Greater flowering and response to flooding in Lythrum virgatum than L. salicaria (purple loosestrife)

Abstract Newly introduced trait diversity can spur rapid evolution and facilitate local adaptation in the introduced plant Lythrum salicaria. The horticultural plant L. virgatum might further introduce meaningful trait variation by escaping into established L. salicaria populations or by hybridizing with L. salicaria. Although many experiments have focused on L. salicaria genotypes, relatively little is known about L. virgatum ecology. We used a greenhouse common garden to compare traits and flood response of L. salicaria and L. virgatum collected from two sources each in their native range. We tested the hypotheses that these two wetland taxa have comparable responses to flooding (inundation), and that flood tolerance correlated to higher fitness. Flooding produced stronger stress responses in L. virgatum. Compared to L. salicaria, L. virgatum shifted more aboveground allocation away from reproduction, decreased inflorescence biomass by 40% more, and produced 7% more stem aerenchymatous phellum, a specialized tissue that maintains aeration. Despite these more pronounced responses to flooding stress, L. virgatum had higher fitness (inflorescence biomass and reproductive allocation) than L. salicaria. Overall, L. virgatum differed from L. salicaria in functionally important ways. Lythrum virgatum persisted under flooding and produced more reproductive biomass than L. salicaria under both flooded and non-flooded conditions. However, inundation stressed L. virgatum more than L. salicaria. Lythrum virgatum is likely able to establish into the wetland habitats in which L. salicaria prevails but may possess broader habitat tolerances.


Introduction
High intraspecific variability in introduced plant species can enable rapid evolution in the non-native range. For species that have been introduced repeatedly from multiple sources, high genetic and trait variation within populations is common (Dlugosch and Parker 2008;Dlugosch et al. 2015;Hodgins et al. 2018), and at the macroscale, such intraspecific trait diversity can stratify across different habitats or even climates (Colautti and Barrett 2013). A diversity of genotypes and phenotypes also provides the raw material for an introduced lineage to evolve in the longer term, with admixture among diverse genotypes creating novel trait combinations (Schierenbeck and Ellstrand 2009;Hovick and Whitney 2014). High diversity and cryptic admixture are especially common in escaped ornamental plants (Culley and Hardiman 2009). Cultivars with desirable traits are often sourced from geographically disparate sources in the native range and selected through breeding. It is thus unsurprising that horticultural escapes are common sources of invasive florae (Reichard and White 2001;Lehan et al. 2013;Beaury et al. 2021), a conservation problem that horticulturists are beginning to recognize (Schnelle and Gettys 2021).
Purple loosestrife (Lythrum salicaria) is a diverse Eurasian perennial introduced to North America both intentionally as an ornamental and unintentionally through ballast water (Stuckey 1980). Seeds rather than asexual propagules facilitate the species' spread over long distances (Yakimowski et al. 2005). Introductions date back to the early 1800s, but L. salicaria remained scarce in much of North America until the mid-1900s (Stuckey 1980). Now, L. salicaria is considered one of the worst invasive plant species in Eastern North America, with estimates of its damage and management costing up to $60-$80 million annually in the U.S. over the past 25 years (estimates adjusted for inflation; ATTRA 1997, as cited in Pimentel et al. 2005;Cusack et al. 2009;Diagne et al. 2020). The rise of L. salicaria has been driven by a variety of factors, including its competitive ability (Gaudet and Keddy 1988;Hovick et al. 2011) and function as an ecosystem engineer (Fickbohm and Zhu, 2006), potentially compounded by its release from natural enemies present in the native range (Blossey and Nötzold 1995;Hunt-Joshi et al. 2004). Perhaps more important than any of these other processes has been local adaptation (Colautti and Barrett 2013;Wu and Colautti 2022). Introduced L. salicaria has adapted across latitudinal gradients (Colautti and Barrett 2013;Wu and Colautti 2022), apparently driven by high genetic and phenotypic diversity in the introduced range (Chun et al. 2009;Colautti and Barrett 2011;Shi et al. 2018;Middleton et al. 2019). Population genetic studies of introduced L. salicaria show evidence of sexual reproduction among multiple introductions from European sources (Chun et al. 2009;Middleton et al. 2019), confirming hypotheses of early botanists who observed the species' patchwork expansion across North America over the 20 th century (Stuckey 1980). Earlier researchers hypothesized that a North American congener, L. alatum (winged loosestrife), might contribute through sexual reproduction to the diversity of invasive L. salicaria populations (summarized in Galatowitsch et al. 1999). However, studies have so far found little evidence of this phenomenon (Strefeler et al. 1996 a;Houghton-Thompson et al. 2005); this is perhaps unsurprising given that L. alatum is uncommon, restricted to high quality prairie fragments.
