Small differences in seasonal and thermal niches influence elevational limits of native and invasive Balsams

Biological Conservation 191 (2015) 682–691

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Small differences in seasonal and thermal niches influence elevational limits of native and invasive Balsams Julia Laube a,b,⁎, Tim H. Sparks a,b,c,d, Claus Bässler e, Annette Menzel a,b a

Ecoclimatology, Department of Ecology and Ecosystem Management, Technische Universität München, Hans-Carl-von-Carlowitz-Platz 2, 85354 Freising, Germany Institute for Advanced Study, Technische Universität München, Lichtenbergstrasse 2a, 85748 Garching, Germany Institute of Zoology, Poznań University of Life Sciences, Wojska Polskiego 71C, 60–625 Poznań, Poland d Sigma, Coventry University, Priory Street, Coventry CV1 5FB, United Kingdom e Bavarian Forest National Park, Freyunger Str. 2, 94481 Grafenau, Germany b c

a r t i c l e

i n f o

Article history: Received 29 April 2015 Received in revised form 7 August 2015 Accepted 8 August 2015 Available online xxxx Keywords: Alien plant species Climate change Field experiment Functional traits Impatiens Mid-mountain range

a b s t r a c t Recent studies suggest that invasive plant species have colonised mountains to previously unobserved elevations, possibly due to ongoing climate change. Thus, they might pose new threats to high-elevation ecosystems, which are often of high conservation value. Current range predictions are primarily based on climate niche models, however many other factors might also contribute to the species' distribution. We studied the species-specific elevational limits of one native (Impatiens noli-tangere) and two invasive balsams (Impatiens glandulifera and Impatiens parviflora) on a mid-mountain range in Germany. We used a combination of trait measurements and a field experiment to assess the relative importance of temperature, trait adaptations, and biotic interactions on elevational limits. Results indicate that concurrent seedling emergence, low frost resistance and, for I. glandulifera, late flowering, are important contributors to elevational limits. Because of a lack of seed bank persistence, erratic spring and autumn frost events coinciding with the plants' annual life-cycles will likely influence the upper limits of the invasive species. The abundance of the species seems to be further limited by herbivory, mainly by molluscs. Given that a highly nuanced interaction between phenological development and erratic frost events are important for range limits, predictions based solely on mean climatic values, such as temperature, are unlikely to accurately predict future invasion limits. Our results indicate that occasional occurrences of the species do not necessarily call for eradication actions, that management efforts might be most effective at intermediate elevations, and that any measure encouraging terrestrial molluscs will help to maintain biotic resistance. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Invasive species are known to impact native biodiversity, and thus pose serious threats to areas of high conservation value (Gurevitch and Padilla, 2004; Vila et al., 2011; Pysek et al., 2012). Many mountain ecosystems in Europe remain pristine or only mildly humaninfluenced, and often harbour rare species. However, they are expected to be highly threatened by climate change due to shifting range limits of both native and invasive species (Thuiller et al., 2005; Engler et al., 2011). Most invasive species worldwide show distribution limits with elevation or lower abundance at higher elevations (Pysek et al., 2002; Becker et al., 2005; McDougall et al., 2005, 2011; Marini et al., 2009). ⁎ Corresponding author at: Ecoclimatology, Department of Ecology and Ecosystem Management, Technische Universität München, Hans-Carl-von-Carlowitz-Platz 2, 85354 Freising, Germany. E-mail address: [email protected] (J. Laube).

http://dx.doi.org/10.1016/j.biocon.2015.08.019 0006-3207/© 2015 Elsevier B.V. All rights reserved.

However, there is evidence that invasive species have colonised formerly unknown elevations, and it remains unclear which mechanisms promote this spread upwards; climate change has been proposed as one possible trigger (Becker et al., 2005; Pauchard et al., 2009; Walther et al., 2009). Therefore, invasive species are expected to colonise endangered habitats at higher elevations (Kleinbauer et al., 2010). Several non-climatic factors that hamper invasion processes at higher elevations are known, such as reduced propagule pressure, lower disturbance levels at higher elevations, less human habitat modification (Tomasetto et al., 2013), as well as time-lag effects, such as delays between first arrival of the species and spread until complete niche filling (Becker et al., 2005; Marini et al., 2009; Pauchard et al., 2009; Alexander et al., 2009b, 2011). Other factors that influence elevational invasion limits are expected to be climate sensitive, such as unsuitable individual thermal niches or inability to adapt to harsher climates due to genetic bottlenecks and lowland genetic filters (Becker et al., 2005; Alexander et al., 2009b; Haider et al., 2010; McDougall et al., 2011). Species climatic niches

