Phenotypic trait differences between Iris pseudacorus in native and introduced ranges support greater capacity of invasive populations to withstand sea level rise

Tidal wetlands are greatly impacted by climate change, and by the invasion of alien plant species that are being exposed to salinity changes and longer inundation periods resulting from sea level rise. To explore the capacity for the invasion of Iris pseudacorus to persist with sea level rise, we initiated an intercontinental study along estuarine gradients in the invaded North American range and the native European range.


| INTRODUC TI ON
Alien plant invasions are global environmental changes that result from anthropogenic movement of species beyond their natural dispersal barriers to new geography and distribution ranges (Vitousek et al., 1997). Global climate change is also dynamically changing habitat conditions in introduced and native ranges of species (IPPC, 2022). Invasion risk is expected to continue to change with climate change because invaders may be better able to succeed in novel and altered environments and benefit from increases in resource availability (e.g. nitrogen and CO 2 ). This highlights a crucial need to improve understanding of invasion risk under global change to support management programmes relevant for the 21st century (Bradley et al., 2010).
The ability to predict what biological attributes drive the invasiveness of plant species can provide foundational support for preventing introductions of alien species with traits that deem them a risk for invasion. Reviews (Pyšek & Richardson, 2008) and metaanalyses (Davidson et al., 2011;Palacio-López & Gianoli, 2011;van Kleunen et al., 2010) suggest functional plant traits related to growth, biomass allocation, physiology, fecundity, and phenotypic plasticity support invasiveness of alien plant species. Environmental variation within intercontinental distributions of invasive alien plant species can influence plant traits that support their fitness, establishment, and spread (Hierro et al., 2005;Pearson et al., 2022). Therefore, investigations conducted through a biogeographical framework coupled with a functional trait approach (Drenovsky et al., 2012) can provide unique insights on how an invasive plant species responds to environmental factors affected by climate change. Incorporating biological traits into conservation biogeography across species ranges can also be crucial for development of risk assessment predictions addressing responses of vulnerable ecosystems to environmental change (Dong et al., 2022;Miatta et al., 2021).

