Spring phenology of temperate trees has advanced worldwide in response to global warming. However, increasing temperatures may not necessarily lead to further phenological advance, especially in the warmer latitudes because of insufficient chilling and/or shorter day length. Determining the start of the forcing phase, that is, when buds are able to respond to warmer temperatures in spring, is therefore crucial to predict how phenology will change in the future. In this study, we used 4,056 leaf‐out date observations during the period 1969–2017 for clones of European beech (Fagus sylvatica L.) and pedunculate oak (Quercus robur L.) planted in 63 sites covering a large latitudinal gradient (from Portugal ~41°N to Norway ~63°N) at the International Phenological Gardens in order to (a) evaluate how the sensitivity periods to forcing and chilling have changed with climate warming, and (b) test whether consistent patterns occur along biogeographical gradients, that is, from colder to warmer environments. Partial least squares regressions suggest that the length of the forcing period has been extended over the recent decades with climate warming in the colder latitudes but has been shortened in the warmer latitudes for both species, with a more pronounced shift for beech. We attribute the lengthening of the forcing period in the colder latitudes to earlier opportunities with temperatures that can promote bud development. In contrast, at warmer or oceanic climates, the beginning of the forcing period has been delayed, possibly due to insufficient chilling. However, in spite of a later beginning of the forcing period, spring phenology has continued to advance at these areas due to a faster satisfaction of heat requirements induced by climate warming. Overall, our results support that ongoing climate warming will have different effects on the spring phenology of forest trees across latitudes due to the interactions between chilling, forcing and photoperiod.     

Differences in atmospheric trees pollen seasons in winter, spring and summer in two European geographic areas, Spain and Italy

The global climate change reported over recent years may prompt changes in the atmospheric pollen season (APS). The aim of this study is to evaluate the possible impact provoked by meteorological conditions variations at different seasons of the year or different geographical areas on APS. Alnus, Betula and Castanea atmospheric pollen seasons and trends during the last 17 years at Ourense and Vigo (Galicia—NW Spain) and Perugia (Italy) were analysed. Possible incidence of the meteorological trends observed in the different cities on the atmospheric pollen seasons and the chill and heat requirements were evaluated. Pollen data from Ourense, Vigo and Perugia (1995–2011) were used. Pollen sampling was performed using LANZONI VPPS 2000 volumetric traps (Hirst in Ann Appl Biol 36:257–265, 1952), placed on top of different buildings at a similar height from the ground. Several methods, dates and threshold temperatures for determining the chill and heat requirements needed to trigger flowering were tested. Different temporary order in the pollination sequence was observed between the three pollen types studied in the three sites. Alnus flowers few days in advance in Ourense respecting to Vigo and 1 month earlier than Perugia. The Betula flowering start date in Ourense and Vigo is almost simultaneous, taking place only 5 days in advance with respect to Perugia. Finally, scarce differences in the APS onset of Castanea were detected between the three cities. The variations observed among the two areas (Umbria, Italy and Galicia, Spain) in the onset of pollen season in the winter or spring flowering trees could be explained by differences in the thermal requirements needed for flowering as consequence of the climatic conditions recorded during the previous period to flowering. The length of the chilling and heat period as well as the thermal requirements obtained showed differences between geographical areas. The chill requirements accumulated were higher in Perugia than Ourense and Vigo. By contrary, the lowest heat accumulation was achieved in Perugia. The observed trends in the APS characteristics and the weather-related parameters were not homogeneous both in the pollen types and sites. The pollen index of Betula and Castanea pollen in Ourense shows a significant trend to increase.     

Climate warming increases spring phenological differences among temperate trees

Climate warming has substantially advanced spring leaf flushing, but winter chilling and photoperiod co‐determine the leaf flushing process in ways that vary among species. As a result, the interspecific differences in spring phenology (IDSP) are expected to change with climate warming, which may, in turn, induce negative or positive ecological consequences. However, the temporal change of IDSP at large spatiotemporal scales remains unclear. In this study, we analyzed long‐term in‐situ observations (1951–2016) of six, coexisting temperate tree species from 305 sites across Central Europe and found that phenological ranking did not change when comparing the rapidly warming period 1984–2016 to the marginally warming period 1951–1983. However, the advance of leaf flushing was significantly larger in early‐flushing species EFS (6.7 ± 0.3 days) than in late‐flushing species LFS (5.9 ± 0.2 days) between the two periods, indicating extended IDSP. This IDSP extension could not be explained by differences in temperature sensitivity between EFS and LFS; however, climatic warming‐induced heat accumulation effects on leaf flushing, which were linked to a greater heat requirement and higher photoperiod sensitivity in LFS, drove the shifts in IDSP. Continued climate warming is expected to further extend IDSP across temperate trees, with associated implications for ecosystem function.     

