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Killing me slowly: Harsh environment extends plant maximum life span
Accepted Manuscript Title: Killing me slowly: harsh environment extends plant maximum life span Authors: Sergey Rosbakh, Peter Poschlod PII: DOI: Reference:
S1439-1791(17)30203-7 https://doi.org/10.1016/j.baae.2018.03.003 BAAE 51095
To appear in: Received date: Revised date: Accepted date:
7-7-2017 10-3-2018 10-3-2018
Please cite this article as: Rosbakh, Sergey., and Poschlod, Peter., Killing me slowly: harsh environment extends plant maximum life span.Basic and Applied Ecology https://doi.org/10.1016/j.baae.2018.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Killing me slowly: harsh environment extends plant maximum life span
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Running headline: plant maximum life span along an elevational gradient
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Sergey Rosbakh* and Peter Poschlod
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University of Regensburg, Ecology and Nature Conservation Biology, Institute
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of Plant Sciences, D-93040 Regensburg, Germany
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*Corresponding author. Tel.: +49-941-943-3105; fax: +49-941-943-3106.
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E-mail address: [email protected].
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Abstract
Life span (the age of death for individuals that survived the establishment phase)
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is a key trait in plant life history. Despite its importance for understanding plantenvironment relationships, there are still numerous substantial knowledge gaps about variation in plant maximum life spans and the ecological processes underlying these patterns.
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Based on plant age data obtained by means of herbochronology, we analysed patterns of intraspecific plant maximum life span variation in three perennial species (Campanula scheuchzeri, Helianthemun nummularium and Lotus
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corniculatus) along environmental gradients of mean annual temperature, soil depth and soil nutrients. This variation was compared with predictions from the ‘death-by-starvation hypothesis’ proposed by Hans Molisch in 1938, an unjustly forgotten ‘extrinsic’ theory on plant life span variation.
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Our study suggests that plant age variation within populations responds
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sensitively to growing conditions. The most important finding is that mean
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annual temperature or environmental conditions related to it seems to be a
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driving factor for intraspecific variation in plant maximum life span in all
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species studied. Despite large within-population variation, individuals of C.
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scheuchzeri, L. corniculatus and H. nummularium generally had a longer life span under colder climates (uplands in our case). In addition, soil depth (as a
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proxy for habitat susceptibility to drought) was found to have a significant
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positive effect on the age values in the case of C. scheuchzeri. These findings, therefore, are in line with Molisch’ “death-by-starvation
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hypothesis”: extended maximum life span results from reduced production of sink tissues and slow vegetative growth. We conclude that the analysis of plant life span adjustments along gradients of environmental factors can considerably
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contribute to our understanding of how plants may cope with changing environmental conditions, e.g., due to global change.
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Keywords: annual ring, elevational gradient, European Alps, herbochronology, intraspecific variation, longevity
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Introduction
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Life span, or the expected age of death for individuals that survived the
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establishment phase, is a key trait in the life history of plants. It is a keystone of
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important basic ecological concepts, such as r- and K-selection (Pianka 1970;
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MacArthur & Wilson 2015), C-S-R model (Grime 2002) and Ramensky-
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Rabotnov V-E-P strategies (Ramensky 1938; Rabotnov 1975). In population biology, this trait (usually based on a classification into ontogenetic stages or
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size classes) has been traditionally used to evaluate the age structure of
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populations and their successional stages (this topic has been reviewed by Schweingruber and Poschlod (2005)). More applied studies employ plant life
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span as an estimate for the potential effects of land use and habitat quality on population structure (Dietz & Ullmann 1998; Rixen et al. 2004; Poschlod et al. 2011). The high potential of this trait to help with reconstruction of historical events in a certain site has also been demonstrated (Bégin & Payette 1991). The 3
recent debate on the potential effects of present and future climate change on plant performance has been drawing researchers’ attention to the importance of (maximal) plant life span. It is believed that the extended longevity of plants is
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able to increase persistence of populations and thus affect community stability and vegetation resilience (von Arx et al. 2006; de Witte & Stöcklin 2010).
Despite its importance, data on the age of a plant or even the potential maximum age of a plant species as a whole are still one of the least accessible parameters
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in the life history of plants, especially in herbaceous plants (Dietz &
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Schweingruber 2002). In the last twenty years, great efforts have been made to
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extract plant age data from scattered literature sources (Bender et al. 2000;
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Poschlod et al. 2003), to establish new methods for age estimation (Dietz &
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Ullmann 1997; de Witte et al. 2010) and collect new data on plant age (Dietz &
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Fattorini 2002; Schweingruber & Poschlod 2005). Based on the actual age estimations, plant life span ranges from a few weeks in some annuals, such as
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Arabidopsis thaliana, to several thousands of years in some clonal herbs and
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trees (e.g. 80600 years for Populus tremuloides; de Woody et al. 2008; Nobis and Schweingruber 2013). When it comes to Central European flora,
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Schweingruber and Poschlod (2005) report that estimated maximum life span for herbs (Trifolium alpinum), dwarf shrubs (Rhododendron hirsutum) and shrubs (Juniperus communis ssp. nana) was 50, 132 and 352 years, respectively. Yet, the underlying reasons for that wide variation in plant life span still remain a puzzle. 4
Several theories have been developed to explain organism senescence in general and physiological and environmental restrictions of maximum life span in particular. Even though these theories have implicitly focused on unitary
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animals, which differ from plants structurally and functionally, or had a human bias in mind (Salguero-Gomez et al. 2013), they could also be used as a foundation for discussions as to why plants die and why plant species differ in
their maximum life span. Among the intrinsic factors that may limit a plant’s
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lifespan are telomere shortening (Kilian et al. 1995; Flanary & Kletetschka
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2005), specific resistance to pathogens (Larson 2001) and somatic mutations
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with negative effects caused by reactive oxygen species or free radicals;
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parameters that tend to build up with age (Warren 2009; de Pinto et al. 2012). Williams (1957) proposed that an organism dies due to trade-offs in gene
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expression between beneficial effects early in life vs. detrimental effects late in
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life (antagonistic pleiotropy). In its turn, Kirkwood’s theory of disposable soma
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(Kirkwood 1977) states that physiological deterioration late in life and consequent organism death is caused by the ‘confluence’ of trade-offs between
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survival and reproduction on the one hand, and the possible limited availability
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of resources on the other. According to this theory, reproduction early in life may exhaust the resources of a plant, thereby making it more prone to stressrelated sources of mortality (Ehrlén & Lehtilä 2002). However, despite the fact that these theories provide a framework for the evolution or maintenance of senescence, they still fail to explain why there are great differences in patterns of 5
senescence among plants and animals that lead to differences in longevity (Borges 2009). In this regard, ‘extrinsic’ theories on senescence are more specific and assume
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that environmental stress in different forms restricts life span. The most central of them, the ‘death-by-starvation hypothesis’, proposed by Molisch (1938), states that a plant dies due to exhaustion or starvation of its source organs (e.g.
photosynthetic leaves and stems) caused by the demands of sink tissues, such as
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flowers, developing seeds, tubers and other structures that accumulate storage
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compounds, such as expanding leaves and branches during vigorous vegetative
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growth (Thomas 2013). According to this theory, environmental stress in the
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form of single events preventing seed set, such as frost or heat spells, drought, etc., greatly promotes vegetative growth and extends longevity (e.g. Noodén &
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Penney 2001). Furthermore, vegetative growth reduction caused by a short
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growing season and/or cold temperatures, or by desiccation and mineral nutrient
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shortage common in stressful environments, such as alpine and arctic habitats, were also thought to extend life span (Körner 1999; Larson 2001). According to
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studies on ‘cost of reproduction’, i.e. trade-offs between reproduction and other
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life-history traits, the positive effect of decreasing environmental favourability on plant longevity is due to reduction in reproductive effort and increase in somatic investment (reviewed in detail by Obeso 2002 and Laiolo and Obeso 2017). Similarly, the dietary restriction theory (Mair & Dillin 2008) states that moderate environmental stress also extends plant life span, by inducing 6
protective mechanisms (e.g. expression of heat shock proteins) that enhance immediate survival but might also extend life more generally by altering the rate of senescence. Chronic severe stress has the opposite effect, possibly through
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continuously elevated metabolism and mobilization of energy reserves (Monaghan et al. 2008). A valuable extension of the aforementioned theories is
a hypothesis that plant size plays an important role in longevity, too. Most perennial plants get bigger as they grow older, and of course organisms that are
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large and old will also be weather-beaten, a visible signifier of aging (Thomas
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2013). Taken together, unfavourable environmental conditions retarding plant
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vegetative growth would also extend plant life span.