Alien populations that become invasive in introduced ranges
are often assumed to be more abundant and grow larger than their conspecific populations in the native range, though most research has been limited to the introduced range (Guo, 2006;Hierro et al., 2005). The few studies focused on plants in their native range that are invasive elsewhere suggest some species are pre-adapted to invasion van Kleunen et al., 2011;Jelbert et al., 2015). Some alien species may acclimate to environmental change and maintain fitness through phenotypic plasticity without evolutionary adaptation (Barrett, 2000;Pearman et al., 2008).
Adaptive phenotypic plasticity can alter functional traits to maintain fitness and alter the ecological niche breadth (Colautti et al., 2017).
In addition, release from environmental stress and natural enemies (e.g., herbivores) in introduced habitats may favour establishment and spread of alien species and may alleviate the negative effects on fitness of depletion of genetic variation derived from demographic bottlenecks (Colautti et al., 2017;Schrieber & Lachmuth, 2017).
Purging genetic loads from founder effects are also now thought to enable alien plants to adapt more quickly via rapid evolution (Marchini et al., 2016). As a result of the continuing evolution of alien plant species, some functional plant traits specifically underlie their success (Bajwa et al., 2016;van Kleunen et al., 2010).
Understanding which plant traits might lead to invasion success is particularly important in sensitive wetland habitats. Wetlands account for less than 6% of Earth's landmass yet have been highly vulnerable to biological invasions due to their landscape sink position, with 24% of plant species identified as the world's worst invaders being wetland plants (Zedler & Kercher, 2004). Of these wetlands, tidal marsh ecosystems have been highly impacted by the invasion of alien species (Adam, 2002). With global climate change, plant species colonizing tidal marshes are being exposed to sea level rise (SLR) and concomitant changes in tidewater salinity and inundation (Morris et al., 2002;Thorne et al., 2018). The modification of salinity and inundation regimes is crucial since these key environmental stressors drive wetland vegetation change in response to SLR (Baldwin & Mendelssohn, 1998). The distribution patterns across freshwater to brackish estuarine gradients are rapidly shifting in response to SLR (Mathiventhan et al., 2022), and rapidly assembled plant communities at these moving fronts will favour alien plant species with superior colonization ability (Grewell et al., 2013). Those plant species with a greater tolerance to modifications in environmental factors and broader ecological niches will influence the future configuration of ecosystems (Thuiller et al., 2005). A key need for conservation of estuarine ecosystem functions is to increase our knowledge of the responses of alien invasive species to changing environmental conditions. Iris pseudacorus L. (yellow flag iris; Iridaceae) is a perennial macrophyte native to the British Isles, Scandinavia, Europe, the Mediterranean Region, and western Asia (Encyclopedia of Life, 2022). Iris pseudacorus has highly attractive yellow flowers which enticed 18th century botanical collectors to import it to North America (e.g., Hayden Reichard & White, 2001). In North America, I. pseudacorus was introduced in Virginia before 1771, and by 1800 it was growing along the tidal Potomac River (Wells & Brown, 2000).
In California tidal marshes, I. pseudacorus reduces species richness and diversity of invaded native plant communities, in contrast to its ecological role as a native species in the Iberian Peninsula, where it co-exists within diverse wetland plant communities (Gallego-Tévar et al., 2022). Recent downstream spread into brackish reaches of the greater San Francisco Bay-Delta Estuary was unexpected, as it had long been assumed this species would be limited to freshwater wetlands. The risk for further spread with increasing salinity and tidal ranges is not clear (Cloern et al., 2011). Given the prevalence of I. pseudacorus in the world's estuarine vegetation, it is important to understand the potential fate or persistence of the species in the native and introduced ranges as SLR continues.
To begin to understand the capacity for the invasion of I. pseudacorus to be sustained with SLR, we carried out a field study at a focused patch scale within populations. I. pseudacorus plants produce many rhizomes, resulting in a dense clonal tussock or clumping growth form. Therefore, hereafter, "patch scale" refers to the finescale assessment level within the study population, and each "patch" is our monitoring plot that spatially encompasses this discrete tussock growth form within a population.Within each assessment patch, we evaluated variation in functional plant traits and environmental variables at peak summer growth along estuarine salinity and inundation gradients in both the introduced and native ranges. Our objective was to determine whether functional traits of I. pseudacorus can explain its invasive success in the introduced range and if current environmental factors can explain phenotypic differences between ranges. We hypothesized that alien I. pseudacorus plants would express enhanced functional traits supporting greater plant vigour, with lower sensitivity to increasing salinity and inundation along estuarine gradients in comparison to native plants.

| Study sites
We established our study at I. pseudacorus population sites distributed along estuarine gradients from the freshwater tidal to brackish tidal marshes in the native range within the Guadalquivir River Estuary (GRE; Southwest Iberian Peninsula), and in the introduced range within the San Francisco Bay-Delta Estuary(SFE; Pacific Coast of North America; Figure 1). The estuaries each occur near continental plate margins, and both are classified as tectonically shaped drowned river estuaries with geomorphic complexity (Atwater, 1979;Rodríguez-Ramírez et al., 2019). Tidal marshes in both estuaries experience mixed semi-diurnal tidal regime with meso-tidal ranges and are influenced by local watershed inflow, freshwater diversions for beneficial uses such as irrigation and municipal water supply, and dam-regulated outflow from upstream reservoir releases. Mediterranean climate prevails at both estuaries, with cool, wet winters and hot, dry summers moderated by Atlantic (GRE) or Pacific (SFE) influence (AEMET, 2020;Kimmerer, 2004).
The native Palearctic biogeographic range of I. pseudacorus is believed to include central and southern Scandinavia, the British Isles, Europe, northern Africa, and western Asia (Gervazoni et al., 2020;Sutherland & Walton, 1990). However, early occurrence records Using standardized methods at all study sites, we evaluated variation in 15 functional plant traits and 11 environmental variables within fixed patch-scale monitoring plots corresponding to discrete plant tussocks (n = 7-8 per population) at five study populations of I. pseudacorus extant along estuarine gradients in both the introduced and native ranges. At all population sites in both estuaries, the team determined apical leaf elongation rates (LER) in the field by marking the base of ten leaves per plot with waterproof sealant and measuring the distance from the mark to the leaf base after 48 h (Castillo et al., 2014).