Another common Lythrum could complicate this story. Lythrum virgatum (European wand loosestrife) gained popularity as an ornamental perennial in North America (Harp 1967) after governments began regulating the sale of L. salicaria. Although often marketed as a sterile and non-invasive alternative to L. salicaria, cultivars are not sterile, they are only self-sterile. Lythrum must reproduce sexually to achieve high seed set (Colautti et al. 2010 b). Lythrum virgatum cultivars produce fertile crosses with L. salicaria experimentally (Anderson and Ascher 1993;Ottenbreit and Staniforth 1994) and in nature under ambient pollinator activity (Lindgren and Clay 1993;Amon et al. 2007). Because they cross readily, many have considered L. salicaria and L. virgatum the same species (Rendall 1989;Anderson and Ascher 1993;Strefeler et al. 1996b;Wilson et al. 2004;Anderson 2019). To date, taxonomic studies have not included precise enough techniques to settle the question (Strefeler et al. 1996 b;Haining and Graham 2007;Morris 2007). Such assessments are also complicated by confusion surrounding the origin of many cultivars labeled or mislabeled L. virgatum (Strefeler et al. 1996 b;Jon Peter and David Galbraith, Royal Botanical Gardens Ontario, pers. comm.). Competing interests may also be at stake. Horticulturists benefit from taxonomic splitting because L. virgatum is less often regulated than L. salicaria (though an increasing number of governments are doing so). Conservationists would benefit from considering them the same species, because any regulations applying to L. salicaria would then also apply to L. virgatum.
Regardless of whether taxonomists determine L. salicaria and L. virgatum to be the same species, L. virgatum's prevalence in landscaping and in naturalized populations in North America (Wherry et al. 1979;USDA, NRCS 2021) represents a unique store of genetic and phenotypic variability. Here we refer to the two taxa as distinct species, while acknowledging the ongoing debate. The native ranges of these Lythrums overlap, but L. salicaria's range extends farther west and north than that of L. virgatum (Fig. 1). Although North American L. salicaria has been introduced from multiple native-range sources (Chun et al. 2009;Middleton et al. 2019), the sources of popular L. virgatum cultivars are unknown (Jon Peter and David Galbraith, Royal Botanical Gardens Ontario, pers. comm.). Native habitats for the two taxa are apparently similar, with L. virgatum described as occupying 'damp places' and L. salicaria 'damp grasslands, banks' (Haining and Graham 2007). Ploidy differs in the two species. Lythrum virgatum is diploid and mostly n = 15 (Graham and Cavalcanti 2001). Lythrum salicaria shows more variation in both base chromosome number and ploidy, with tetraploids (n = 15) most common in the native range (Graham and Cavalcanti 2001;Kubátová et al. 2008) and the only cytotype detected in North America (Kubátová et al. 2008). Several morphological traits differ between the two species. Lythrum virgatum has traditionally been reported as being overall less robust and having smaller flowers, narrower leaves, shorter height, and less pubescence overall than L. salicaria (Haining and Graham 2007).