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It is a hard task to test these theories, because accurate quantitative data are scarce in the current landscape of research into plant senescence and longevity.
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Although some information on plant life span variation and corresponding traits
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(e.g. telomere length, frequency of somatic mutations; Kilian et al. 1995;
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Flanary et al. 2005; Warren 2009; de Pinto et al. 2012) support several ‘intrinsic’ theories, inadequate efforts have been made to estimate environmental
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effects on maximum plant life span (however, see Schweingruber and Poschlod
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2005; von Arx et al. 2006; Nobis and Schweingruber 2013). Therefore, the main objective here was to estimate maximal plant life span variation along environmental gradients. More specifically, we analysed patterns of intraspecific plant maximum life span variation in three perennial species along environmental gradients of temperature, soil depth and soil nutrients, comparing 7
this variation with predictions from the ‘death-by-starvation hypothesis’ (Molisch 1938). In line with our hypothesis, we expected maximum plant life span to increase in harsh environments characterized by low temperatures and/or
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nutrient-poor soils, as well as in habitats with shallow soils (soil depth is considered to be a proxy for habitat susceptibility to drought).
In order to test our hypothesis, an elevational gradient was selected as a study system, for the reason that it is one of the most powerful ‘natural experiments’
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for exploring macroecological mechanisms of plant–climate interactions
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(Körner 2007). The large elevational ranges occurring across comparatively
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small distances in high mountains represent complex gradients of several
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environmental factors, such as atmospheric pressure, CO2 content, temperature,
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the length of the vegetation period, and nutrient availability, all of which
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decrease with increasing elevation. At the same time, annual precipitation, frequency of frost during the vegetation period, and solar radiation tend to
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increase (Körner 1999; von Arx et al. 2006). Taken together, these changes
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usually make plant growing conditions more severe at higher elevations
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(Tranquillini 1964; Billings & Mooney 1968; Körner 1999).
Materials and methods Study site
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Fieldwork was carried out in the Berchtesgaden National Park (Bavaria, Germany), which is located in the Bavarian Alps (northern part of the Calcareous Alps) and is approximately 200 km2 in size. The National Park has a
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typical alpine relief, with steep mountain peaks composed of Triassic lime and dolomite rocks (Marke et al. 2013). The climate in the region is a typical
mountain climate, showing a large decrease in mean annual air temperatures from +7 to -2 °C (from 603 to 2713 m a.s.l., respectively). Mean annual
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1500 to 2600 mm per year (Marke et al. 2013).
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precipitation in the region varies from year to year, ranging from approximately
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Study species
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For the purpose of this study, three native perennial species with permanent main roots were investigated: Campanula scheuchzeri, Lotus corniculatus and
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Helianthemun nummularium. Campanula scheuchzeri is a small-sized forb
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(height: up to 30 cm) with a comparatively thin creeping rhizome located mainly
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in the upper soil layer and an upright stem. Lotus corniculatus (height: 5-40 cm) is a herbaceous plant with a strong well-developed taproot; it is typically
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sprawling at the height of the surrounding vegetation. Helianthemum
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nummularium (height: 10-20 cm) is an evergreen clonal dwarf shrub with creeping and upright stems; it has a strong taproot originating from a woody base. In Central Europe, all three species are largely confined to open vegetation on calcareous, dry, nutrient-poor, often skeletal soils.
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The species were chosen for three reasons. First, they all develop clearlydemarcated and reliable annual rings in the secondary root and rhizome xylem (Schweingruber & Poschlod 2005). Second, the species are long-lived, with a
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maximum life span of 18, 16 and 55 years for C. scheuchzeri, L. corniculatus, and H. nummularium, respectively (Schweingruber & Poschlod 2005). Third, in
the study area, all species are abundant and occur over a broad elevational range from 600 to 2300 m a.s.l. Thus, the variation in plant growing conditions along
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with a comparatively high variation in the species’ life span provides a good
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basis for the study of plant age-environment relationships.
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Collection sites
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In the National park, we selected 17, 16 and 11 sample sites for C. scheuchzeri,
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L. corniculatus, and H. nummularium, respectively; the sites represented distinct
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populations distributed along the elevational range (see Appendix A). In order to
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extend the elevational gradient into the lowlands, three additional study sites, Kallmünz (364 m a.s.l.; L. corniculatus, and H. nummularium), Riedenburg (350
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m a.s.l.; L. corniculatus, and H. nummularium) and St. Johann (760 m a.s.l.; H. nummularium), were selected. All the additional study sites represent the same
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type of vegetation (open, nutrient-poor calcareous grasslands), as the alpine sites. Each site, approximately one hectare in size, was characterised in terms of its mean annual temperature, soil depth (as a proxy for moisture content in the upper soil layer (0-20 cm)) and soil fertility (nitrogen, potassium, phosphorous). 10
Due to the fact that C. scheuchzeri is replaced in lower elevations by the closelyrelated C. rotundifolia, great care was taken to determine species identity of collected individuals of the former species, particularly in the sites H1, H2, H3,
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HO1, WM1-1 and WM2-2 (all located below 1200 m a.s.l.; see Appendix A). Temperature data were obtained from 15 weather stations located in the
National Park (604–1919 m a.s.l.) and five stations from the adjacent region (360–1100 m a.s.l., distances did not exceed 50 km). Mean annual temperature
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(MAT) at each station was calculated over the years 2000–2008; from these data
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lapse rates between elevation and MAT in the study region were determined
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(0.33 °C per 100 m of elevation) to define the MAT at all the collection sites.
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MAT was selected as a proxy for site temperature environment, since it strongly
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correlates with growing season length, mean growing season temperature and
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number of frost days during vegetation period (see Appendix B), factors that potentially affect plant maximum life span (Molisch 1938).
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All soil samples were collected within 2 weeks at the end of the vegetation
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season (end of August to end of September, depending on elevation). At each site, six random wholes were dug, and the soil from a depth of 5–10 cm was
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collected, thoroughly mixed, air dried, sieved (2 mm) and subsequently analysed. Plant available phosphorus and potassium were extracted from the collected soil samples with a calcium acetate lactate solution (VDLUFA 1991). Phosphorus contents were determined using a UV–visible spectrophotometer (Thermo-Spectronic UV-1 model, Thermo Electron Corporation, USA) after 11
visualising the phosphorus content with ammonium heptamolybdate. Potassium contents were determined using an atomic absorption spectrometer (Solaar AA model, Thermo Elemental, USA). Total soil nitrogen content was quantified via
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gas chromatographic measurement with an element analyser (Vario EL III model, Elementar Analysensysteme GmbH, Germany).
Data on soil nitrogen, phosphorus and potassium are presented as the mean values of six replicates per site. Soil depth was estimated by the repeated
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sticking of an iron rod of 0.6 mm diameter into the soil.
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Age determination
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The age of the largest individuals growing in a collection site was used as a
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proxy for maximal plant life span, because previous studies (e.g. Dietz et al. 1998; Erschbamer et al. 2009; Powolny et al. 2016) have clearly demonstrated
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that herbaceous plant size is positively and linearly related to plant age. Owing
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to the special nature conservation status of the study area, it was not possible to
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study the plant size-age relationship for the study species, since it would have required a much larger sample size. We, thus, assumed that such relationship
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also held true for C. scheuchzeri, L. corniculatus and H. nummularium.
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Depending on population size, 10 to 20 of the largest individuals of each target species were selected from each site between 2009 and 2014, carefully excavated and stored in 40% ethanol. In total, 262, 258 and 151 individuals of C. scheuchzeri, L. corniculatus and H. nummularium, respectively, were analysed. 12
The age of the collected individuals was determined by means of herbochronology, a technique adapted from dendrochronology using growth rings in the secondary xylem of the root collars (Schweingruber & Poschlod
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2005). The development of annual rings in the roots of herbaceous species, especially dicotyledonous species outside the tropics with differential growth in different seasons of the year, was found to be rather common (Schweingruber & Poschlod 2005).
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In the laboratory, the main roots (L. corniculatus, H. nummularium) or main
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rhizomes (C. scheuchzeri) were cut using a sledge microtome (Reichert, Austria)
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to produce thin transverse sections (ca.10-40 nm thick). The sections were first
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stained with 1% water solution of Astra blue (Sigma-Aldrich, Germany) for 3 minutes. After rinsing with distilled water, the sections were stained with 1%
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water solution of Safranin (Sigma-Aldrich, Germany) for 30 seconds, followed
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by rinsing with ethanol. The staining causes a red coloration of the lignified cell
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walls (mainly secondary xylem) and a blue coloration of the cell walls rich in pectin (mainly secondary phloem), thus making the annual rings visible. The
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coloured growth rings were counted under a microscope (TS100, Nikon, Japan).