J. Laube et al. / Biological Conservation 191 (2015) 682–691

might be of lesser importance in determining elevational range limits, since several studies on different invasive species have shown that growth and reproduction are maintained above their actual limits (Willis and Hulme, 2002; Paiaro et al., 2007; Poll et al., 2009; Trtikova et al., 2010). Biotic interactions could equally contribute to elevational limits (Alexander et al., 2009b), since the success of many invasive species is linked to strong competitive abilities (Vila and Weiner, 2004; van Kleunen et al., 2010; Vila et al., 2011). The competitive ability of species is known to be context-sensitive, and can change drastically along gradients (Choler et al., 2001; White et al., 2001; Daehler, 2003; Walther et al., 2009; Mason et al., 2012; He et al., 2013). Both competitive abilities of invasive species as well as changed biotic resistance of native plant communities are expected to respond to climate change (Brooker, 2006; Vila et al., 2007; Hellmann et al., 2008). In addition, rapid acclimatisation and adaptation is an important factor for successful spread into new environments (Vila et al., 2007; Whitney and Gabler, 2008; Clements and Ditommaso, 2011), and adaptive evolution is thought to play an important role for species to become successful invaders (Lambrinos, 2004), although, so far, studies have found little evidence for this hypothesis (Williams and Black, 1993; Parker et al., 2003; Trtikova et al., 2010, 2011; Pahl et al., 2013). Nevertheless, apart from the rather broad ecological niches of invasive species (Dukes and Mooney, 1999; Vila et al., 2007; Hellmann et al., 2008), they often show a high phenotypic plasticity (Daehler, 2003; Davidson et al., 2011; but see Godoy et al., 2011; Palacio-Lopez and Gianoli, 2011), which might contribute to a flexible response of elevational range limits to climate change. Given these multiple, interacting factors that possibly contribute to the actual range limits of invasive species, the predictability of elevational range expansions under climate change remains limited. This limitation decreases the potential identification of future invasion risk, as well as implementation of management strategies, and might lead to devaluation of rare habitats on the one hand, or a misallocation of resources on the other. In this study we aim to disentangle the effects of several possible factors on current elevational limits for two invasive balsam species (Impatiens glandulifera Royle and Impatiens parviflora DC.) on a steep gradient (300–1200 m a.s.l.) in a mid-mountain range in Bavaria, Germany, and compared them to those of the native congener (Impatiens noli-tangere L.). We used a combination of a field survey and a field experiment to study the influence of trait adaptation, biotic interactions and thermal niche on the elevational limits of the species. The field survey of existing populations focused on changes in plant functional traits along the elevational gradient. The field experiment concentrated on possible germination and establishment barriers, as well as on the role of biotic interactions along the gradient. The main driving force of the actual elevational limits of the species will influence invasion risks under future climate conditions, and consequently optimal management options might also differ. Thus, the main question of this study was whether climate variables, missing trait adaptation and trait plasticity of the local populations or biotic interactions were the main drivers of the observed elevational limits. 2. Materials and methods

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below (Bässler et al., 2010). The lowlands completing the elevational gradient towards the Danube valley are mainly alluvial forests with European ash. Higher elevation habitats harbour rare and threatened mountain plant species, e.g. Gentiana pannonica Scop. or Botrychium multifidum (S. G. Gmel.) Rupr., and likewise species of other taxonomic groups (e.g. fungi, birds, bats) that are threatened in Germany or in Bavaria occur in the area (Bässler, 2008). The forests were only extensively used for forestry in previous centuries (Müller et al., 2008; Röder et al., 2010), and thus are now close to natural. These Central European montane mixed forests belong to the most threatened natural systems worldwide (Hannah et al., 1995). Overall, the gradient stretches from an elevation of 300 m to 1200 m a.s.l. (highest peak at 1350 m), and comprises a wide range of mean annual temperature (from 8.3 to 4.6 °C) and annual precipitation (from 1100 to 1700 mm). The number of frost days (from 106 to 163) as well as the duration of snow cover (from 5 to 7 months) increase considerably with elevation (Elling et al., 1987). Soils in the area are nutrient poor and with low pH-values (pH mainly between 3 and 4); they show little variation except for soil depth, which is mainly related to the steepness of slopes. However, since balsams in the area occur at flat and moist sites, these differences in soil depth and slope are unlikely to be of relevance. 2.2. Species Both invasive balsams are very common in Central Europe. I. glandulifera (IG), in particular, is known to suppress native species (Beerling and Perrins, 1993; Pysek and Prach, 1995; Hulme and Bremner, 2006; but see Hejda and Pysek, 2006), while I. parviflora (IP) also tends to locally dominate species communities (Hejda, 2012). In the study area, both species are highly abundant, and often dominate the understorey vegetation in the lowlands. At higher elevations, the abundance of both invasive species declines, and when present, they exhibit lower density than native I. noli-tangere (IN). IN reaches elevations up to 1100 m, invasive IG is found up to 900 m, and invasive IP has an upper limit of around 700 m a.s.l. Assuming linear responses of plants to temperature change, a scenario of a 2 °C temperature increase relates to an upward shift of species by 400 m (current lapse rate 0.5 °C/100 m). For invasive IG, this might lead to an invasion up to the mountain tops, and invasive IP might almost reach them. At the current time, the species are found in riverside habitats, but also in forest communities (Pahl et al., 2013). Thus, with climate change a spread into close-to natural high-elevation forest communities is of concern. These three congeners represent a good study system, since they share comparable niche requirements, and often co-occur at the same sites (Skalova and Pysek, 2009; Vervoort and Jacquemart, 2012; Skalova et al., 2013; Cuda et al., 2014). They are close relatives, which minimises phylogenetic bias (MacDougall et al., 2009; van Kleunen et al., 2010), and share the same life-cycle (all three are annual herbs), with comparable pollination by Bumblebees and other Hymenoptera (Klotz et al., 2002), and comparable dispersal habits (self-dispersed via ballochory). Furthermore, both invasive species established in Germany more than 150 years ago (Nehring et al., 2013), hence current distribution limits probably reflect a current climate steady state, which should allow a meaningful analysis of trait differences (Pysek et al., 2015).

2.1. Study area 2.3. Field survey The Bavarian Forest National Park is situated on the border between Germany and the Czech Republic. Most of the area is covered by forest, which is, together with neighbouring Šumava in the Czech Republic, part of the largest contiguous forest in Central Europe (Bässler et al., 2009). The upper elevations are mainly dominated by spruce (high montane forest), with mixed montane forests of spruce, beech, and fir

We sampled plant traits in populations of invasive and native balsams at different elevations (Table 1). The occupied sites were highly comparable: flat, moist, and either under canopy gaps, or at the edges of larger open areas within forest stands, e.g. in windthrown gaps. Due to a low number of occurrences at higher elevations, three populations