The trait diversity L. virgatum could contribute, through hybridization or establishment into the wild, may be relevant to the evolution and ecology of L. salicaria, particularly in the context of invasions. Trait variation in L. salicaria facilitates local adaptation in the introduced range (Colautti and Barrett 2013; Wu and Colautti 2022). The most relevant components of such variation are those that translate into ecologically pertinent phenotypes, which may facilitate invasiveness (Dlugosch et al. 2015). An important ecological context for these wetland species is flooding. Many researchers have studied effects of flooding on L. salicaria from the introduced range (Merendino et al. 1990;Haworth-Brockman et al. 1993;Weiher et al. 1996;Mal et al. 1997;Stevens et al. 1997Stevens et al. , 2002Lempe et al. 2001;Ferrarese and Garono 2011), native range (Bastlová et al. 2004), and both (Chun et al. 2007;Chun 2011). Lythrum salicaria responds to flooding by producing adventitious roots from stems and aerenchymatous phellum on stem and roots (Schenck 1889;Stevens et al. 1997Stevens et al. , 2002Lempe et al. 2001). Studies of L. salicaria have shown these adaptations function to maintain aeration of both root and aboveground cells experiencing inundation (Stevens et al. 2002), and, along with other trait shifts (Haworth-Brockman et al. 1993;Mal et al. 1997;Stevens et al. 1997Stevens et al. , 2002, facilitate flood tolerance. Flood response of L. salicaria has not been compared to that of L. virgatum. Assertions that the two taxa are the same species (Rendall 1989;Anderson and Ascher 1993;Strefeler et al. 1996 b;Wilson et al. 2004;Anderson 2019)-combined with observations that L. virgatum produces aerenchyma (Schenck 1889) and grows in wet conditions in the native range (Haining and Graham 2007;Lehner and Grill 2013)-suggest L. virgatum and L. salicaria should show comparable responses to flooding. Similarities in the two taxa's responses to moisture gradients would suggest that introduced L. virgatum could escape into the same habitats long occupied by L. salicaria, potentially introducing new trait diversity. Differences in flood response might indicate some differentiation in the types of sites L. virgatum could colonize. We compared traits and flood responses of L. salicaria and L. virgatum collected from sources in their native range. We used morphological data from a greenhouse experiment to test the hypotheses that: 1) the two species have comparable flooding response, and 2) that flood tolerance is associated with higher fitness (quantified here as inflorescence biomass) under flooded conditions in both species.

Plant material
We obtained seeds of two different accessions for each species from Europe. All four accessions originally hailed from local natural collections rather than horticultural sources, an important requirement given the unknown origins of many cultivars (Strefeler et al. 1996 b). Two accessions of L. salicaria, one each from Cornwall and Surrey, UK; were provided by the Millennium Seed Bank Partnership, Kew Gardens. We obtained two L. virgatum accessions, one from Rostock Botanical Garden, Rostock, Germany, and one from Anastasie Fatu Botanic Garden, Iaşi, Romania. Purported species identities of each accession matched identifying traits described by Haining and Graham (2007). Compared to the L. salicaria accessions, L. virgatum plants were overall smaller with smaller flowers (note flower size differences in Fig. 2A,B) and were glabrous rather than hairy ( Fig. 2A,B). We also measured calyx appendages and calyx lobes; L. salicaria has long appendages, while those of L. virgatum are much shorter (Haining and Graham 2007). Ratios of calyx appendage length to calyx lobe length ( Fig. 2A

Experimental design
Seeds from all accessions had high survivability (~90%) when germinated in petri dishes on moist filter paper. We transplanted germinants into fertilizer-free propagation mix (LM-AP All Purpose Mix, Lambert, Rivère-Ouelle, Quebec, Canada). We retained four robust genets of each of the four accessions to serve as maternal plants for our experiment. Within accessions, these genets were presumably distinct genotypes, although no information was provided regarding how our source seeds were originally collected. We grew and acclimated genets for about one year in a greenhouse at The Ohio State University (Columbus, Ohio, USA) under 16 h/d of light. One L. salicaria (from Cornwall, UK) died during this time, bringing the total to 15 genets. We cloned genets by planting stem cuttings dipped in rooting hormone (Garden Safe TakeRoot, Spectrum Brands, Middleton, Wisconsin, USA) in germination trays. We allowed cuttings at least 20 d to establish, then selected robust individuals to be transplanted into 20 cm diameter × 13 cm tall pots with drainage holes, filled with propagation mix (LM-ORG, Lambert, Rivière-Ouelle, Québec, Canada) mixed with ~1% solution of dish detergent (Ajax, Colgate-Palmolive, New York, New York, USA) to aid rewetting. Prior to initiation of treatments, these pots were kept in trays and bottom-watered every other day by filling the tray to a depth of ~5 cm (8 cm below the top of the pot).