| Plant traits
Reproductive traits were also recorded. The number of capsules (0 to X) produced on each flowering stem within each discrete patch was counted. At two sites in the introduced range (C1, C3) where it was not possible to distinguish a discrete individual patch given extensive contiguous linear bands of plants, we counted capsules within a 2 × 2 m subplot which was representative of the average area sampled for discrete patches. Mature capsules were collected (c)

Invaded Range
Native Range randomly based on capsules present (n = 1 to a maximum of 40 capsules per plot) and evaluated for seed count per capsule, then airdried, and weighed to obtain mean seed mass (n = 4-10 capsules per plot; 10 seeds per capsule). In the native range, seed traits were determined for a subset of population patches given early dispersal or herbivory. Rhizome samples were randomly hand-excavated from shallow soil. Sample sizes were stratified based on scale of the occupied area of the study patch, with one sample from small patches (<1.5 m 2 ), two samples from medium plots (1.5-4.0 m 2 ), and three from large plots (>4.0 m 2 ).Rhizomes were stored in coolers with blue ice and transported to the laboratory. Rhizome samples (2-3 cm diameter × 5 cm length) were dried, ground to pass through 40-mesh sieve in preparation for analysis of total nonstructural carbohydrate concentrations (TNC). TNC concentration in rhizome samples was analysed with a colorimetric assay of reducing sugars following ethanol extraction (Chow & Landhäusser, 2004) and enzymatic digestion of the starch residue (Quentin et al., 2015).

| Environmental variables
In situ environmental conditions including soil physico-chemical characteristics and plant species cover, were sampled and assessed in each study patch of I. pseudacorus simultaneous withplant trait measurements. The presence associated plant species within I. pseudacorus patches was recorded as previously explained for I. pseudacorus using the same transects.
Soil cores (4.5 cm diameter × 10 cm depth) were collected from each monitoring plot using the same patch size-stratified sampling scheme previously described for rhizome sampling. Cores were placed in coolers with blue ice and transported to the laboratory.
Samples were oven-dried at 60°C for 48 h and then ground to pass through a 40-mesh sieve. Soil bulk density (BD) was calculated from soil dry mass and volume of the core. Soil organic matter (OM) content was determined standard loss on ignition (Nelson & Sommers, 1996).
Total soil C and N concentrations were measured using the same methodology reported above for leaves. A set of soil cores (4.5 cm diameter × 5.0 cm depth) were also collected at each plot for determination of soil pH and electrical conductivity (EC) of interstitial soil water. These samples were air-dried, ground, and passed through a 20-mesh sieve. Saturated paste extracts were obtained through vacuum filtration, and the extract was measured for soil pH (introduced range: Accumet AB15 Plus, Thermo Fisher Scientific; native range: Crison pH-meter Basic 20, Crison Instruments) and EC (mS cm −1 ) (in-

| Data analyses
Standard error (SE) was calculated for each arithmetic mean. Prior to conducting the analyses, data series were tested for normality with the Shapiro-Wilk test, for homoscedasticity with the Levene's test, and for redundancy with correlation analysis using the software SigmaPlot v. 12 (Systat Software). The variable elevation was transformed using the function 1/x and two variables (leaf N and soil organic matter) using the function log(x) to address the assumptions needed for parametric tests. Multivariate analysis of variance (MANOVA) using the Pillai's Trace test statistic were conducted for plant traits and environmental conditions recorded in the field, separately, using population as grouping factor. The analyses of multivariate variance protect subsequent analyses from type I error (Scheiner, 2001). Once multivariate significance was established, General Linear Models (GLM) with Bonferroni-Dunn's test as post hoc analysis were used to assess the main univariate differences of each variable recorded in the field using range (native or introduced) and population as grouping factors. When homogeneity of variance was not accomplished after data transformation, univariate differences were analysed using the Gamma Generalized Linear Model (GGLM) with Chi-square (χ 2 ) de Wald (Ng & Cribbie, 2017).
In addition, we also used plant traits and environmental condi-