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Table 1 Numbers of samples/measurements per date, site and species in the field study. Ele: elevation (m a.s.l.); IN: Impatiens noli-tangere, IG: I. glandulifera, IP: I. parviflora. Full: full set of trait measurements, with records on phenological development stage (N = 25), plant height (N = 25), specific leaf area (N = 20), seed samples (N = 10), and frost samples (N = N Frost) taken for the given dates. Add. Dates Frost: dates when additional samples were taken for leaf frost measurements, N Frost: number of leaf frost measurements per species (same order as in Species column), numbers vary due to damage or loss of samples during the procedure (broken glass, failure of frost). Site

Alt

Species

Date

Full

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

300 300 300 300 300 600 600 600 600 600 600 900 900 900 900 900

IN, IG IP IN, IG, IP IN, IG, IP IN, IG, IP IN, IG IP IN, IG, IP IN, IG, IP IN, IG, IP IN, IG IN, IG IN IG IN, IG IN, IG

09.09.2011 09.09.2011 10.08.2011 28.08.2011

x x xa x

09.09.2011 09.09.2011 10.08.2011 28.08.2011

x x xb x

09.09.2011 10.08.2011 10.08.2011

x x xc

28.08.2011

x

a b c

Add. Dates Frost

13.07.2012 17.05.2012 13.07.2012 13.07.2012 17.05.2012 13.07.2012 13.07.2012 13.07.2012 13.07.2012

13.07.2012 17.05.2012 13.07.2012

N Frost 5,5 5 8,6,6 9,11,10 3,1,4 5,5 11 8,10,11 11,11,11 5,4,6 2,5 11,11 3 5 6,5 13,16

Seed: only IP. Seed: only IN, IP. N SLA = 19.

per species were studied at 300, 600 and 900 m a.s.l., with IP absent at 900 m (no known populations). We investigated several functional traits possibly showing important adaptations to elevation (description in Table 2), such as phenological development stage, plant height, specific leaf area, seed mass and leaf frost resistance (Cornelissen et al., 2003; Alexander et al., 2009a). Some of these traits relate to the competitive ability of species, and thus influence biotic interactions (plant height, specific leaf area, seed mass), or are of direct relevance for plant–climate-relationships (phenological development, frost resistance). The number of samples per population follows Cornelissen et al. (2003) (see Table 1), the individuals sampled were chosen randomly and were all fully developed.

2.4. Experiment A germination experiment was set up along the gradient, with three experimental sites at each of 300, 600, 900 and 1200 m a.s.l. (12 sites in total, a short description of the sites available for analysis is given in Table 3). The highest elevations were above the current range limits of all three species. The experimental sites comprised pots containing 20 seeds of one of the balsam species either with or without competition, with five replicates each. Thus, each site consisted of a total of 30 pots (3 balsam species × 2 competition treatments × 5 replicates). Positions of species and competition treatments were fully randomised per site. The sown density yielded a lower, but still comparable, seed density than in natural, dense communities of about 1400 seeds/m2 (Skalova and Pysek, 2009). The competition treatment used a fixed amount of seed of Deschampsia cespitosa (L.) P. Beauv. (0.10 g, about 330 seeds), Carex brizoides L. (0.08 g, about 270 seeds) and Senecio ovatus (G. Gaertn., B. Mey. & Scherb.) Willd. (0.03 g, about 40 seeds). These species all co-occur with the balsams in rather moist, semi-open habitats in the area. Seeds of all species used were collected locally in late summer 2011. Seeds were kept dry at room temperature until mid-October, then the pots were prepared in a greenhouse, and buried in the field in autumn 2011 (15/16.10.2011). During the seedling emergence phase (10.04.2012–15.05.2012), weekly observations were undertaken (one week missing). Then until the start of flowering, fortnightly observations were made, with a weekly check in between (i.e. without recording phenological stage or number of individuals). Due to very uneven seedling emergence and initial survival of seedlings, the number of balsam individuals per pot was reduced to a maximum of three at the second consecutive visit without further seedling emergence. The vegetation between pots was regularly clipped to reduce shading from surrounding grasses and forbs. We harvested aboveground biomass of focal individuals and competing species at the start of flowering (BBCH 60), which depending on elevation was between 29.06.2012 and 22.08.2012. The variables assessed during the experiment are given in Table 2. Out of the 12 (unprotected) experimental sites, two were lost completely (300 m/900 m elevation) due to trampling, most likely by wild boar, during winter 2011/2012, and one at 600 m lacked harvest data due to mass herbivory by (mainly) snails and slugs. Seedling emergence was poor at another site at 600 m elevation (only 8%, in total 46

Table 2 Short description of sampling and recording methods used in the field study and field experiment. FS: Field study; Exp Reg: regular observations during experiment, Exp Harv: assessed at harvest. Variable

Method description

FS

Exp Reg

Exp Harv

Phenological development stage

BBCH-code (Meier, 2001). Fine-scaled recording scheme encompassing the complete annual life-cycle with seedling emergence and unfolding of cotyledons (BBCH 10), vegetative growth stages (BBCH 11–49), and reproductive stages, e.g. flower bud development (BBCH 50–59), or flowering (BBCH 60–69). Height of the highest vegetative organ (Cornelissen et al., 2003), accuracy of about 0.5 cm in the field. Sampling of undamaged, sun-exposed leaves from the second topmost fully developed leaf pair, one leaf per individual. Leaves were kept in moist plastic bags until scanned to obtain leaf area using the Software ImageJ (Schneider et al., 2012). After scanning, they were oven-dried for 48 h at 60 °C, and weighed (Sartorius balance with a precision of 0.1 mg) to obtain the specific leaf area (SLA). Sampling of seeds out of five individuals, weighted with Sartorius balance with a precision of 0.1 mg. Sampling of undamaged, sun-exposed leaves. Frost damage was measured as the percentage of electrolyte leakage (PEL) of small leaf samples exposed to frost in a freezer (Gurvich et al., 2002; Cornelissen et al., 2003). Frost damage is calculated as the percentage of total ion losses from the frosted sample in comparison to an unfrosted control sample taken from the same leaf, expressed in %. Total ion losses are measured as electrolyte conductance (μS cm−1) after treatment/control and a second time after complete cell denaturation in boiled samples. In the year 2011, samples were exposed to −17 °C for 8 h in 1 ml deionised water, in 2012 samples were exposed to −8 °C for 8 h in 6 ml deionised water. Number of Impatiens individuals per pot. Number of damaged individuals with damage type: frost, herbivory by snails, aphids etc. Number of flower buds, flowers and capsules. Dry biomass (oven-dried at 60 °C for at least 48 h).