We used a randomized block design with 64 individuals total: two greenhouse rooms × two species × two seed accessions × four genets (with the contribution of one salicaria Cornwall mother doubled) × two experimental treatments. This procedure resulted in eight replicates per species (two accessions × four clones) per treatment in each of two blocks (i.e., greenhouse room). We randomized placement of individuals within replicates.

Treatments-
After a period of 60 d post-transplant, we began preparing plants for the start of our two treatments: flooded and nonflooded conditions. We first spread a ~3 cm layer of coarse sand (Paver Base, Quikrete, Atlanta, Georgia, USA) across the top of all pots to prevent flooded individuals from floating. All individuals, flooded and non-flooded, received sand to control for any effect this media addition might introduce. We trimmed aboveground biomass to standardize individuals to a similar size prior to the start of treatments. We retained the single largest stem, trimming any additional stems flush to the sand. We trimmed the single retained stem to a height of 30 cm above the sand and removed any lateral branches > 1 cm long. We collected the removed plant material, separated it into vegetative and inflorescence fractions, measured the dry mass (after drying at 60°C), and used these data as model  (Haining and Graham 2007) used to confirm species identities for each accession. Photos and measurements taken with Olympus SZX7, SC100, and cellSens (Olympus, Tokyo, Japan). (A) Collection from Cornwall, UK, consistent with L. salicaria: hairy with calyx appendages nearly twice as long as calyx lobes. (B) Collection from Rostock, Germany, consistent with L. virgatum: glabrous with calyx appendages shorter than calyx lobes. (C) Example of preserved stem cross section used to measure aerenchyma production, or the ratio of stem aerenchyma area to total stem area (sample from flooded L. virgatum, Iaşi, Romania).
covariates to control for pre-experiment size differences among individuals. We also noted at this time which individuals were flowering prior the start of the experiment. We then nested all 13 cm tall pots within larger ~19 L pots. For the flooded treatment, the outer pots were 28 cm in height and did not contain drainage holes. For the non-flooded controls, the outer pots were holed pots with heights of 24-31 cm. These heights varied due to product availability, but we spread pots of different heights evenly across different replicates and accessions. Nine days after these preparations were completed, we initiated the flooding treatment. For flooded individuals, we lined the unholed outside pots with 2 mm contractor bags (Up & Up, Target, Minneapolis, Minnesota, USA) to ensure they were watertight and filled them to a water depth of ~27 cm (14 cm above the top of the inner pot containing the plant). An inundation depth of 14 cm is near the upper limit reported for L. salicaria in the wild, and the species is seen across a wide range of moisture regimes, from irregular soil saturation to permanent inundation (NCHRP 1996). For non-flooded individuals, bottom-watering proceeded as previously described. We topped off water every other day and replaced flooded individuals' water every 7 d to limit algae growth. We ran the experiment for a total of 53 d, at which point some flooded individuals were showing signs of senescence. Given these species' invasive tendencies in the area we conducted this study, we did not cross-pollinate individuals, meaning they did not set seed (these heterostylous species require crossing to set seed [Colautti et al. 2010 b]).