Chilling and heat requirements for leaf unfolding in European beech and sessile oak populations at the southern limit of their distribution range

With global warming, an advance in spring leaf phenology has been reported worldwide. However, it is difficult to forecast phenology for a given species, due to a lack of knowledge about chilling requirements. We quantified chilling and heat requirements for leaf unfolding in two European tree species and investigated their relative contributions to phenological variations between and within populations. We used an extensive database containing information about the leaf phenology of 14 oak and 10 beech populations monitored over elevation gradients since 2005. In parallel, we studied the various bud dormancy phases, in controlled conditions, by regularly sampling low- and high-elevation populations during fall and winter. Oak was 2.3 times more sensitive to temperature for leaf unfolding over the elevation gradient and had a lower chilling requirement for dormancy release than beech. We found that chilling is currently insufficient for the full release of dormancy, for both species, at the lowest elevations in the area studied. Genetic variation in leaf unfolding timing between and within oak populations was probably due to differences in heat requirement rather than differences in chilling requirement. Our results demonstrate the importance of chilling for leaf unfolding in forest trees and indicate that the advance in leaf unfolding phenology with increasing temperature will probably be less pronounced than forecasted. This highlights the urgent need to determine experimentally the interactions between chilling and heat requirements in forest tree species, to improve our understanding and modeling of changes in phenological timing under global warming.     

Tree Diversity Limits the Impact of an Invasive Forest Pest

The impact of invasive herbivore species may be lower in more diverse plant communities due to mechanisms of associational resistance. According to the “resource concentration hypothesis” the amount and accessibility of host plants is reduced in diverse plant communities, thus limiting the exploitation of resources by consumers. In addition, the “natural enemy hypothesis” suggests that richer plant assemblages provide natural enemies with more complementary resources and habitats, thus promoting top down regulation of herbivores. We tested these two hypotheses by comparing crown damage by the invasive Asian chestnut gall wasp (Dryocosmus kuriphilus) on chestnut trees (Castanea sativa) in pure and mixed stands in Italy. We estimated the defoliation on 70 chestnut trees in 15 mature stands sampled in the same region along a gradient of tree species richness ranging from one species (chestnut monocultures) to four species (mixtures of chestnut and three broadleaved species). Chestnut defoliation was significantly lower in stands with higher tree diversity. Damage on individual chestnut trees decreased with increasing height of neighboring, heterospecific trees. These results suggest that conservation biological control method based on tree species mixtures might help to reduce the impact of the Asian chestnut gall.     

Increased heat requirement for leaf flushing in temperate woody species over 1980–2012: effects of chilling,precipitation and insolation

Recent studies have revealed large unexplained variation in heat requirement‐based phenology models, resulting in large uncertainty when predicting ecosystem carbon and water balance responses to climate variability. Improving our understanding of the heat requirement for spring phenology is thus urgently needed. In this study, we estimated the species‐specific heat requirement for leaf flushing of 13 temperate woody species using long‐term phenological observations from Europe and North America. The species were defined as early and late flushing species according to the mean date of leaf flushing across all sites. Partial correlation analyses were applied to determine the temporal correlations between heat requirement and chilling accumulation, precipitation and insolation sum during dormancy. We found that the heat requirement for leaf flushing increased by almost 50% over the study period 1980–2012, with an average of 30 heat units per decade. This temporal increase in heat requirement was observed in all species, but was much larger for late than for early flushing species. Consistent with previous studies, we found that the heat requirement negatively correlates with chilling accumulation. Interestingly, after removing the variation induced by chilling accumulation, a predominantly positive partial correlation exists between heat requirement and precipitation sum, and a predominantly negative correlation between heat requirement and insolation sum. This suggests that besides the well‐known effect of chilling, the heat requirement for leaf flushing is also influenced by precipitation and insolation sum during dormancy. However, we hypothesize that the observed precipitation and insolation effects might be artefacts attributable to the inappropriate use of air temperature in the heat requirement quantification. Rather than air temperature, meristem temperature is probably the prominent driver of the leaf flushing process, but these data are not available. Further experimental research is thus needed to verify whether insolation and precipitation sums directly affect the heat requirement for leaf flushing.     