x

x

x

Plant height Specific leaf area (SLA)

Seed mass Frost damage (PEL)

Germination & Establishment Survival Reproduction Biomass

x x

x

x x

x x x x

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Table 3 Description of the experimental sites. Ele: elevation, Exp: exposition, Incl: inclination, Tree cover: tree cover above experimental plots, Open area: estimated size of canopy gap, T mean: mean soil temperatures during mean balsam growing period (DOY 101–DOY 180), WC mean: mean volumetric soil water content (DOY 101–DOY 180), N pots germination/harvest: respective number of pots with germination and harvest data available for each of the species (IN: I. noli-tangere, IG: I. glandulifera, IP: I. parviflora). Site

Ele

Exp

Incl (%)

Tree cover (%)

Open area m2

Dominant canopy species

T mean (°C)

WC mean (%)

N pots germination IN/IG/IP

N pots harvest IN/IG/IP

Isar 1 Isar 2 Schleicher Kolbersbach Hasenloher Bach Scheuereck Spiegelhütte Ruckowitz Höllbachgspreng Goldquelle

300 300 600 600 600 900 900 1200 1200 1200

– – W E SW NW W N SSE SE

0 0 b1 b1 b1 3 2 3 b1 1

10 30 0 0 0 10 0 0 0 10

b50 600 b50 2800 1800 b50 b50 1800 3900 b50

Fraxinus excelsior, Quercus robur Quercus robur Fagus sylvatica, Picea abies Alnus glutinosa Picea abies Fagus sylvatica, Picea abies Fagus sylvatica, Picea abies Picea abies Picea abies Fagus sylvatica, Picea abies

14.4 – 10.9 12.2 11.9 – 10.3 7.4 8.1 6.7

0.40 – 0.34 0.31 0.34 – 0.52 0.52 0.50 0.46

8/7/10 10/6/9 6/8/10 9/10/9 3/3/9 8/10/10 4/7/9 7/9/6 8/10/10 7/7/5

5/5/8 6/2/4 5/3/10 0/0/0 0/0/5 7/10/10 4/7/7 6/6/4 6/9/6 6/6/5

individuals). For the remaining sites, losses, mostly due to trampling, a wind-thrown tree, herbivory, two frost events, and maybe also fungal infections and insect infestations were recorded. The actual numbers of pots used in analysis are given in Table 3. Considerable death rates during the course of the experiment reduced the number of replicates of harvested individuals for biomass measurements. However, we have no indication that death rates during the experiment were higher than under natural conditions. 2.5. Climate data Soil temperatures (one sensor per site, 5 cm depth) and volumetric soil moisture (three sensors per site, 5 cm depth) were logged at 10 minutes intervals (HOBO Micro Station Data Logger) at all experimental sites. The number of stratification days per experimental site was calculated as the number of days with mean temperatures below b5 °C (Perglova et al., 2009), as recorded by the soil temperature loggers. Unfortunately, some loggers were completely damaged at two sites (300 and 900 m). Since onsite temperature measurements were therefore not given for all sites, we used elevation as a proxy for temperature. The mean logged temperature during the balsam growing period (from first seedling emergence 10.04.2012 to first harvest 29.06.2012) at the eight sites showed that elevation explained 94% of the variance in temperatures (linear regression, p b 0.001). Thus, the simplified use of elevation instead of temperatures seems reasonable. For calculation of frost event probabilities, air temperature data were obtained from the DWD (German meteorological service), which ran four stations at 313 m, 615 m, 940 m, and 1307 m a.s.l. in the area. For analysis of frost probability, we used data from 1971–2004 (the station at 940 m was abandoned in 2004, the station at 1307 m in 1982). Frost probability was calculated with a threshold of −2 °C of daily minimum temperatures at 2 m height, which coincided with observed frost damage dates at the experimental sites, and corresponded to about −4 °C minimum temperature at ground level.

were log-transformed, and PEL was power-transformed to meet normality assumptions of residuals. Residual plots were inspected for deviations from normality. P-values of fixed effects were calculated from ttests with Satterthwaite approximation for degrees of freedom (R package lmerTest (Kuznetsova et al., 2015)). We used likelihood-ratio tests to select minimal adequate models (based on recalculated loglikelihood models, with backward model-selection) (R package lme4 (Bates et al., 2015)). For the experiment, we used linear mixed effect models with species identity, elevation, competition treatment and the interaction term of species identity and elevation as fixed effects. Competition treatment was used as a binary factor, since neither biomass of competitors nor the percentage of competitors of the total pot biomass showed a trend with elevation (both p N 0.05). We used mean values per pot as responses to avoid pseudo-replication (Hurlbert, 1984), and experimental site nested in elevation as a random term to account for site-specific differences not primarily related to elevation (see above). For response variables related to harvest (biomass, reproductive organs, plant height, phenological development stage at harvest), the number of species per pot was used as a weight. We analysed seedling emergence and death rates, day of the year since January 1st (DOY) of seedling emergence, beginning of reproductive phase (BBCH 59, which indicates that flower buds are close to opening), and the development time needed between seedling emergence and beginning of reproductive phase as response variables. The number of reproductive organs was pooled (sum of flower buds, flowers, and capsules) prior to analysis. Models included BBCHcode as a random predictor, since due to practical constraints, a daily harvest was not feasible, and harvested individuals differed slightly in stages from BCCH 59 (flower buds close to opening) to BBCH 65 (50% of flowers open). BBCH at harvest was analysed as an ordered response. Biomass, height and number of reproductive organs were square-roottransformed prior to analysis to meet normality assumptions of residuals. 3. Results