Data collection
To quantify each species' response to flooding, we collected aboveground biomass and sampled stem aerenchymatous phellem at the end of the experiment. We separated the aboveground material into vegetative and inflorescence biomass and measured dry masses. Fractioning the biomass allowed us to examine potential differences in allocation strategies, which have been shown to be genetically controlled in L. salicaria (Olsson 2004;Colautti and Barrett 2011). We investigated total aboveground biomass as a metric of overall plant size and vigor, as well as two indicators of fitness. The first was inflorescence biomass, to gauge total reproductive output. The other fitness metric was the ratio of inflorescence biomass to total aboveground biomass, or an individuals' reproductive allocation. We also quantified the aerenchymatous phellum that formed around the interior vascular cylinder of the stem (Fig. 2C)-a flood tolerance response well-characterized in L. salicaria (Schenck 1889;Stevens et al. 1997Stevens et al. , 2002Lempe et al. 2001) and also observed for L. virgatum (Schenck 1889). From the largest stem of each individual, we cut a 4 cm portion starting 1 cm from the top of the soil and submerged it in FAA (Formalin-Alcohol-Acetic Acid, 10%: 50%: 5% + 35% water). Then, we hardened the tissue by storing it in 70% ethanol. From each stem section, we used a razor blade to take a cross section from the middle (~3 cm from the top of the soil). We photographed cross sections (Olympus SZX7, SC100, and cellSens, Olympus, Tokyo, Japan) and used winFOLIA (Regent Instruments, Sainte-Foy, Quebec, Canada) to draw measurement polygons around the inner vascular cylinder and the outer aerenchyma. The aerenchyma area equaled the area inside the outer polygon minus that inside the inner polygon. We calculated aerenchyma production: the ratio of aerenchyma area to total stem area. Aerenchyma measurements excluded data from one flooded German L. virgatum individual that died a few days before harvest and for which the aerenchyma had become visibly deflated. Our dataset is available as Supporting Information 1.

Data analysis
We tested Hypothesis 1 by comparing all traits across species by treatments and Hypothesis 2 by testing whether our two fitness indicators (inflorescence biomass and reproductive allocation) were predicted by our flood tolerance metric (aerenchyma production). To compare how many individuals of each species were flowering prior to the start of the experiment, we used a Pearson's Χ 2 test (stats::chisq.test; R v. 4.0.3, R Development Core Team 2020). For all other analyses, we used linear mixed models with general purpose optimization (Pinheiro et al. 2020). For Hyp. 1 models, we tested whether trait values were predicted by species × treatment interactions. Hypothesis 2 models had a fitness metric as a response variable and aerenchyma production as a predictor. Species was also included as a predictor in Hyp. 2 models, because the relationship between aerenchyma production and fitness might be moderated by species. Hypothesis 2 models included only individuals that produced aerenchyma (flooded treatment). All Hyp. 1 and 2 linear mixed models included random effects for block (i.e., replicate) and accession, and we weighted variance by accession to address variance heterogeneity. Analyses of the three biomass-based responses included as covariates their respective pretreatment measurements. These covariates accounted for the fact that some plants had grown larger than others prior to being trimmed to a standard size at the start of the experiment, and some had initiated flowering while others had not. Analyses of traits other than total aboveground biomass also included that trait as a covariate, so that model results could be interpreted independent of potential size-based trait differences. The statistics we present for each predictor from the linear mixed models are P-values for significance, calculated from type III sums of squares (Fox and Weisberg 2019), and β coefficients to describe the strength of relationships. For continuous predictors, β coefficients are interpreted as regression coefficients or slopes of the relationship of that predictor to the response (e.g., for Hyp. 2, the relationship of aerenchyma production to a fitness metric). For categorical predictors, β coefficients contrast means of each category (e.g., for Hyp. 1, the mean trait value for L. virgatum is compared to the mean for L. salicaria).

RESULTS
Tests of Hyp. 1 revealed that the two species differed in their trait values and responses to flooding (Table 1.1). Comparisons of flowering prior to the start of the experiment showed that L. virgatum's 28% flowering rate did not differ statistically from L. salicaria's 9% flowering rate (Χ 2 = 2.564, P = 0.109). After adjusting for pre-treatment biomass via the covariate, aboveground biomass response to flooding also did not differ between species (Table 1.1, Fig. 3A). Lythrum virgatum did produce significantly more total inflorescence biomass, 30% more than L. salicaria (Table 1.1, Fig. 3B), and allocated a 76% higher proportion of its aboveground biomass to inflorescences (Table 1.1, Fig. 3C). Averaged across species, flooding decreased aboveground biomass by 29% (Fig. 3A, Table 1.1). Flooding decreased total inflorescence biomass by 40% more in L. virgatum than in L. salicaria ( Fig. 3B; interaction, Table 1.1). Lythrum virgatum also decreased reproductive allocation over six times more strongly in response to flooding than did L. salicaria, which did not shift its allocation under flooding (Fig. 3C, Table 1.1). Every flooded plant produced stem aerenchymatous phellum, with L. virgatum producing 7% more aerenchyma per stem area than L. salicaria (Fig. 3D). These species differences in traits and fitness in response to flooding were independent of differences in overall size (total aboveground biomass covariate, Table 1.1).