| Plant traits
Iris pseudacorus presented approximately 80% absolute cover in the native and introduced ranges, though lower abundance (60% cover) was recorded in native population A5 ( Figure S1). In addi- when the environmental matrix is included, the differences were not significant. In contrast, patch area and the cover of iris only showed significant differences between ranges when environmental factors were included as covariables (Table S3).
Principal Components Analysis grouped plant traits into four factors that together explained 64.3% of the total variance in functional plant trait values that separated relative to intercontinental ranges. The first factor (PC1) explaining 24.7% of the variance was positively related to the number of capsules per stem, LWC and leaf length, and was negatively related to the number of senescent leaves per fan (Table S4) PC2 explaining 15.5% of the variance was negatively related to leaf N concentration and SLA (Table S4), separating the native populations along the estuarine gradient more than alien populations ( Figure 3).
Live leaves in the native range presented c. 20% higher leaf N concentration than in the introduced range (Tables 1 and S2). Leaf N concentration was the highest for the most inland population in the native range (A1). Mean SLA was consistent among populations in the introduced range and 50% higher than SLA in the native range where SLA decreased by five times moving downstream in the estuarine gradient.

F I G U R E 2 (a) Plant trait responses and (b) environmental variation in
Iris pseudacorus populations in the introduced (California, C; cyan bars) and native (Andalusia, A; yellow bars) ranges along estuarine gradients (1, upstream; 5, seaward). Data are mean ± SE (n = 7-8).
In the introduced range, mean elevation was 1.67 ± 0.06 m, increasing to a maximum of 2.65 m seawards. Mean elevation in the native range was 1.88 ± 0.03 m, with a range of 0.62 m between the highest and the lowest values. Maximum HyDx was 141 km in the introduced range and 95 km in the native range. Maximum inundation depth was 75% greater in the introduced range than in the native range ( Figure 2; Tables 1 and S2).

| Relationships between plant traits and environmental conditions
Plant traits and associated environmental conditions clearly distinguished I. pseudacorus populations in the introduced range from those in the native range (CCA ordination, Figure 4). The first two axes of the CCA explained 82.5% of the total variance in the relationships between recorded iris plant traits and the range of environmental variables associated with the monitoring plots. Axis 1 explained 60.3% of the variance and was negatively correlated with MID, soil OM, the number of capsules per stem and rhizome TNC, and positively with soil pH, BD, and leaf N concentration. Almost all the monitoring plots in the introduced range were negatively related and most plots in the native range were positively related to Axis 1 ( Figure 4; Table S5). Axis 2 explained 22.2% of the variance and was negatively correlated with HyDx and LER and positively with soil EC.
More upstream monitoring plots tended to show more negative values along Axis 2 than those plots located closer to the sea. Axes 3, 4, 5, and 6 represented just 16.4% of total variance (Figure 4; Table S5).
Simple regression analyses illustrate the relationships between soil EC and plant traits, highlighting marked differences between TA B L E 1 Mean and standard error, F-statistic and p-values of GLMs and GGLMS for plant traits and environmental conditions recorded in the field comparing between geographic ranges (introduced range, California, USA; native range, Andalusia, Spain) and between populations in both geographic ranges (N = 77) as fixed factors. geographic ranges ( Figure 5). In the native range, I. pseudacorus was more sensitive to increasing interstitial soil EC than plants in the introduced range as expressed by several key traits. Soil EC was negatively related to LWC and LER for both alien and native plants; however, the decreases at higher salinities were doubled for native than alien plants. In contrast, soil EC was positively related with senescent leaf number per fan and negatively with leaf length and width, leaf N concentration, and SLA only for native plants ( Figure 5).
The number of live leaves per fan increased with increasing IP and MID in the introduced range but not in the native range ( Figure 5).
In fact, all plant traits recoded in the native range were independent of IP and MID (Pearson correlation,p > .05), except the number of senescent leaves per fan that increased together with MID (r = .344, p = .034, n = 38).