2.6. Analysis 3.1. Field study For the field study, we analysed trends in trait values with elevation. We used linear mixed effect models with species identity, elevation, and their interaction term as fixed effects. Site nested in elevation was included as a random effect for all traits to account for site-specific differences not primarily related to elevation, e.g. differences in moisture, possible small differences in soil pH or light availability. For percentage of electrolyte leakage (PEL), sampling date was included as an additional random factor, and due to differences in methods, year was included as a fixed effect. BBCH (phenological development stage) was analysed as an ordered response, all other trait values as scalar responses. Due to problems with supercooling during PEL measurements, we excluded (outlier) measurements from the analysis which exceeded two standard deviations in either frost or control values. Height and seed mass

We found several differences in plant functional traits between species and as a response to elevation (Fig. 1). Not surprisingly, a difference in plant height between species was found, with invasive IG larger (mean 73.3 cm, p = 0.003), and invasive IP smaller (mean 40.6 cm, p b 0.001) than native IN (mean 66.9 cm). The height of all three species decreased with elevation (overall 4.9 cm/ 100 m, p = 0.03). The decrease in height was least pronounced for native IN (4.2 cm/100 m), in comparison to invasive IG (5.3 cm/100 m) and IP (6.5 cm/100 m), but the species × elevation interaction term was only close to significance (p = 0.056). Invasive IG generally had smaller specific leaf areas (mean 55.9 mm2/mg), and invasive IP larger SLA (mean 73.9 mm2/mg) than

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Fig. 1. Bean plots of the traits measured per species and elevation. Shown are medians (thick black horizontal lines), beanlines (grey lines) corresponding to the values of each observation, and beans (shapes) showing nonparametric density estimates. 300: 300 m, 600: 600 m, 900: 900 m a.s.l. IN: Impatiens noli-tangere, IG: I. glandulifera, IP: I. parviflora. NB no IP at 900 m.

native IN (mean 69.5 mm2/mg) (p b 0.001). With respect to elevation, all species differed in response, with native IN having slightly higher SLA with elevation, IG showing no change, and IP showing an increase in SLA with elevation (species × elevation interaction p b 0.001). For seed mass, we found significantly lighter seeds for native IN in comparison to the two congeners (IG on average more than three times heavier, IP almost double, both p b 0.001), but no influence of elevation. Both invasive species showed an overall higher leaf frost damage than native IN (IN: 65.5%, IG: 72.8%, IP: 71.2%, p b 0.001), but we found no effect of elevation. Probes at −17 °C treatment had PEL values of 77.0% (2011) while probes exposed to −8 °C (2012) had an overall mean of 63.4%, which was accounted for statistically (differing methodologies accounted for as fixed effects). With respect to phenological development, both invasive species generally lagged behind native IN (lower BBCH-values, IG p b 0.001, IP p = 0.002, see Fig. 2). While native IN populations at higher elevations were generally further advanced than populations at lower elevations, the difference from the other two congeners significantly widened (species x elevation interaction, IP: p b 0.001, IG: p = 0.003). Overall the levels of explained variance for most of the traits were reasonable to high (R2 0.61–0.79), except for height and PEL with R2 values of only 0.56 and 0.53. 3.2. Experiment Seedlings emerged almost all at once per site (mean 3.3 days between first seedling emergence and 50% emergence), with, on average, one month difference between the 300 m and 1200 m sites (mean DOY 102 at 300 m, mean DOY 134 at 1200 m, Table 4). Overall, there was no significant difference in seedling emergence dates for the different species (species effect p N 0.05), although IG was slightly earlier at higher elevations (species × elevation interaction p = 0.013). Overall seedling emergence rates were high (50%), lowest for native IN (38%), and higher

Fig. 2. Bean plots of the phenological phase recorded per species and elevations in the field study (upper panel). Proportion of balsam individuals of the experiment in different development stage (July 11th 2012, lower panel), the darker, the further developed. White: early vegetative development up to BBCH 20, light grey: vegetative development up to BBCH 49, grey: start flower bud development up to BBCH 55, dark grey: late flower bud development up to BBCH 59, black: start flowering BBCH 60. 300: 300 m, 600: 600 m, 900: 900 m, 1200: 1200 m a.s.l. IN: Impatiens noli-tangere, IG: I. glandulifera, IP: I. parviflora. NB no IP at 900 m (upper panel), no 300 m because all individuals were harvested earlier (lower panel).

for both invasive congeners (IG: 45%, IP: 66%). There was no significant overall influence of elevation (p N 0.05) on seedling emergence, but germination of IG was poorer than that of native IN at lower elevations (species effect p b 0.001), and IG showed a tendency towards higher seedling emergence rates with higher elevation (species × elevation interaction p b 0.001). Seedling emergence rates of IP were generally higher than of IN, but the effect was only close to significant (p = 0.056). From seedling emergence to flowering, overall death rates were rather high (average 50.6%), with comparable, but highly variable values for all three species (IN: 47.8%, IG: 61.3%, IP: 43.6%). Overall, death rates for native IN were slightly lower at higher elevations (p b 0.001), but differences between species were not significant (p N 0.05). However, the decrease in death rates with elevation was slightly smaller for IG (p = 0.053) than for native IN. With respect to the most obvious damage types (herbivory and frost), almost one third (26.3%) of individuals showed severe herbivore damage, mainly by molluscs, at least once. The number of individuals with herbivore damage decreased with elevation (p b 0.001), and overall did not differ between species (p N 0.05). However, IN was least affected at highest elevations (900 m and 1200 m), and the species × elevation interaction was significant for IG (p = 0.016) and close to significant for IP (p = 0.059). Apart from herbivory, both invasive species showed severe damage after two frost events (DOY 135 and DOY 158), which caused damage mainly at 900 m and 1200 m, but also partly at 600 m. At 900 and 1200 m, on average 14% of IG individuals and 44% of IP individuals were affected. In comparison, no frost damage was recorded for any individual of native IN. While there was no significant difference between IN and IG, the species × elevation interaction was significant for IP (p = 0.001).