Tests of Hyp. 2 allowed us to determine whether propensity to produce aerenchyma was adaptive (Table 1.2). We found no relationship between aerenchyma production and total fitness (inflorescence biomass, Table 1.2, Fig. 4A) or reproductive allocation (Table 1.2, Fig. 4B). Any apparent relationships were explained by differences in overall size (total aboveground biomass covariate, Table 1.2).  (Pinheiro et al. 2020) testing Hypothesis (Hyp.) 1, that each species has comparable traits and flooding tolerances (

DISCUSSION
We sought to investigate whether the little-studied ornamental species L. virgatum is similar to or different from L. salicaria in ecologically meaningful traits and in response to flooded conditions. We revealed similarities as well as important differences that challenge the assumption that these two taxa are indistinguishable (Rendall 1989;Anderson and Ascher 1993;Strefeler et al. 1996 b;Wilson et al. 2004;Anderson 2019). Our findings are relevant in the applied context of L. salicaria invasions. L. virgatum may be an underappreciated store of adaptive trait diversity.
Higher fitness in L. virgatum than L. salicaria One of our main findings was that L. virgatum had higher fitness than L. salicaria based on inflorescence biomass and reproductive allocation. This finding does not support the Hyp. 1 prediction of overall similarity between the two species. Fitness differences between the two species might be explained by constraints between flowering time and overall size. Flowering time is a genetically-based trait that governs adaptive responses in L. salicaria (Olsson 2004;Barrett 2011, 2013;Wu and Colautti 2022). Flowering time also experiences a tradeoff with size, where smaller L. salicaria genotypes flower earlier, and larger genotypes flower later, putting on more vegetative biomass before shifting their allocation to reproduction (Bastlová and Květ 2002;Olsson and Ågren 2002;Bastlová et al. 2004;Montague et al. 2008;Colautti et al. 2010a;Barrett 2011, 2013;Middleton et al. 2019;Wu and Colautti 2022). Consistent with the flowering time-size tradeoff, we saw trends wherein L. virgatum plants were smaller than L. salicaria and had more individuals flowering at the start of the experiment (though these differences were not statistically significant). By the end of the experiment, the larger L. salicaria consistently allocated more of its aboveground  (Table 1). (A) Total aboveground biomass. (B) Inflorescence biomass. (C) Reproductive allocation (the ratio of inflorescence biomass to total aboveground biomass). (D) Aerenchyma production (the ratio of stem aerenchyma area to total stem area, measured from stem cross sections), for which all standard errors < 0.012. biomass to vegetative growth while the smaller L. virgatum allocated more to reproduction. As a result, L. virgatum was more fit based on our fitness metrics under both flooded and non-flooded conditions. If given more days to grow, our L. salicaria might have reached total fitness comparable to L. virgatum. For consistency, we harvested all individuals at the same time, when the flooded individuals were starting to senesce but potentially before some non-flooded individuals had reached their full reproductive output. The timing of our experiment is however reflected in relevant real-world scenarios. At more northern latitudes, earlier frosts might cut the growing season short. Earlier flowering might allow L. virgatum to thrive in the north, while L. salicaria might be more suited to establishing in all but the most northerly parts of the range. In L. salicaria, the early-flowering strategy predominates in the north (Olsson and Ågren 2002;Bastlová et al. 2004;Montague et al. 2008;Colautti et al. 2010a) and can be adaptive (Shadel and Molofsky 2002;Colautti and Barrett 2013;Wu and Colautti 2022). At southern latitudes with longer growing seasons, the late-flowering strategy is more common (Olsson and Ågren 2002;Bastlová et al. 2004;Colautti et al. 2010a) and adaptive (Montague et al. 2008;Colautti and Barrett 2013;Middleton et al. 2019;Wu and Colautti 2022). Studies are needed to examine potential local adaptation and parse variation in flowering time strategies across these species. Variation at the within-species level should also be examined by separately examining multiple genotypes of each species.