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Three separate radii were analysed for each specimen to account for tangential variation in growth ring width. Means of the three measurements were used as the age of the collected individual (Dietz & Fattorini 2002). Statistical analysis
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To test the relative effects of environmental factors on the estimated ages of the collected largest individuals (proxy for maximum plant life span; response variable), we calculated linear mixed-effects models for each species separately.
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As fixed effects (predictors), we included the MAT of each collection site (both simple and quadratic terms), soil depth, soil phosphorous and soil potassium
contents. Collection site was included in each model as a random effect. Total soil nitrogen content was excluded from the statistical analysis, due to high
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autocorrelation with the MAT, which would violate the model assumptions.
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Collinearity between the remaining predictors was relatively low. The strongest
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correlation was between soil phosphorous and soil potassium in Campanula
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scheuchzeri plots (-0.64), which could be considered as acceptable (Dormann et al. 2013). All predictors were scaled to zero mean and unit variance prior to
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analyses to directly infer the relative effects of the environmental variables from
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their model estimates. Power transformation with an exponent 0.3 was applied
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to the data on plant maximum life span, in order to obtain more normal residuals.
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Models were fit with R software version 3.3.2 (R core development team 2016),
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using the package lme4 (Bates et al. 2015). P-values were calculated with the package lmerTest based on Satterthwaite approximations of the degrees of freedom to test for significance of the fixed effects (Kuznetsova et al. 2014). Additionally, marginal R2 (proportion of variance explained by the fixed effects only) and conditional R2 (proportion of variance explained by both the fixed and 14
random factors) were calculated as a measure of goodness-of-fit of each model (Nakagawa & Schielzeth 2013).
Environmental characteristics of the collection sites
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Results
The data on environmental factors measured are presented in Appendix A.
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Across all sites, MAT decreased along the elevational gradient from 7.04 °C
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(WM1-1 site, 766 m a.s.l.) to 2.00 °C (SchSt site, 2276 m a.s.l.) and had an
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average of 4.52 °C. Soil depth ranged from 8 to 56 cm and did not show any
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relationship with elevation. Soil was most shallow in site HO5 and deepest in
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site HO1. As for soil potassium content, sites ranged from 18.3 mg/kg of soil
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(WM1-1) to 141.1 mg/kg of soil (SchSt), with an average of 60.3 mg/kg of soil; no elevational trend was observed. Values for soil phosphorus content varied
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among the collection sites 10-fold, from 4.1 (WM1-1 site) to 41.8 mg/kg of soil
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(PFK site) and averaged of 15.3 mg/kg of soil. The total soil nitrogen content ranged from 4.6 mg/kg (HO1 site) to 19.0 (PFK site) and averaged of 10.4
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mg/kg of soil. Age variation of the study species The maximum age of collected individuals (‘maximum life span’) was found to be highest in H. nummularium (34 years; HO4 plot) and lowest in C. 15
scheuchzeri (1 year; a few individuals of that age were found mainly in lowland sites). As for single species, maximum and minimum values of plant age were as follows: C. scheuchzeri: 1-11 (average 3.2 years), L. corniculatus: 2-30 (average
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9.0 years), H. nummularium: 3-34 (average 13.4 years). The population medians of the age of collected individuals ranged from 1.5 (WM2-2 site) to 4.5 years
(HO1 site) for C. scheuchzeri, from 3.5 years (H1 site) to 16 years (H6 site) for
L. corniculatus and from 7.5 years (H2 site) to 19 years (HO4 site) for H.
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nummularium.
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Effects of environment on plant age
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A general decrease in maximum age of collected individuals was found along
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the gradient of mean annual temperature (simple term only) in all species
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studied (Table 1; Fig. 1). The decrease per 1 °C MAT was 0.32, 0.83 and 1.57
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years for C. scheuchzeri, L. corniculatus and H. nummularium, respectively.
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MAT explained 7%, 7% and 9% of the variation in age in C. scheuchzeri, L. corniculatus and H. nummularium, respectively. In addition, soil depth was
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found to have a significant positive effect on the age values in the case of C. scheuchzeri (5% of total variation in plant maximum life span; Table 1).
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Variation in soil phosphorous and soil potassium contents did not show any significant effects on age values.
Discussion 16
There is an urgent need for knowledge about variation in plant maximum life spans in general and ecological processes underlying these patterns in particular (Schweingruber & Poschlod 2005; von Arx et al. 2006). To our knowledge, this
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study is one among the very few studies (e.g. Neuffer and Hurka 1986; von Arx et al. 2006; Poschlod et al. 2011) that suggest that age of perennial herbaceous plants at intraspecific level responds sensitively to environmental conditions. Here, we find supporting evidence that high variation in population medians of
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the maximum life span among populations of C. scheuchzeri (1.5 to 4.5 years),
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L. corniculatus (3.5 to 16 years) and H. nummularium (7.5 to 19 years) is due to
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differences in the mean annual temperature of habitats where the plants grow.
Temperature effect on plant life span
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Our results suggest that temperature has a significant effect on intraspecific
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variation in plant maximum life span in C. scheuchzeri, H. nummularium and L.
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corniculatus. Despite large within-population fluctuations, individuals of these three species generally had a longer life span under colder climates, in our case
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in higher elevations. These results are in line with a previously detected general
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trend of increasing adult age with increasing low-temperature stress at intraspecific (Neuffer & Hurka. 1986; von Arx et al. 2006), interspecific (Nobis & Schweingruber 2013), community (Körner 1999) and ecosystem levels (Larson 2001). The findings, therefore, can be interpreted as strong support for the ‘death-by-starvation hypothesis’ (Molisch 1938; Thomas 2002). More 17
specifically, habitats with low MATs (uplands) are characterized by comparatively low temperatures and very high probability of late-frost events during the vegetation period (see Appendix B). Therefore, we think that, in
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order to reduce mortality, plants growing in such habitats might allocate a higher proportion of their resources to vegetative growth, especially in early life stages (Körner 1999; von Arx et al. 2006). In addition, in order to increase the chances
of surviving hazardous low-temperature events, plants from high elevations tend
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to store considerable carbohydrate reserves (Sakai 1987; Rosbakh et al. 2017).
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Because of the trade-off in resource allocation between current plant
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performance and future population fitness (Willems & Dorland 2000; Ehrlén &
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Lehtilä 2002; García & Zamora 2003), such a conservative or stress-tolerant life strategy could lead to a reduced reproductive effort (García et al. 2003; Laiolo et
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al. 2017). Accordingly, limited irregular production of sink tissues (e.g. flowers,
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developing seeds, tubers) would promote vegetative growth and extend the
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longevity of plants growing in cold habitats (Molisch 1938). A long life span is considered to be a compensation for erratic and hazardous seed production,
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which is quite common in alpine and arctic areas (Billings & Mooney. 1968;
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Körner 1999; de Witte et al. 2010). In contrast, when moving along the temperature gradient, environmental favourability gradually increases, thereby leading to a shift in plant life strategy from persistence by longevity to regeneration by seeding (García et al. 2003; Laiolo et al. 2017). Sexual reproduction considerably exhausts the resources of a plant (Willems & Dorland 18
2000; Ehrlén & Lehtilä 2002), thereby making it more prone to stress-related sources of mortality (Obeso 2002). Yet, this conclusion remains speculative as we lack direct measurements of generative and vegetative traits for the study
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species.
Soil nutrient effect on plant life span
The ‘death-by-starvation’ hypothesis (Molisch 1938; Thomas 2002) postulates
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that mineral nutrient shortages should extend plant life span by reduced
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production of sink tissues and slow vegetative growth. However, the pattern of
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increasing plant adult age towards the ‘poorer’ end of the soil nutrient gradient
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was not detected for any of the study species; comparatively old individuals were found in nearly all plots, regardless of their soil phosphorous and/or soil
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potassium contents. There are three possible explanations for the lack of soil
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nutrient effects on plant life span in our study. Firstly, in Central Europe, all the
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tested species occur mainly in calcareous, comparatively nutrient-poor grasslands (see Appendix A). Accordingly, these environmental conditions
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select for plants able to survive and function under low-nutrient stress (Rosbakh
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et al. 2015). Consequently, such slow-growing plants with low resource acquisition are able to sustain their physiological processes with minimal amounts of soil nutrients (Rosbakh et al. 2017). Furthermore, these plants are characterized by the low level of response to increased soil nutrients. We assume, therefore, that neither vegetative growth nor sexual reproduction and in 19
its turn plant life span were affected by soil phosphorus and soil potassium variation among the study populations (8-fold and 11-fold for P and K, respectively). Secondly, soil nutrient deficiency in such habitats usually leads to
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a mycorrhizal infection which confers nutritional benefits to plants (Read & Haselwandter 1981; Bernhardt-Römermann et al. 2011). It is also remarkable
that the levels of mycorrhizal infection tend to increase along the gradients of
environmental stress (e.g. temperature, soil nutrients, competition), thereby
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proportionally reducing these negative effects on plant functioning (e.g. Read
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and Haselwandter 1981; Bernhardt-Römermann et al. 2011).