Daylength helps temperate deciduous trees to leaf‐out at the optimal time

Global warming has led to substantially earlier spring leaf‐out in temperate‐zone deciduous trees. The interactive effects of temperature and daylength underlying this warming response remain unclear. However, they need to be accurately represented by earth system models to improve projections of the carbon and energy balances of temperate forests and the associated feedbacks to the Earth's climate system. We studied the control of leaf‐out by daylength and temperature using data from six tree species across 2,377 European phenological network ( www.pep725.eu ), each with at least 30 years of observations. We found that, in addition to and independent of the known effect of chilling, daylength correlates negatively with the heat requirement for leaf‐out in all studied species. In warm springs when leaf‐out is early, days are short and the heat requirement is higher than in an average spring, which mitigates the warming‐induced advancement of leaf‐out and protects the tree against precocious leaf‐out and the associated risks of late frosts. In contrast, longer‐than‐average daylength (in cold springs when leaf‐out is late) reduces the heat requirement for leaf‐out, ensuring that trees do not leaf‐out too late and miss out on large amounts of solar energy. These results provide the first large‐scale empirical evidence of a widespread daylength effect on the temperature sensitivity of leaf‐out phenology in temperate deciduous trees.     

Airborne olive pollen in Vigo (Northwest Spain): a survey to forecast the onset and daily concentrations of the pollen season

Temperature is one of the main factors affecting the start of flowering in tree species that flower at the beginning of spring. Knowledge of the chilling and heat needs required by plants to overcome the period of dormancy enables us to determine the onset of pollination, which is of great importance to allergy sufferers. This study attempted to obtain behaviour models with a view to determining both the onset of the olive pollen season and daily pollen concentrations during the pollination period in Vigo. Monitoring was carried out using a Lanzoni VPPS 2000 pollen trap, from 1995 to 2002 inclusive.

Olea pollen is mainly detected during the spring, principally in May. Given the geographical location, the very limited presence of this tree in the study area and the low Olea pollen concentrations detected in northern Spain as a whole, the values recorded here in the atmosphere of Vigo are particularly striking. A strong correlation was observed between total quantity olive pollen collected over the season and rainfall recorded during the second fortnight in February. According to the proposed model, an average of 680 Chilling Hours (CH) are necessary to overcome the chill period and break the state of bud dormancy, and 481 Growth Degree Days (GDD) °C are needed to induce flowering. Models for predicting daily mean pollen concentrations combine temperature and the previous days' pollen concentrations as predictor variables to provide a high level of prediction.     

From observations to experiments in phenology research: investigating climate change impacts on trees and shrubs using dormant twigs

Background and Aims Climate change is advancing the leaf-out times of many plant species and mostly extending the growing season in temperate ecosystems. Laboratory experiments using twig cuttings from woody plant species present an affordable, easily replicated approach to investigate the relative importance of factors such as winter chilling, photoperiod, spring warming and frost tolerance on the leafing-out times of plant communities. This Viewpoint article demonstrates how the results of these experiments deepen our understanding beyond what is possible via analyses of remote sensing and field observation data, and can be used to improve climate change forecasts of shifts in phenology, ecosystem processes and ecological interactions.Scope The twig method involves cutting dormant twigs from trees, shrubs and vines on a single date or at intervals over the course of the winter and early spring, placing them in containers of water in controlled environments, and regularly recording leaf-out, flowering or other phenomena. Prior to or following leaf-out or flowering, twigs may be assigned to treatment groups for experiments involving temperature, photoperiod, frost, humidity and more. Recent studies using these methods have shown that winter chilling requirements and spring warming strongly affect leaf-out and flowering times of temperate trees and shrubs, whereas photoperiod requirements are less important than previously thought for most species. Invasive plant species have weaker winter chilling requirements than native species in temperate ecosystems, and species that leaf-out early in the season have greater frost tolerance than later leafing species.Conclusions This methodology could be extended to investigate additional drivers of leaf-out phenology, leaf senescence in the autumn, and other phenomena, and could be a useful tool for education and outreach. Additional ecosystems, such as boreal, southern hemisphere and sub-tropical forests, could also be investigated using dormant twigs to determine the drivers of leaf-out times and how these ecosystems will be affected by climate change.