Differences in flowering between L. salicaria and L. virgatum might be controlled by a simple mechanism: whole genome duplication. Lythrum virgatum is diploid (2x = 30; Graham and Cavalcanti 2001), and L. salicaria from the source locations we used are most likely tetraploids (4x = 60; Kubátová et al. 2008). Consistent with our observations for the likely-polyploid, later-flowering L. salicaria, polyploids can show slowed growth and delayed flowering (del Pozo and Ramirez-Parra 2015; Laport et al. 2016). If ploidy does constrain flowering time, L. virgatum might be able to flower earlier than any L. salicaria (except perhaps for rare diploid L. salicaria, Kubátová et al. 2008). Higher fitness in L. virgatum versus L. salicaria might be generalizable beyond our experimental conditions because polyploids commonly show decreased allocation to sexual reproduction (Levin 1975;Van Drunen and Husband 2018). It is also likely, however, that diverse genotypes of both species contribute more to trait variation than ploidy alone, encompassing much more diversity than was captured by the two sets of genotypes per species that we used.
We quantified fitness as inflorescence biomass and reproductive allocation. Lythrum virgatum fitness traits are previously unexamined, but L. salicaria researchers have used fitness proxies including seed set (Montague et al. 2008;Colautti et al. 2010a), fruit number (Colautti et al. 2010a, b;Colautti and Barrett 2013), flower number (Chun et al. 2007;Chun 2011), and inflorescence biomass (Bastlová and Květ 2002;Bastlová et al. 2004;Colautti and Barrett 2011;Middleton et al. 2019;this study). We intentionally did not allow individuals to create mature fruits and to set seeds, given their invasive status in the region where we conducted this study. Using floral biomass as our fitness metric assumes that having more flowers is adaptively important. Rather than reproduction, environmental factors might limit these long-lived perennials.

Different responses to flooding in L. virgatum and L. salicaria
Flooding is one factor with the potential to impact Lythrum performance. In our experiment, flooding caused both species to decrease total biomass and inflorescence biomass. Some studies of L. salicaria have seen opposite effects of flooding stress, including increased allocation either to floral production or to aboveground vegetative biomass (Mal et al. 1997;Bastlová et al. 2004;Chun et al. 2007). Our differing results likely result from our use of a relatively intense duration and degree of inundation, near the upper tolerance recorded for L. salicaria in the wild (NCHRP 1996). This intense flooding stressed L. virgatum more than L. salicaria, evidenced by L. virgatum's stronger decreases in both fitness metrics. The mechanism behind these responses could be differences in flowering time, the key trait that controls adaptive responses in L. salicaria (Olsson 2004;Barrett 2011, 2013;Wu and Colautti 2022). The smaller, earlier-flowering L. virgatum might respond more strongly to stress than the larger, later-flowering L. salicaria. We did not measure whether flooding affected flowering time but did qualitatively observe other phenological effects: flooding resulted in earlier onset of senescence and, in one case, resulted in mortality. Although L. virgatum was more stressed by flooding, it still maintained higher fitness than L. salicaria in both conditions of our experiment. Under stress, which suppressed fitness of both species, any fitness differences that are present could be of particular adaptive importance-i.e., the early-flowering strategy might be more advantageous in the face of stress. Overall, these differential stress responses add another piece of evidence against the Hyp. 1 prediction of similarity between the two species.