Der
Heijden
2006; C.
scheuchzeri: Read
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Van
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Since all the study species possess mycorrhiza (L. corniculatus: Scheublin & &
Haselwandter
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1981; H.nummularium: Massaccesi et al. 2015), we suggest that physiological
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processes of the study species were unaffected by the among-site variation in soil P and K levels, which in its turn did not affect plant life span. Thirdly, our
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study does not account for variation in soil ammonium and nitrate contents
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among the populations of the study species, two other important soil nutrients, which affect plant growth and reproduction. In their study on plant life span
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variation at species level, Schweingruber and Poschlod (2005) found a negative relationship between plant age and soil nutrient content (expressed as Ellenberg indicator values). Since it was not feasible to obtain data on the nutrient status of the sites where the populations were sampled, we could not test the hypothesis.
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Therefore, further studies are required to determine the effect of soil nitrogen on plant life span variations at population level. Soil depth (drought) effect on plant life span
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Abiotic stress associated with drought may also affect plant age. Drought limits
plant photosynthetic capacity and therefore growth (Kreyling et al. 2008), and it may as well induce flower sterility and reductions in grain yield (Ekanayake et al. 1989), which, according to Molisch (1938), could also lead to an extended
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plant life span. Despite our prediction that plants growing in habitats with
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shallow soils should live longer, maximum life span in none of the study species
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was negatively affected by soil depth. Surprisingly, trait values of C.
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scheuchzeri were significantly positively affected by soil depth; individuals of
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the species tended to live longer in deeper soils. This positive relationship can be
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explained by high levels of precipitation (960 to 2100 mm precipitation per year; Marke et al. 2013) uniformly distributed over the vegetation period in all the
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sampled populations (except for Kallmünz and Riedenburg; see Appendix A).
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The result of this is that the soil in the majority of the study populations remains continually moist and plants growing there never or very seldom experience
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drought events. Therefore, the question as to whether water limitation extends plant maximum life span at population level remains to be tested in another study system with a more pronounced drought gradient. As for C. scheuchzeri, we speculate that the positive effect of increasing soil depth on maximum life span of this species can be explained by higher competition pressures that the 21
species experiences in sites with deeper soils. Plant distribution in calcareous grasslands, our study system, is limited, among other factors, by shallow soils; more competitive tall fast-growing plants, which usually require larger soil
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volume for their extensive root systems, tend to occur in deeper soils. When occurring in such sites, short-stature slow-growing individuals of C. scheuchzeri
(Rosbakh et al. 2015) are overgrown by taller plants, which can negatively affect their sexual reproduction by limiting the nutrients in soils and worsening the
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light conditions. In order to compensate for reduced reproductive effort and to
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reduce mortality (García et al. 2003), the plant growing in such stressful
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conditions allocates a higher proportion of its resources to vegetative growth
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leading (according to Molisch’s ‘death-by-starvation’ hypothesis) to an extended
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Conclusions
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life span.
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Our study strongly suggests that fluctuations in plant growing conditions affect plant maximum life span, in our case at the population level. These results have
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several theoretical and practical implications. From a theoretical perspective, our
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study is a modest, but important contribution to the ongoing discussion on what factors, intrinsic or extrinsic, restrict whole plant life span. Particularly, we believe that the pattern of increasing plant age along the gradient of MAT is highly supportive to the ‘death-by-starvation’ hypothesis (Molisch 1938; Thomas 2002), an important but, unfortunately, unjustly forgotten concept of 22
plant life span variation. If variations in plant maximum age are indeed generally related to variations in plant growth conditions, the plant ageenvironment relationship could be widely used in plant ecology, including plant
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ecophysiology, plant population ecology, community ecology and conservation biology (Dietz & Fattorini 2002; Salguero-Gomez et al. 2013). For example, the
analysis of plant life span adjustments along gradients of environmental factors
can considerably contribute to our understanding of how plants may cope with
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changing environmental conditions, e.g. due to global change. It has been
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predicted that ongoing temperature rises and increased nitrogen deposition,
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especially in cold habitats, will increase flowering, increase senescence of old
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individuals and alter internal resource ratios (de Witte et al. 2010), factors that according to Molisch (1938) restrict plant life span. Decreased environmental
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stress will inevitably lead to shortened longevity of plants that in its turn could
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potentially decrease the persistence of populations and thus affect community
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stability and vegetation responses to present and future climate change (e.g.
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Ehrlén and van Groenendael 1998; Borges 2009).
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Authors' Contributions PP conceived the ideas and designed methodology; both authors collected and analysed the data; SR led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.
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Data accessibility All data used in this manuscript are available upon request.
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Acknowledgments
We thank participants of the course “Ecology and nature conservation” and
Theresa Lehmair for the help with the plant age identifications and Günter Kolb for the soil analysis. Accommodation and access to the collection sites was
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provided by the Berchtesgaden National Park. The research was funded by
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FORKAST project (TP 12 Poschlod). We thank editor of the Special Issue Niek
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Scheepens, managing editor Klaus Hövemeyer and two anonymous referees for
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their very helpful comments on earlier versions of the manuscript.
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Figures
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Fig. 1
Fig 1. Regressions of back-transformed age of largest individuals (proxy for plant maximum life span; see MM for details) to site mean annual temperatures
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with regard to the three study species. For each species, the regression lines are given (solid line: Helianthemum nummularium, dotted line: Campanula scheuchzeri and dashed line Lotus corniculatus; Table 1.) For the sake of clarity, population means of age of largest individuals were plotted.
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Tables
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Table 1. Effects of environmental factors on maximum life span of Campanula scheuchzeri, Lotus corniculatus and Helianthemum nummularium as deduced from linear mixed-effects models fit by REML t test using Satterthwaite approximations to degrees of freedom. The variables were scaled prior to analysis. The power transformation with an exponent 0.3 was applied to data on plant maximum life span, in order to obtain more normal residuals. SE: standard error; SD: standard deviation. Significance levels: *** p< 0.001, ** p<0.01, * p<0.05. Marginal R² indicates proportion of variance explained by the fixed effects only, whereas conditional R² indicates proportion of variance explained by both the fixed (see Parameter) and random factors (Site). Random effects Species
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Marginal R² = 0.19 Conditional R² = 0.33
Lotus corniculatus
Variance
SD
Parameter
Estimate
SE
P-value
Site Residual
0.01 0.04
0.09 0.19
Intercept MAT MAT² Soil depth Soil phosphorus Soil potassium
1.36 -0.11 0.02 0.09 0 -0.06
0.04 0.04 0.03 0.02 0.04 0.04
<0.01 *** 0.01 ** 0.61 0.01 ** 0.99 0.17
Site Residual
0.01 0.05
0.11 0.22
Intercept MAT MAT² Soil depth Soil phosphorus Soil potassium
1.91 -0.11 -0.02 0.03 -0.04 0.04
0.06 0.05 0.05 0.04 0.05 0.07
<0.01 ***
Site Residual
0.02 0.04
0.13 0.2
Intercept MAT MAT²
2.13 -0.24 -0.07
0.08 0.07 0.07
<0.01 *** 0.01 ** 0.35
ED
Variable
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Campanula scheuchzeri
Fixed effects
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Marginal R² = 0.20 Conditional R² = 0.35
Helianthemum nummularium
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0.03 * 0.74 0.45 0.46 0.55
N U SC RI PT Soil depth Soil phosphorus Soil potassium
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PT
ED
M
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Marginal R² = 0.37 Conditional R² = 0.56
33
-0.06 -0.09 0.14
0.06 0.09 0.08
0.38 0.34 0.12
S1439-1791(17)30203-7 https://doi.org/10.1016/j.baae.2018.03.003 BAAE 51095
To appear in: Received date: Revised date: Accepted date:
7-7-2017 10-3-2018 10-3-2018
Please cite this article as: Rosbakh, Sergey., and Poschlod, Peter., Killing me slowly: harsh environment extends plant maximum life span.Basic and Applied Ecology https://doi.org/10.1016/j.baae.2018.03.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Killing me slowly: harsh environment extends plant maximum life span
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Running headline: plant maximum life span along an elevational gradient
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Sergey Rosbakh* and Peter Poschlod
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University of Regensburg, Ecology and Nature Conservation Biology, Institute
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of Plant Sciences, D-93040 Regensburg, Germany
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*Corresponding author. Tel.: +49-941-943-3105; fax: +49-941-943-3106.