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Table 4 Results of linear mixed effect models for the different response variables in the field experiment. Intercepts refer to native Impatiens noli-tangere. Ele: elevation, IG: I. glandulifera, IP: I. parviflora, Ele × IG: elevation IG interaction, Ele × IP: elevation IP interaction, Comp: competition treatment, N Obs: number of observations, REML: restricted maximum likelihood, N Seedlings: number of emerged individuals, N Herbivore damage: number of individuals with herbivore damage, N Frost damage: number of individuals with frost damage, N Reproductive organs: mean number of reproductive organs per plant, sqrt: square-root-transformed, DOY: day of year. Response N Seedlings Death rate (%) N Herbivore damage N Frost damage Biomass (sqrt) Height (sqrt) N Reproductive organs (sqrt) DOY Seedling emergence DOY Start reproductive phase Days seedling emergence to repr. phase

Est. p Est. p Est. p Est. p Est. p Est. p Est. p Est. p Est. p Est. p

Ele

IG

IP

Ele × IG

Ele × IP

Comp

N Obs

R2

REML

0.06 0.876 −0.05 b0.001 −0.66 b0.001 0.01 0.937 −0.04 0.056 −0.14 0.094 −0.13 0.134 4.07 b0.001 3.46 b0.001 −0.38 0.555

−9.35 b0.001 −0.02 0.868 −1.91 0.105 −1.71 0.414 0.58 b0.001 2.17 b0.001 −1.11 0.036 2.73 0.412 13.51 0.010 12.07 0.035

3.37 0.056 0.03 0.736 −0.14 0.901 −1.53 0.392 −0.08 0.339 −0.68 0.053 −1.45 0.002 4.45 0.156 −1.93 0.667 −8.26 0.095

1.39 b0.001 0.02 0.053 0.33 0.016 0.31 0.169 −0.03 0.002 −0.03 0.462 −0.05 0.334 −0.96 0.013 −0.02 0.966 0.82 0.170

0.28 0.200 −0.01 0.590 0.25 0.059 0.68 0.001 0.00 0.946 0.07 0.075 −0.02 0.648 −0.57 0.132 0.26 0.610 1.15 0.039

−0.13 0.828 0.00 0.959 −0.16 0.672 −0.94 0.110 −0.03 0.328 0.01 0.957 0.15 0.288 0.97 0.354 −1.09 0.451 −3.27 0.040

277

0.52

1672.5

234

0.56

640.1

234

0.43

1748.8

177

0.39

1390.2

152

0.78

68.1

152

0.77

489.3

149

0.74

551.4

234

0.74

1641.2

152

0.76

1217.8

152

0.61

1249.4

Bold values indicate significant at p-values.

Due to targeting harvest to coincide with the start of flowering, harvest dates differed for the different species, with an average harvest date for native IN at DOY 208, invasive IP at DOY 206, and invasive IG about 10 days later (DOY 219). In effect, IN and IP were further developed than IG at harvest, with a median development phase (BBCH) of 63 for IN and IP (between beginning of flowering and full flowering) in comparison to BBCH 60 for IG (start of flowering), due to practical constraints during harvest. Overall, individuals reached their reproductive phase later at higher elevations, with overall means of 34.5 days delay at 1200 m compared to 300 m. IG generally reached the reproductive phase almost 3 weeks later than IN (mean 17 days, p = 0.010, see Fig. 2). More interestingly, the time interval between seedling emergence and reproductive phase was also longer for IG (mean 99.4 days) than for IN (mean 79.1 days) (p = 0.035), while IP (mean 81.5 days) was delayed most with increasing elevation (species × elevation interaction p = 0.039). For the individuals that survived until harvest, there was a very slight tendency towards overall lower biomass with elevation (p = 0.056). IG had heaviest individuals at 300 m, and reduced most with elevation (species p b 0.001, species × elevation interaction p = 0.002). Results for plant height were highly comparable to the results for biomass (Table 4). The number of reproductive organs decreased with elevation, but the effect was not significant (p N 0.05). Both invasive congeners had a smaller number of reproductive organs than native IN (IG: p = 0.036, IP: p = 0.002) with overall mean numbers for IN of 22.4, IG of 6.1 and IP of 7.9. Competition treatments had no significant effect on any of the assessed response variables (p N 0.05, see Table 4), except that competition treatments advanced phenological development (p = 0.040). We found no significant effect of competition treatments on Impatiens biomass, or of Impatiens species and elevation on the harvested biomass of competitors (all p N 0.05).

conditions such as inclination, tree cover, or size of canopy gap (all p N 0.05). In relation to increasing precipitation with elevation, water content increased slightly (Table 3).

3.3. Climate data For the sites where soil temperature data are available, the number of stratification days increased linearly with elevation, from 129 days at 300 m to 171 days at 1200 m (linear regression R2 = 0.89, n = 8). Similarly, soil temperatures decreased linearly with elevation (linear regression R2 = 0.94, n = 8), while trends were absent for other site

Fig. 3. Patterns of frost events (minimum temperature below −2 °C) in spring and autumn for the different elevations (1971–2004). Lines indicate different probabilities for the dates of the latest frost in spring (left) and earliest frost in autumn (right) given as day of year (DOY). The number of days gives the number of days between the two frost events. Light grey: probability of 50% for no later/no earlier frost (expected every second year), grey: probability of 80% for no later/no earlier frost (expected every fifth year), dark grey: probability of 93% for no later/no earlier frost (expected every 15th year).