| DISCUSS ION
We Iris pseudacorus has been described as a salt-sensitive species in a greenhouse study , coinciding with its LWC and LER decreasing seaward where soils were more saline (higher EC) in both intercontinental ranges. In fact, most of plant trait values sampled at soil salinities c. 2 ppt in the field were in the range of those recorded at salinity as high as 15 ppt in a greenhouse experiment . This result suggests I. pseudacorus plants were growing in suboptimal conditions with elevated soil salinity at both study locations. In this context, native plants were more sensitive to increasing salinity along the estuarine gradient than alien plants.
This result is informative regarding the response of I. pseudacorus to global warming and SLR in Mediterranean climate zones where soil salinity in tidal marshes is increasing (Vicente & Boscaiu, 2020).
Physiological traits that underlie water loss and carbon uptake and allocation by plants are highly plastic in response to environmental heterogeneity and are key determinants of growth and fitness (Ackerly et al., 2000;Sage, 1994). Thus, native I. pseudacorus plants tended to show lower SLA, but higher leaf N concentrations than alien plants in the introduced range, and higher numbers of senescent leaves per sprout with increasing salinity. Previous studies have recorded an increase in salt tolerance related to ion accumulation in senescent leaf tissue (Reddy et al., 2017) and increased SLA Zong et al., 2021). In contrast, alien plants, exposed to similar soil salinities as the native plants, also had decreased LWC and LER with increasing salinity gradient, but responded with about half the decrease in these leaf traits than observed for native plants.
Plant traits considered to represent variation in life history relevant to predicting invasiveness include high SLA (Hamilton et al., 2005). In this regard, the greater performance of alien I. pseudacorus plants exposed to increasing salinity was supported by leaves with 50% higher SLA and greater hydration (LWC) and growth (LER) than those in the native range. Greater SLA has been associated with higher relative growth rates and greater competitive ability in productive environments, while a lower SLA indicative of a lower relative growth rate, can provide a selective advantage in unfavourable habitats (Lambers & Poorter, 2004). It is interesting to note that alien plants maintained very similar SLA along the estuarine gradient, while SLA in native populations decreased 5-fold, corresponding to a seaward increase in interstitial soil salinity. In contrast, alien I.