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E-mail address: [email protected].
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Abstract
Life span (the age of death for individuals that survived the establishment phase)
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is a key trait in plant life history. Despite its importance for understanding plantenvironment relationships, there are still numerous substantial knowledge gaps about variation in plant maximum life spans and the ecological processes underlying these patterns.
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Based on plant age data obtained by means of herbochronology, we analysed patterns of intraspecific plant maximum life span variation in three perennial species (Campanula scheuchzeri, Helianthemun nummularium and Lotus
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corniculatus) along environmental gradients of mean annual temperature, soil depth and soil nutrients. This variation was compared with predictions from the ‘death-by-starvation hypothesis’ proposed by Hans Molisch in 1938, an unjustly forgotten ‘extrinsic’ theory on plant life span variation.
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Our study suggests that plant age variation within populations responds
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sensitively to growing conditions. The most important finding is that mean
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annual temperature or environmental conditions related to it seems to be a
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driving factor for intraspecific variation in plant maximum life span in all
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species studied. Despite large within-population variation, individuals of C.
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scheuchzeri, L. corniculatus and H. nummularium generally had a longer life span under colder climates (uplands in our case). In addition, soil depth (as a
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proxy for habitat susceptibility to drought) was found to have a significant
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positive effect on the age values in the case of C. scheuchzeri. These findings, therefore, are in line with Molisch’ “death-by-starvation
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hypothesis”: extended maximum life span results from reduced production of sink tissues and slow vegetative growth. We conclude that the analysis of plant life span adjustments along gradients of environmental factors can considerably
2
contribute to our understanding of how plants may cope with changing environmental conditions, e.g., due to global change.
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Keywords: annual ring, elevational gradient, European Alps, herbochronology, intraspecific variation, longevity
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Introduction
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Life span, or the expected age of death for individuals that survived the
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establishment phase, is a key trait in the life history of plants. It is a keystone of
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important basic ecological concepts, such as r- and K-selection (Pianka 1970;
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MacArthur & Wilson 2015), C-S-R model (Grime 2002) and Ramensky-
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Rabotnov V-E-P strategies (Ramensky 1938; Rabotnov 1975). In population biology, this trait (usually based on a classification into ontogenetic stages or
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size classes) has been traditionally used to evaluate the age structure of
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populations and their successional stages (this topic has been reviewed by Schweingruber and Poschlod (2005)). More applied studies employ plant life
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span as an estimate for the potential effects of land use and habitat quality on population structure (Dietz & Ullmann 1998; Rixen et al. 2004; Poschlod et al. 2011). The high potential of this trait to help with reconstruction of historical events in a certain site has also been demonstrated (Bégin & Payette 1991). The 3
recent debate on the potential effects of present and future climate change on plant performance has been drawing researchers’ attention to the importance of (maximal) plant life span. It is believed that the extended longevity of plants is
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able to increase persistence of populations and thus affect community stability and vegetation resilience (von Arx et al. 2006; de Witte & Stöcklin 2010).
Despite its importance, data on the age of a plant or even the potential maximum age of a plant species as a whole are still one of the least accessible parameters
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in the life history of plants, especially in herbaceous plants (Dietz &
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Schweingruber 2002). In the last twenty years, great efforts have been made to
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extract plant age data from scattered literature sources (Bender et al. 2000;
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Poschlod et al. 2003), to establish new methods for age estimation (Dietz &
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Ullmann 1997; de Witte et al. 2010) and collect new data on plant age (Dietz &
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Fattorini 2002; Schweingruber & Poschlod 2005). Based on the actual age estimations, plant life span ranges from a few weeks in some annuals, such as
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Arabidopsis thaliana, to several thousands of years in some clonal herbs and
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trees (e.g. 80600 years for Populus tremuloides; de Woody et al. 2008; Nobis and Schweingruber 2013). When it comes to Central European flora,
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Schweingruber and Poschlod (2005) report that estimated maximum life span for herbs (Trifolium alpinum), dwarf shrubs (Rhododendron hirsutum) and shrubs (Juniperus communis ssp. nana) was 50, 132 and 352 years, respectively. Yet, the underlying reasons for that wide variation in plant life span still remain a puzzle. 4
Several theories have been developed to explain organism senescence in general and physiological and environmental restrictions of maximum life span in particular. Even though these theories have implicitly focused on unitary
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animals, which differ from plants structurally and functionally, or had a human bias in mind (Salguero-Gomez et al. 2013), they could also be used as a foundation for discussions as to why plants die and why plant species differ in
their maximum life span. Among the intrinsic factors that may limit a plant’s
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lifespan are telomere shortening (Kilian et al. 1995; Flanary & Kletetschka
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2005), specific resistance to pathogens (Larson 2001) and somatic mutations
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with negative effects caused by reactive oxygen species or free radicals;
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parameters that tend to build up with age (Warren 2009; de Pinto et al. 2012). Williams (1957) proposed that an organism dies due to trade-offs in gene
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expression between beneficial effects early in life vs. detrimental effects late in
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life (antagonistic pleiotropy). In its turn, Kirkwood’s theory of disposable soma
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(Kirkwood 1977) states that physiological deterioration late in life and consequent organism death is caused by the ‘confluence’ of trade-offs between
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survival and reproduction on the one hand, and the possible limited availability
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of resources on the other. According to this theory, reproduction early in life may exhaust the resources of a plant, thereby making it more prone to stressrelated sources of mortality (Ehrlén & Lehtilä 2002). However, despite the fact that these theories provide a framework for the evolution or maintenance of senescence, they still fail to explain why there are great differences in patterns of 5
senescence among plants and animals that lead to differences in longevity (Borges 2009). In this regard, ‘extrinsic’ theories on senescence are more specific and assume
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that environmental stress in different forms restricts life span. The most central of them, the ‘death-by-starvation hypothesis’, proposed by Molisch (1938), states that a plant dies due to exhaustion or starvation of its source organs (e.g.
photosynthetic leaves and stems) caused by the demands of sink tissues, such as
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flowers, developing seeds, tubers and other structures that accumulate storage
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compounds, such as expanding leaves and branches during vigorous vegetative
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growth (Thomas 2013). According to this theory, environmental stress in the
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form of single events preventing seed set, such as frost or heat spells, drought, etc., greatly promotes vegetative growth and extends longevity (e.g. Noodén &
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Penney 2001). Furthermore, vegetative growth reduction caused by a short
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growing season and/or cold temperatures, or by desiccation and mineral nutrient
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shortage common in stressful environments, such as alpine and arctic habitats, were also thought to extend life span (Körner 1999; Larson 2001). According to
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studies on ‘cost of reproduction’, i.e. trade-offs between reproduction and other
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life-history traits, the positive effect of decreasing environmental favourability on plant longevity is due to reduction in reproductive effort and increase in somatic investment (reviewed in detail by Obeso 2002 and Laiolo and Obeso 2017). Similarly, the dietary restriction theory (Mair & Dillin 2008) states that moderate environmental stress also extends plant life span, by inducing 6
protective mechanisms (e.g. expression of heat shock proteins) that enhance immediate survival but might also extend life more generally by altering the rate of senescence. Chronic severe stress has the opposite effect, possibly through
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continuously elevated metabolism and mobilization of energy reserves (Monaghan et al. 2008). A valuable extension of the aforementioned theories is
a hypothesis that plant size plays an important role in longevity, too. Most perennial plants get bigger as they grow older, and of course organisms that are
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large and old will also be weather-beaten, a visible signifier of aging (Thomas
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2013). Taken together, unfavourable environmental conditions retarding plant
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vegetative growth would also extend plant life span.
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It is a hard task to test these theories, because accurate quantitative data are scarce in the current landscape of research into plant senescence and longevity.