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An analysis of long-term daily minimum air temperatures showed that the average lengths of the frost-free period (threshold −2 °C) generally decreased with elevation (Fig. 3). The mean interval between last spring and first autumn frost at 300 m was 205 days, but only 155 days at 1300 m. 4. Discussion The combination of trait measurements and field experiment led to several important insights into the elevational range margins of the Genus, which are needed to reliably predict range expansions of these invasive balsams with climate change. 4.1. Trait plasticity and competition For the functional traits of natural populations, plastic responses to elevation were only partly detectable. We found no signs of adaptation of seed mass, which is in contrast to earlier studies (Willis and Hulme, 2004), or changes in leaf frost resistance of the balsam species with elevation. Plant height clearly decreased with elevation for all three congeners, and native IN tended to reduce plant height least, thus this species responded in the least plastic way. Consistent with this, we also found a tendency of decreasing individual size and biomass with elevation in the experimental sites. Invasive IG reduced biomass mostly with elevation. We found no flexible response of leaf traits to elevation for IN, while specific leaf area of IP increased with elevation, which can be interpreted as an adaptation to shorter growing seasons (Milla et al., 2008). These patterns of plant height and SLA cannot be attributed to changes in light availability at high elevation sites, since the size of open-canopy area, or canopy cover, showed no trend with elevation. Moreover, site-specific differences were taken into account by including site as a random effect in all models. Likewise, varying harvest dates probably did not affect our results, since balsams usually cease growth with the start of reproduction (Kollmann and Banuelos, 2004), and thus only small effects of harvest dates on plant height or biomass have to be assumed. Often, a high plasticity of traits is related to higher performance, and thus, a high trait plasticity might translate into establishment success over a large range of elevations. However, the species with least plastic responses, native IN, currently occurs up to the highest elevations. Moreover, a high trait plasticity of plant size does not translate into higher fitness (Davidson et al., 2011). It is well known that, in particular, invasive IG owes much of its success to high competitive ability (Skalova et al., 2012, 2013) which is related to high growth rates and plant size (Beerling and Perrins, 1993; Pysek and Prach, 1995; Cuda et al., 2014), although other factors, such as allelopathy, are also important (Vrchotova et al., 2011). A highly flexible reduction of plant size might reduce competitive superiority of IG at high elevations, which has been suggested to also influence the latitudinal limits of this species (Kollmann and Banuelos, 2004). However, results of our field experiment do not support this idea, since we found no effect of competition treatments on performance (size and biomass). It is possible that the effects of our competition treatments, although using a high density of competing species, were reduced by background competition, which the non-competition pots also experienced, e.g. possible root intrusion of surrounding plants. Furthermore, the competing species were sown and, thus, balsams experienced competition from seedlings and one year old individuals, which are generally less competitive than adult individuals, and priority effects, that is, temporal competitive advantages due to earlier development might have confounded results (Mangla et al., 2011). On the other hand, invasive balsams primarily establish at sites with recent soil disturbance, while allelopathic effects might play a prior role only in subsequent years. Thus, the experimental settings are probably rather close to natural establishment conditions. Yet, we cannot be entirely

sure whether competition truly plays a minor role, as our experimental results suggest. 4.2. Germination and establishment High seedling emergence rates in the field experiment indicated that seed survival during winter does not restrict the occurrence of any congener at high elevations, and indeed IG even showed higher seedling emergence rates with elevation. Most likely, the high cold-wet stratification needs of the species contribute to this result, with smallest requirements in IG (about 120 days), IP intermediate (195 days), and IN longest (210 days) of temperatures below 5 °C (Perglova et al., 2009). These are higher than we found at 300 m (129 days), and thus, the stratification requirements of species were probably not completely fulfilled at 300 m. Slightly lower seedling emergence rates of IN in comparison to the other congeners are in accordance with previous studies (Perglova et al., 2009; Skalova and Pysek, 2009), which might point towards lower seed fecundity and survival, or relate to the capability of IN to persist in short-term seed banks for up to three years, while the invasive congeners do not have a seed bank beyond one winter (Perglova et al., 2009). Seedlings emerged highly simultaneously, and dates overall did not differ between the three congeners. Only small species-specific differences, as well as a concurrent seedling emergence have been reported for this Genus previously (Beerling and Perrins, 1993; Kollmann and Banuelos, 2004; Andrews et al., 2009). The tendency of IG to germinate earlier at higher elevations is also supported by a previous study, which found an earlier seedling emergence of this species in the laboratory, although not under field conditions (Perglova et al., 2009). Although trait measurements showed that leaf frost sensitivity does not change with elevation, it revealed that native IN was more frost resistant than both invasive congeners. In accordance with this, we noted frost damage for both invasive species, but none for native IN during the experiment. This is in accordance with laboratory results for Czech balsam populations, where invasive IP also showed highest frost sensitivity, followed by invasive IG, and native IN being least vulnerable (Skalova et al., 2011). Although PEL measurements in populations overall indicated that both invasive congeners were more susceptible to frost, it did not identify that IP leaves were more susceptible than IG. However, we can't exclude that seedlings show different susceptibilities compared to adult individuals. Data from the experimental sites clearly indicated that herbivory, mainly by snails and slugs, influenced seedling survival considerably. About a third of the individuals were affected, which locally (at one site) led to a complete establishment failure. In contrast to expectations (enemy release hypothesis, for which highly mixed empirical support is given (Jeschke et al., 2012)), we found the invasive congeners to be equally affected as native IN. 4.3. Reproduction and life-cycle With respect to phenological development, the results of the experiment supported the results of the field study. For the natural populations, native IN showed an advanced development, and thus entered the reproductive phase earlier than the invasive congeners, especially at higher elevations. This pattern was repeated in the experimental sites, with invasive IG overall delaying the reproductive phase most (Fig. 2), and invasive IP delaying development more than native IN at higher elevations. In combination with the rather low frost tolerances of adult individuals of the invasive species, as shown by the field study, these findings suggest that both the invasive species are less likely to successfully reproduce at higher elevations, at least in years with early autumn frosts. As analysis of climate data suggests (Fig. 3), years with very short growing seasons (120 days) between last spring and first autumn frost are expected to recur within a few decades. In comparison, it took invasive