F I G U R E 4 Ordination diagram of a Canonical Correspondence
Analysis (CCA) with plant traits (red circles), monitoring plots in the invaded range (purple circles) and native range (green circles), and environmental variables (blue arrows). Plant traits: C, carbon concentration; LER, leaf elongation rate; LWC, leaf water content; N, nitrogen concentration; SLA, specific leaf area; TNC, rhizome total non-structural carbohydrates. Environmental variables: BD, bulk density; C, carbon content; EC, electrical conductivityN, nitrogen content; OM, organic matter.
pseudacorus acquired sufficient resources to support robust growth while also allocating 50% more carbon storage reserves in rhizomes than native plants. In this context, the relatively low leaf N concentrations recorded for invading plants could indicate re-translocation of N from leaves to sexual reproduction and subterranean storage (Sinkkonen, 2006;Wright & Dorken, 2014). Thus, the significantly higher soil N availability observed in the introduced range may likely affect the vigour and reproductive output in alien plants (Pearson et al., 2022). Evolution of traits related to resource uptake can be relevant for alien populations (Burns et al., 2013). SFE has higher F I G U R E 5 Relationships between soil electrical conductivity (EC) and plant traits in the introduced range (a, c, e, g, i, k) and native range (b, d, f, h, j, l). Relationships between maximum inundation depth and the number of live leaves per leaf fan for Iris pseudacorus in ( anthropogenic loadings of both N and P concentrations than those in most other global estuaries impaired by nutrient pollution (Cloern et al., 2020). In addition, climate change is also increasing atmospheric nitrogen deposition, enhancing the growth of fast-growing alien plant species (Suddick et al., 2013).
Extreme meteorological events, such as torrential rains, droughts and heat waves, are more frequent now in the actual scenario of climate change (Payne et al., 2020). The water depths we recorded in the introduced range reflected the atmospheric river storm flooding that occurred in SFE in 2017 (Thorne et al., 2022). Even though the year was atypical, if we exclude analysis of the above average data from 2017, maximum inundation depth was still considerably higher in the introduced range when compared to the native range. Even under the extreme conditions which are recurring more frequently, I.
pseudacorus in the invasive range had greater functional trait capacity to counter environmental stress.
Iris pseudacorus has been experimentally shown to be highly tolerant of inundation . In this sense, most expressed functional traits by native plants were independent of inundation period and depth, except the number of dead leaves per leaf fan that increased with increasing inundation. In the introduced range, alien I. pseudacorus plants were exposed to deeper inundation than native plants, which illustrated a niche shift between the study areas in the native and introduced ranges. Yuan et al. (2021) also recorded niche shift between intercontinental introduced and native ranges for Spartina alterniflora Loisel., also a tidal marsh invader. In this context, alien I. pseudacorus plants increased the number of live leaves when exposed to deeper inundation. This response seems to reflect the capacity of I. pseudacorus to respond to rising inundation levels since increasing resource allocation to greater leaf biomass production can facilitate carbon acquisition by increasing photosynthetic area (Zhao et al., 2015).
In this sense, Grewell et al. (2021) recorded an increase in leaf mass ratio under deeper inundation in a greenhouse experiment. Capsule and seed traits such as high seed output and germination rates can be critical for dispersal and establishment success of alien plants (Rejmánek & Richardson, 1996;Pyšek, 1998). In the introduced range, I. pseudacorus had 40% more capsules per stem than the native counterparts, suggesting greater propagule pressure. Furthermore, seed viability and seed germination rates were found to exceed 95% for I. pseudacorus in our SFE study populations (Gillard et al., , 2022, which should lead to more invasive colonization according to the hypothesis of propagule pressure (Carr et al., 2019).
Some plant traits showing significant differences between geographic ranges were markedly related to variation in environmental conditions and, in contrast, other plant traits were independent of every recorded environmental factor. Ecologically important functional traits of some alien plant species have undergone rapid evolution as they naturalize during range expansion, increasing genetic variation of traits along environmental gradients that may support broadened niche shifts beyond those of founder populations (Colautti et al., 2017). Growing empirical evidence indicates adaptive traits have evolved in introduced plant populations that have become invasive (e.g. Dlugosch & Parker, 2008;Lavergne et al., 2010;Molina-Montenegro et al., 2011, and some of these evolutionary adaptations have been quite rapid (Colautii & Barrett, 2013;Colautti et al., 2017;Leger & Rice, 2007;Molina-Montenegro et al., 2018). In this sense, our results suggest there are genetic differences in iris populations between the studied biogeographical ranges since six plant traits showed significant differences between ranges only when environmental variables were excluded as covariates. Also, only two plant traits differed between ranges only when the environmental matrix was included in the analysis, which pointed to limited environmental influence on plant trait differences between geographical ranges. Explanatory mechanisms for the differences we documented between ranges of I. pseudacorus cannot be fully interpreted without support of common garden experiments and molecular evaluations (Bufford & Hulme, 2021;Colautti & Lau, 2015) to elucidate the potential genetic and environmental contributions to the variation we observed, but our results provide an important foundation for future studies.

| CON CLUS IONS
Study of Iris pseudacorus through a biogeographic framework in both native and introduced population field sites has provided insights on variation in environmental conditions and functional plant trait responses that support the fitness and spread of invasions in tidal wetlands facing SLR. Our results show that alien I. pseudacorus plants in the introduced range were more robust than plants in the native range. Alien I. pseudacorus plants were less sensitive to increasing salinity than native plants and were positively affected by higher inundation levels, reflecting a niche shift compared to our study sites in the native range. In summary, our comparative results suggest alien populations in SFE are currently better able to adjust to increasing salinity and inundation with SLR than native populations in GRE.
Biogeographic knowledge of these functional trait responses can support improved risk assessments addressing both management of invasive species and conservation of native species in tidal wetland ecosystems vulnerable to impacts of climate change.

ACK N O WLE D G E M ENTS
We thank Caryn J. Futrell, Rebecca Reicholf, Reina Neilsen, Anita Arenas, Matt Connors, Francisca Real-Castro, and Lourdes Bernal-Galiano for field assistance and Caryn J. Futrell for laboratory analyses. We also thank California State Parks for wetland access at C2 and C5. We wish to thank the Associate Editor and two anonymous referees for their constructive and valuable suggestions that improved the draft of this manuscript. USDA is an equal opportunity provider and employer. Mention of trade names or commercial products is solely to provide specific information and does not imply recommendation or endorsement by USDA and USGS.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study will be openly available in DRYAD.
"Data: Phenotypic trait differences between Iris pseudacorus in native and introduced ranges support greater capacity of invasive populations to withstand sea level rise" (doi:10.25338/B8FP72).