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Although some information on plant life span variation and corresponding traits
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(e.g. telomere length, frequency of somatic mutations; Kilian et al. 1995;
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Flanary et al. 2005; Warren 2009; de Pinto et al. 2012) support several ‘intrinsic’ theories, inadequate efforts have been made to estimate environmental
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effects on maximum plant life span (however, see Schweingruber and Poschlod
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2005; von Arx et al. 2006; Nobis and Schweingruber 2013). Therefore, the main objective here was to estimate maximal plant life span variation along environmental gradients. More specifically, we analysed patterns of intraspecific plant maximum life span variation in three perennial species along environmental gradients of temperature, soil depth and soil nutrients, comparing 7
this variation with predictions from the ‘death-by-starvation hypothesis’ (Molisch 1938). In line with our hypothesis, we expected maximum plant life span to increase in harsh environments characterized by low temperatures and/or
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nutrient-poor soils, as well as in habitats with shallow soils (soil depth is considered to be a proxy for habitat susceptibility to drought).
In order to test our hypothesis, an elevational gradient was selected as a study system, for the reason that it is one of the most powerful ‘natural experiments’
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for exploring macroecological mechanisms of plant–climate interactions
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(Körner 2007). The large elevational ranges occurring across comparatively
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small distances in high mountains represent complex gradients of several
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environmental factors, such as atmospheric pressure, CO2 content, temperature,
D
the length of the vegetation period, and nutrient availability, all of which
TE
decrease with increasing elevation. At the same time, annual precipitation, frequency of frost during the vegetation period, and solar radiation tend to
EP
increase (Körner 1999; von Arx et al. 2006). Taken together, these changes
CC
usually make plant growing conditions more severe at higher elevations
A
(Tranquillini 1964; Billings & Mooney 1968; Körner 1999).
Materials and methods Study site
8
Fieldwork was carried out in the Berchtesgaden National Park (Bavaria, Germany), which is located in the Bavarian Alps (northern part of the Calcareous Alps) and is approximately 200 km2 in size. The National Park has a
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typical alpine relief, with steep mountain peaks composed of Triassic lime and dolomite rocks (Marke et al. 2013). The climate in the region is a typical
mountain climate, showing a large decrease in mean annual air temperatures from +7 to -2 °C (from 603 to 2713 m a.s.l., respectively). Mean annual
N
1500 to 2600 mm per year (Marke et al. 2013).
U
precipitation in the region varies from year to year, ranging from approximately
A
Study species
M
For the purpose of this study, three native perennial species with permanent main roots were investigated: Campanula scheuchzeri, Lotus corniculatus and
D
Helianthemun nummularium. Campanula scheuchzeri is a small-sized forb
TE
(height: up to 30 cm) with a comparatively thin creeping rhizome located mainly
EP
in the upper soil layer and an upright stem. Lotus corniculatus (height: 5-40 cm) is a herbaceous plant with a strong well-developed taproot; it is typically
CC
sprawling at the height of the surrounding vegetation. Helianthemum
A
nummularium (height: 10-20 cm) is an evergreen clonal dwarf shrub with creeping and upright stems; it has a strong taproot originating from a woody base. In Central Europe, all three species are largely confined to open vegetation on calcareous, dry, nutrient-poor, often skeletal soils.
9
The species were chosen for three reasons. First, they all develop clearlydemarcated and reliable annual rings in the secondary root and rhizome xylem (Schweingruber & Poschlod 2005). Second, the species are long-lived, with a
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maximum life span of 18, 16 and 55 years for C. scheuchzeri, L. corniculatus, and H. nummularium, respectively (Schweingruber & Poschlod 2005). Third, in
the study area, all species are abundant and occur over a broad elevational range from 600 to 2300 m a.s.l. Thus, the variation in plant growing conditions along
U
with a comparatively high variation in the species’ life span provides a good
N
basis for the study of plant age-environment relationships.
A
Collection sites
M
In the National park, we selected 17, 16 and 11 sample sites for C. scheuchzeri,
D
L. corniculatus, and H. nummularium, respectively; the sites represented distinct
TE
populations distributed along the elevational range (see Appendix A). In order to
EP
extend the elevational gradient into the lowlands, three additional study sites, Kallmünz (364 m a.s.l.; L. corniculatus, and H. nummularium), Riedenburg (350
CC
m a.s.l.; L. corniculatus, and H. nummularium) and St. Johann (760 m a.s.l.; H. nummularium), were selected. All the additional study sites represent the same
A
type of vegetation (open, nutrient-poor calcareous grasslands), as the alpine sites. Each site, approximately one hectare in size, was characterised in terms of its mean annual temperature, soil depth (as a proxy for moisture content in the upper soil layer (0-20 cm)) and soil fertility (nitrogen, potassium, phosphorous). 10
Due to the fact that C. scheuchzeri is replaced in lower elevations by the closelyrelated C. rotundifolia, great care was taken to determine species identity of collected individuals of the former species, particularly in the sites H1, H2, H3,
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HO1, WM1-1 and WM2-2 (all located below 1200 m a.s.l.; see Appendix A). Temperature data were obtained from 15 weather stations located in the
National Park (604–1919 m a.s.l.) and five stations from the adjacent region (360–1100 m a.s.l., distances did not exceed 50 km). Mean annual temperature
U
(MAT) at each station was calculated over the years 2000–2008; from these data
N
lapse rates between elevation and MAT in the study region were determined
A
(0.33 °C per 100 m of elevation) to define the MAT at all the collection sites.
M
MAT was selected as a proxy for site temperature environment, since it strongly
D
correlates with growing season length, mean growing season temperature and
TE
number of frost days during vegetation period (see Appendix B), factors that potentially affect plant maximum life span (Molisch 1938).
EP
All soil samples were collected within 2 weeks at the end of the vegetation
CC
season (end of August to end of September, depending on elevation). At each site, six random wholes were dug, and the soil from a depth of 5–10 cm was
A
collected, thoroughly mixed, air dried, sieved (2 mm) and subsequently analysed. Plant available phosphorus and potassium were extracted from the collected soil samples with a calcium acetate lactate solution (VDLUFA 1991). Phosphorus contents were determined using a UV–visible spectrophotometer (Thermo-Spectronic UV-1 model, Thermo Electron Corporation, USA) after 11
visualising the phosphorus content with ammonium heptamolybdate. Potassium contents were determined using an atomic absorption spectrometer (Solaar AA model, Thermo Elemental, USA). Total soil nitrogen content was quantified via
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gas chromatographic measurement with an element analyser (Vario EL III model, Elementar Analysensysteme GmbH, Germany).
Data on soil nitrogen, phosphorus and potassium are presented as the mean values of six replicates per site. Soil depth was estimated by the repeated
U
sticking of an iron rod of 0.6 mm diameter into the soil.
N
Age determination
A
The age of the largest individuals growing in a collection site was used as a
M
proxy for maximal plant life span, because previous studies (e.g. Dietz et al. 1998; Erschbamer et al. 2009; Powolny et al. 2016) have clearly demonstrated
D
that herbaceous plant size is positively and linearly related to plant age. Owing
TE
to the special nature conservation status of the study area, it was not possible to
EP
study the plant size-age relationship for the study species, since it would have required a much larger sample size. We, thus, assumed that such relationship
CC
also held true for C. scheuchzeri, L. corniculatus and H. nummularium.
A
Depending on population size, 10 to 20 of the largest individuals of each target species were selected from each site between 2009 and 2014, carefully excavated and stored in 40% ethanol. In total, 262, 258 and 151 individuals of C. scheuchzeri, L. corniculatus and H. nummularium, respectively, were analysed. 12
The age of the collected individuals was determined by means of herbochronology, a technique adapted from dendrochronology using growth rings in the secondary xylem of the root collars (Schweingruber & Poschlod
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2005). The development of annual rings in the roots of herbaceous species, especially dicotyledonous species outside the tropics with differential growth in different seasons of the year, was found to be rather common (Schweingruber & Poschlod 2005).
U
In the laboratory, the main roots (L. corniculatus, H. nummularium) or main
N
rhizomes (C. scheuchzeri) were cut using a sledge microtome (Reichert, Austria)
A
to produce thin transverse sections (ca.10-40 nm thick). The sections were first
M
stained with 1% water solution of Astra blue (Sigma-Aldrich, Germany) for 3 minutes. After rinsing with distilled water, the sections were stained with 1%
D
water solution of Safranin (Sigma-Aldrich, Germany) for 30 seconds, followed
TE
by rinsing with ethanol. The staining causes a red coloration of the lignified cell
EP
walls (mainly secondary xylem) and a blue coloration of the cell walls rich in pectin (mainly secondary phloem), thus making the annual rings visible. The
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coloured growth rings were counted under a microscope (TS100, Nikon, Japan).