J. Laube et al. / Biological Conservation 191 (2015) 682–691

IG on average 100 days from seedling emergence to start of flowering, and thus this species likely fails to reproduce once within every few decades. At harvest, the individuals of invasive IG also displayed, on average, a lower number of reproductive organs, which expectedly results in reduced seed output, at least at high-elevation sites, where the reproductive period will be considerably shorter than in the lowlands due to rather low frost tolerances of the species. Growing season length was suggested to also limit the latitudinal range of IG (Beerling, 1993). It has to be noted that lower numbers of reproductive organs for IG might relate to an unintended, slightly delayed development phase at the time of harvest, although statistical models accounted for this. 4.4. Main drivers of elevational limits To conclude, our results suggest that the occurrences of the three congeneric balsam species along the elevational gradient are linked to differing stratification and seedling emergence patterns, seed bank, frost resistance, and last, but not least, time needed for life-cycle completion. More specifically, invasive IG is probably not able to successfully complete its life-cycle and produce abundant seed, on a highly regular, yearly basis. The very long period needed from seedling emergence until the start of flowering suggests a high vulnerability to autumn frosts. For invasive IP, a simultaneous seedling emergence pattern, combined with a very low frost resistance, is likely to result in establishment failures in years with severe or late spring frosts. Since both congeners lack a seed bank, each year of reduced establishment or reproduction success is likely to decrease next years' population size considerably. In the case of severe and late spring frosts, or very early autumn frosts, complete diebacks of populations at high elevations for the invasive balsams seem likely. Although the trait measurements suggested that a reduced competitive ability of invasive congeners at higher elevations might also contribute to upper range limits, our experiment did not support this idea. Likewise, a lack of trait plasticity seems unlikely to be of major importance. We generally found small responses of traits to elevation in all three congeners, and the least plastic response was found for native IN, which is the most abundant at highest elevations. 4.5. Expectation with climate change

689

need to be of concern primarily if highly valuable habitats are affected immediately. Since the surrounding lowlands are completely invaded, eradication measures are not feasible, although propagule pressure is seen as a prior driver of invasion risk (Lockwood et al., 2007; Pysek et al., 2015). However, climate data analysis indicates that serious climatic constraints relate to an elevation of about 600 m a.s.l. (Fig. 3). Therefore, we suggest that eradication measures at infestations above these elevations might be most cost-effective. Herbivory by terrestrial molluscs seems to be an important establishment barrier for the invasive balsams, and thus, any management in favour of this species group will contribute to maintain a high biotic resistance. In the area, both abundance and number of molluscs are highly linked to less disturbed sites in terms of tree age, habitat continuity and the amount of woody debris (Müller et al., 2009). Therefore, protection of the existing unmanaged sites, as well as an expansion of these protected sites is also of high value with respect to balsam invasions.

4.7. Conclusions Our results indicate that the current elevational range limits are linked to the thermal niche of the species, but nevertheless are unlikely to be predictable only by changes in mean temperatures. Unfortunately, information on both “traits” that are highly influential for upper range limits of the species (phenology and leaf frost resistance) are usually missing in trait databases, and key climate data for these species distributions (timing of frost events) are not always fully available. We argue that a prediction of species occurrences based on mean climatic input variables needs to consider carefully whether the critical development phases are included, especially if these are annual plants with a poor seed bank. This applies not solely to invasive species or herbs, but has been shown to be of importance also for elevational limits of tree species (Kollas et al., 2014; Vitasse et al., 2014a, 2014b). Our results also highlight the necessity to better include life-cycle timing, and phenological development of species into distribution models (Chuine, 2010), especially as these themselves are sensitive to climatic change (Menzel, 2002; Menzel et al., 2006; Cleland et al., 2007; Polgar and Primack, 2011). We agree that improved methods to incorporate temporal aspects are needed (Wolkovich et al., 2014), and suggest that the development of a temporal component of climate envelope models is urgently needed.

Since both invasive congeners seem to be directly limited by climatic conditions, upward shifts have to be expected with climate change. With an increase in growing season length and temperature, the reproduction success of invasive IG is expected to increase and stabilise at higher elevations. Yet, these effects will probably counteract increased establishment failure due to an increase in late spring frost frequency, which is expected with warming and with decreasing snow cover (Inouye, 2008; Kreyling, 2010; Augspurger, 2013). Furthermore, the first autumn frosts are generally expected to occur later, but trends are spatially highly diverse, and in parts even insignificant (Menzel et al., 2003). For invasive IP, an increase of growing season length and temperature will likely cause little effect. In contrast, an increased frequency of late spring frost events will challenge existing populations at higher elevations. Taken together, our data suggest that an upward shift of both invasive balsams will likely be smaller than the changes in mean temperatures suggest.

We thank Kathrin Ziegler for her help with setting up the experimental sites, and Michael Matiu for his statistical advice. Our special thank is to Helga Merxmüller and Michael Teig for their kind help with collecting jumpy seed, to Amanda Gallinat for her valuable comments on an earlier draft of this manuscript, and to two anonymous Reviewers for their very helpful suggestions. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement n° [282250]. With the support of the Technische Universität München — Institute for Advanced Study, funded by the German Excellence Initiative.

4.6. Management implications

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