That prediction was also challenged by our findings regarding another aspect of flood response, production of aerenchymatous phellum on the stem. Aerenchyma had been documented in L. virgatum (Schenck 1889) but had never been compared to L. salicaria. We found L. virgatum produced more aerenchyma than L. salicaria (irrespective of differences in aboveground biomass). Aerenchyma is a specialized tissue produced under inundation to maintain aeration (Stevens et al. 2002) and is presumably costly to create. That L. virgatum produced more aerenchyma further illustrates our flooded condition as more stressful to L. virgatum than L. salicaria. Amount of aerenchyma, however, did not correlate with fitness in our study (Hyp. 2), suggesting aerenchyma production was a homeostatic response, rather than being adaptive (bolstering fitness) or maladaptive (at the cost of fitness). Other researchers have likewise hypothesized that presence of aerenchyma has not conferred an adaptive advantage to L. salicaria over other aerenchyma-producing Lythraceae species (Lempe et al. 2001). However, aerenchyma might bolster fitness over longer durations of flooding than those we explored. Previous work in L. salicaria has shown that stem aerenchymatous phellum functions to maintain overall aboveground biomass, a benefit which increases over the duration of flooding (Stevens et al. 2002).
Longer or repeated inundations might also affect fitness through interactions with flowering time differences. When allowed to grow for longer, the later-flowering pattern of our L. salicaria might be more advantageous, and is predicted to be increasingly favored as climate change lengthens growing seasons (Colautti et al. 2017). Novel inundation regimes are also resulting from climate change and human land use (AghaKouchak et al. 2020). Some areas are experiencing more inundation, including the core of the invasive range in Eastern North America, which is showing higher flood frequencies (Mallakpour and Villarini 2015) and higher maximum daily stream flows (Do et al. 2017). These changes will make differential flood tolerance increasingly relevant for these species.
Habitat tolerances in L. virgatum and L. salicaria The finding that L. virgatum was more stressed by flooding than was L. salicaria suggests that, in nature, moisture gradients might structure habitat differentiation between the two species. Such effects would likely operate at a withinecosystem scale because individual wetlands often span substantial moisture gradients. In the context of invasion, such stratification might indicate L. virgatum would preferentially naturalize into less inundated wetland zones than those occupied by L. salicaria. Other environmental factors surely control establishment and persistence of L. virgatum in nature. For example, specialist beetles incur greater damage on early-flowering genotypes (Lehndal and Ågren 2015) and might prefer L. virgatum, especially because it is less hairy. Examining potential interactions with these beetles, which are widely used for biocontrol in the invasive range, should be prioritized (Colautti et al. 2017).
Although the two taxa show important differences, both are adapted to flooded environments and, when found in local proximity, hybridization may be a factor affecting plant habitat tolerances. Studies indicate that hybridization is not only possible (Anderson and Ascher 1993;Lindgren and Clay 1993;Ottenbreit and Staniforth 1994;Amon et al. 2007) but likely, meaning the unique trait variation we have documented for L. virgatum could introduce adaptive novelty into long-established L. salicaria invasions. Future studies should further examine hybridization in nature, although ploidy differences might make hybridization rare and thus difficult to detect. Climate change might provide more chances for hybridization by virtue of lengthened growing seasons, which would extend both flowering and pollinator activity (Colautti et al. 2017). Examining hybridization could start with observations of traits and genetics in natural populations in the introduced range. Genetics in this system have thus far only been studied with methods that have limited reproducibility or low resolution (Strefeler et al. 1996 a, b;Houghton-Thompson et al. 2005;Kubátová et al. 2008;Chun et al. 2009;Middleton et al. 2019;Jocienė et al. 2022), which-along with hybridizationleave Lythrum taxonomy largely unresolved. As genetic tools become increasingly accessible, biologists are creating a more generalized understanding rapid evolution in introduced species. This system, for which local adaptation in the invasive range has already been demonstrated, could provide an excellent model.

Supporting Information
The following additional information is available in the online version of this article -Supporting Information 1. The dataset of all measured traits and experimental design variables.

Sources of Funding
This work was supported by funding from The Ohio State University to SMH and KZM, and grants to KZM from the Botanical Society of America, Ohio Invasive Plants Council, and Society of Wetland Scientists.