A
Three separate radii were analysed for each specimen to account for tangential variation in growth ring width. Means of the three measurements were used as the age of the collected individual (Dietz & Fattorini 2002). Statistical analysis
13
To test the relative effects of environmental factors on the estimated ages of the collected largest individuals (proxy for maximum plant life span; response variable), we calculated linear mixed-effects models for each species separately.
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As fixed effects (predictors), we included the MAT of each collection site (both simple and quadratic terms), soil depth, soil phosphorous and soil potassium
contents. Collection site was included in each model as a random effect. Total soil nitrogen content was excluded from the statistical analysis, due to high
U
autocorrelation with the MAT, which would violate the model assumptions.
N
Collinearity between the remaining predictors was relatively low. The strongest
A
correlation was between soil phosphorous and soil potassium in Campanula
M
scheuchzeri plots (-0.64), which could be considered as acceptable (Dormann et al. 2013). All predictors were scaled to zero mean and unit variance prior to
D
analyses to directly infer the relative effects of the environmental variables from
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their model estimates. Power transformation with an exponent 0.3 was applied
EP
to the data on plant maximum life span, in order to obtain more normal residuals.
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Models were fit with R software version 3.3.2 (R core development team 2016),
A
using the package lme4 (Bates et al. 2015). P-values were calculated with the package lmerTest based on Satterthwaite approximations of the degrees of freedom to test for significance of the fixed effects (Kuznetsova et al. 2014). Additionally, marginal R2 (proportion of variance explained by the fixed effects only) and conditional R2 (proportion of variance explained by both the fixed and 14
random factors) were calculated as a measure of goodness-of-fit of each model (Nakagawa & Schielzeth 2013).
Environmental characteristics of the collection sites
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Results
The data on environmental factors measured are presented in Appendix A.
U
Across all sites, MAT decreased along the elevational gradient from 7.04 °C
N
(WM1-1 site, 766 m a.s.l.) to 2.00 °C (SchSt site, 2276 m a.s.l.) and had an
A
average of 4.52 °C. Soil depth ranged from 8 to 56 cm and did not show any
M
relationship with elevation. Soil was most shallow in site HO5 and deepest in
D
site HO1. As for soil potassium content, sites ranged from 18.3 mg/kg of soil
TE
(WM1-1) to 141.1 mg/kg of soil (SchSt), with an average of 60.3 mg/kg of soil; no elevational trend was observed. Values for soil phosphorus content varied
EP
among the collection sites 10-fold, from 4.1 (WM1-1 site) to 41.8 mg/kg of soil
CC
(PFK site) and averaged of 15.3 mg/kg of soil. The total soil nitrogen content ranged from 4.6 mg/kg (HO1 site) to 19.0 (PFK site) and averaged of 10.4
A
mg/kg of soil. Age variation of the study species The maximum age of collected individuals (‘maximum life span’) was found to be highest in H. nummularium (34 years; HO4 plot) and lowest in C. 15
scheuchzeri (1 year; a few individuals of that age were found mainly in lowland sites). As for single species, maximum and minimum values of plant age were as follows: C. scheuchzeri: 1-11 (average 3.2 years), L. corniculatus: 2-30 (average
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9.0 years), H. nummularium: 3-34 (average 13.4 years). The population medians of the age of collected individuals ranged from 1.5 (WM2-2 site) to 4.5 years
(HO1 site) for C. scheuchzeri, from 3.5 years (H1 site) to 16 years (H6 site) for
L. corniculatus and from 7.5 years (H2 site) to 19 years (HO4 site) for H.
U
nummularium.
N
Effects of environment on plant age
A
A general decrease in maximum age of collected individuals was found along
M
the gradient of mean annual temperature (simple term only) in all species
D
studied (Table 1; Fig. 1). The decrease per 1 °C MAT was 0.32, 0.83 and 1.57
TE
years for C. scheuchzeri, L. corniculatus and H. nummularium, respectively.
EP
MAT explained 7%, 7% and 9% of the variation in age in C. scheuchzeri, L. corniculatus and H. nummularium, respectively. In addition, soil depth was
CC
found to have a significant positive effect on the age values in the case of C. scheuchzeri (5% of total variation in plant maximum life span; Table 1).
A
Variation in soil phosphorous and soil potassium contents did not show any significant effects on age values.
Discussion 16
There is an urgent need for knowledge about variation in plant maximum life spans in general and ecological processes underlying these patterns in particular (Schweingruber & Poschlod 2005; von Arx et al. 2006). To our knowledge, this
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study is one among the very few studies (e.g. Neuffer and Hurka 1986; von Arx et al. 2006; Poschlod et al. 2011) that suggest that age of perennial herbaceous plants at intraspecific level responds sensitively to environmental conditions. Here, we find supporting evidence that high variation in population medians of
U
the maximum life span among populations of C. scheuchzeri (1.5 to 4.5 years),
N
L. corniculatus (3.5 to 16 years) and H. nummularium (7.5 to 19 years) is due to
M
A
differences in the mean annual temperature of habitats where the plants grow.
Temperature effect on plant life span
D
Our results suggest that temperature has a significant effect on intraspecific
TE
variation in plant maximum life span in C. scheuchzeri, H. nummularium and L.
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corniculatus. Despite large within-population fluctuations, individuals of these three species generally had a longer life span under colder climates, in our case
CC
in higher elevations. These results are in line with a previously detected general
A
trend of increasing adult age with increasing low-temperature stress at intraspecific (Neuffer & Hurka. 1986; von Arx et al. 2006), interspecific (Nobis & Schweingruber 2013), community (Körner 1999) and ecosystem levels (Larson 2001). The findings, therefore, can be interpreted as strong support for the ‘death-by-starvation hypothesis’ (Molisch 1938; Thomas 2002). More 17
specifically, habitats with low MATs (uplands) are characterized by comparatively low temperatures and very high probability of late-frost events during the vegetation period (see Appendix B). Therefore, we think that, in
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order to reduce mortality, plants growing in such habitats might allocate a higher proportion of their resources to vegetative growth, especially in early life stages (Körner 1999; von Arx et al. 2006). In addition, in order to increase the chances
of surviving hazardous low-temperature events, plants from high elevations tend
U
to store considerable carbohydrate reserves (Sakai 1987; Rosbakh et al. 2017).
N
Because of the trade-off in resource allocation between current plant
A
performance and future population fitness (Willems & Dorland 2000; Ehrlén &
M
Lehtilä 2002; García & Zamora 2003), such a conservative or stress-tolerant life strategy could lead to a reduced reproductive effort (García et al. 2003; Laiolo et
D
al. 2017). Accordingly, limited irregular production of sink tissues (e.g. flowers,
TE
developing seeds, tubers) would promote vegetative growth and extend the
EP
longevity of plants growing in cold habitats (Molisch 1938). A long life span is considered to be a compensation for erratic and hazardous seed production,
CC
which is quite common in alpine and arctic areas (Billings & Mooney. 1968;
A
Körner 1999; de Witte et al. 2010). In contrast, when moving along the temperature gradient, environmental favourability gradually increases, thereby leading to a shift in plant life strategy from persistence by longevity to regeneration by seeding (García et al. 2003; Laiolo et al. 2017). Sexual reproduction considerably exhausts the resources of a plant (Willems & Dorland 18
2000; Ehrlén & Lehtilä 2002), thereby making it more prone to stress-related sources of mortality (Obeso 2002). Yet, this conclusion remains speculative as we lack direct measurements of generative and vegetative traits for the study
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species.
Soil nutrient effect on plant life span
The ‘death-by-starvation’ hypothesis (Molisch 1938; Thomas 2002) postulates
U
that mineral nutrient shortages should extend plant life span by reduced
N
production of sink tissues and slow vegetative growth. However, the pattern of
A
increasing plant adult age towards the ‘poorer’ end of the soil nutrient gradient
M
was not detected for any of the study species; comparatively old individuals were found in nearly all plots, regardless of their soil phosphorous and/or soil
D
potassium contents. There are three possible explanations for the lack of soil
TE
nutrient effects on plant life span in our study. Firstly, in Central Europe, all the
EP
tested species occur mainly in calcareous, comparatively nutrient-poor grasslands (see Appendix A). Accordingly, these environmental conditions
CC
select for plants able to survive and function under low-nutrient stress (Rosbakh
A
et al. 2015). Consequently, such slow-growing plants with low resource acquisition are able to sustain their physiological processes with minimal amounts of soil nutrients (Rosbakh et al. 2017). Furthermore, these plants are characterized by the low level of response to increased soil nutrients. We assume, therefore, that neither vegetative growth nor sexual reproduction and in 19
its turn plant life span were affected by soil phosphorus and soil potassium variation among the study populations (8-fold and 11-fold for P and K, respectively). Secondly, soil nutrient deficiency in such habitats usually leads to
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a mycorrhizal infection which confers nutritional benefits to plants (Read & Haselwandter 1981; Bernhardt-Römermann et al. 2011). It is also remarkable
that the levels of mycorrhizal infection tend to increase along the gradients of
environmental stress (e.g. temperature, soil nutrients, competition), thereby
U
proportionally reducing these negative effects on plant functioning (e.g. Read
N
and Haselwandter 1981; Bernhardt-Römermann et al. 2011).
Der
Heijden
2006; C.
scheuchzeri: Read
M
Van
A
Since all the study species possess mycorrhiza (L. corniculatus: Scheublin & &
Haselwandter
D
1981; H.nummularium: Massaccesi et al. 2015), we suggest that physiological
TE
processes of the study species were unaffected by the among-site variation in soil P and K levels, which in its turn did not affect plant life span. Thirdly, our
EP
study does not account for variation in soil ammonium and nitrate contents
CC
among the populations of the study species, two other important soil nutrients, which affect plant growth and reproduction. In their study on plant life span
A
variation at species level, Schweingruber and Poschlod (2005) found a negative relationship between plant age and soil nutrient content (expressed as Ellenberg indicator values). Since it was not feasible to obtain data on the nutrient status of the sites where the populations were sampled, we could not test the hypothesis.
20
Therefore, further studies are required to determine the effect of soil nitrogen on plant life span variations at population level. Soil depth (drought) effect on plant life span
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Abiotic stress associated with drought may also affect plant age. Drought limits
plant photosynthetic capacity and therefore growth (Kreyling et al. 2008), and it may as well induce flower sterility and reductions in grain yield (Ekanayake et al. 1989), which, according to Molisch (1938), could also lead to an extended
U
plant life span. Despite our prediction that plants growing in habitats with
N
shallow soils should live longer, maximum life span in none of the study species
A
was negatively affected by soil depth. Surprisingly, trait values of C.
M
scheuchzeri were significantly positively affected by soil depth; individuals of
D
the species tended to live longer in deeper soils. This positive relationship can be
TE
explained by high levels of precipitation (960 to 2100 mm precipitation per year; Marke et al. 2013) uniformly distributed over the vegetation period in all the
EP
sampled populations (except for Kallmünz and Riedenburg; see Appendix A).
CC
The result of this is that the soil in the majority of the study populations remains continually moist and plants growing there never or very seldom experience
A
drought events. Therefore, the question as to whether water limitation extends plant maximum life span at population level remains to be tested in another study system with a more pronounced drought gradient. As for C. scheuchzeri, we speculate that the positive effect of increasing soil depth on maximum life span of this species can be explained by higher competition pressures that the 21
species experiences in sites with deeper soils. Plant distribution in calcareous grasslands, our study system, is limited, among other factors, by shallow soils; more competitive tall fast-growing plants, which usually require larger soil
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volume for their extensive root systems, tend to occur in deeper soils. When occurring in such sites, short-stature slow-growing individuals of C. scheuchzeri
(Rosbakh et al. 2015) are overgrown by taller plants, which can negatively affect their sexual reproduction by limiting the nutrients in soils and worsening the
U
light conditions. In order to compensate for reduced reproductive effort and to
N
reduce mortality (García et al. 2003), the plant growing in such stressful
A
conditions allocates a higher proportion of its resources to vegetative growth
M
leading (according to Molisch’s ‘death-by-starvation’ hypothesis) to an extended
TE
Conclusions
D
life span.
EP
Our study strongly suggests that fluctuations in plant growing conditions affect plant maximum life span, in our case at the population level. These results have
CC
several theoretical and practical implications. From a theoretical perspective, our
A
study is a modest, but important contribution to the ongoing discussion on what factors, intrinsic or extrinsic, restrict whole plant life span. Particularly, we believe that the pattern of increasing plant age along the gradient of MAT is highly supportive to the ‘death-by-starvation’ hypothesis (Molisch 1938; Thomas 2002), an important but, unfortunately, unjustly forgotten concept of 22
plant life span variation. If variations in plant maximum age are indeed generally related to variations in plant growth conditions, the plant ageenvironment relationship could be widely used in plant ecology, including plant
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ecophysiology, plant population ecology, community ecology and conservation biology (Dietz & Fattorini 2002; Salguero-Gomez et al. 2013). For example, the
analysis of plant life span adjustments along gradients of environmental factors
can considerably contribute to our understanding of how plants may cope with
U
changing environmental conditions, e.g. due to global change. It has been
N
predicted that ongoing temperature rises and increased nitrogen deposition,
A
especially in cold habitats, will increase flowering, increase senescence of old
M
individuals and alter internal resource ratios (de Witte et al. 2010), factors that according to Molisch (1938) restrict plant life span. Decreased environmental
D
stress will inevitably lead to shortened longevity of plants that in its turn could
TE
potentially decrease the persistence of populations and thus affect community
EP
stability and vegetation responses to present and future climate change (e.g.
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Ehrlén and van Groenendael 1998; Borges 2009).
A
Authors' Contributions PP conceived the ideas and designed methodology; both authors collected and analysed the data; SR led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.
23
Data accessibility All data used in this manuscript are available upon request.
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Acknowledgments
We thank participants of the course “Ecology and nature conservation” and
Theresa Lehmair for the help with the plant age identifications and Günter Kolb for the soil analysis. Accommodation and access to the collection sites was
U
provided by the Berchtesgaden National Park. The research was funded by
N
FORKAST project (TP 12 Poschlod). We thank editor of the Special Issue Niek
A
Scheepens, managing editor Klaus Hövemeyer and two anonymous referees for
A
CC
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TE
D
M
their very helpful comments on earlier versions of the manuscript.
24
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Figures
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Fig. 1
Fig 1. Regressions of back-transformed age of largest individuals (proxy for plant maximum life span; see MM for details) to site mean annual temperatures
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with regard to the three study species. For each species, the regression lines are given (solid line: Helianthemum nummularium, dotted line: Campanula scheuchzeri and dashed line Lotus corniculatus; Table 1.) For the sake of clarity, population means of age of largest individuals were plotted.
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Tables
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Table 1. Effects of environmental factors on maximum life span of Campanula scheuchzeri, Lotus corniculatus and Helianthemum nummularium as deduced from linear mixed-effects models fit by REML t test using Satterthwaite approximations to degrees of freedom. The variables were scaled prior to analysis. The power transformation with an exponent 0.3 was applied to data on plant maximum life span, in order to obtain more normal residuals. SE: standard error; SD: standard deviation. Significance levels: *** p< 0.001, ** p<0.01, * p<0.05. Marginal R² indicates proportion of variance explained by the fixed effects only, whereas conditional R² indicates proportion of variance explained by both the fixed (see Parameter) and random factors (Site). Random effects Species
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Marginal R² = 0.19 Conditional R² = 0.33
Lotus corniculatus
Variance
SD
Parameter
Estimate
SE
P-value
Site Residual
0.01 0.04
0.09 0.19
Intercept MAT MAT² Soil depth Soil phosphorus Soil potassium
1.36 -0.11 0.02 0.09 0 -0.06
0.04 0.04 0.03 0.02 0.04 0.04
<0.01 *** 0.01 ** 0.61 0.01 ** 0.99 0.17
Site Residual
0.01 0.05
0.11 0.22
Intercept MAT MAT² Soil depth Soil phosphorus Soil potassium
1.91 -0.11 -0.02 0.03 -0.04 0.04
0.06 0.05 0.05 0.04 0.05 0.07
<0.01 ***
Site Residual
0.02 0.04
0.13 0.2
Intercept MAT MAT²
2.13 -0.24 -0.07
0.08 0.07 0.07
<0.01 *** 0.01 ** 0.35
ED
Variable
PT
Campanula scheuchzeri
Fixed effects
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Marginal R² = 0.20 Conditional R² = 0.35
Helianthemum nummularium
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0.03 * 0.74 0.45 0.46 0.55
N U SC RI PT Soil depth Soil phosphorus Soil potassium
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PT
ED
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Marginal R² = 0.37 Conditional R² = 0.56
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-0.06 -0.09 0.14
0.06 0.09 0.08
0.38 0.34 0.12