Long-term patterns and mechanisms of plant invasions in forests: the role of forest age and land-use history

It has become increasingly apparent that even mature forests are susceptible to plant invasions. However, invasive plants are often more abundant in younger forest stands. It is di�cult to disentangle possible mechanisms that would explain this pattern due to the scarcity of long-term studies in successional forests. Several mechanisms have been proposed to explain patterns of invasions as forests age, including biotic resistance, window of opportunity, historical legacies, and invasion debt. We explored patterns and potential mechanisms of plant invasions over 70-years in a regenerating forest with different land use histories in the Bolleswood Natural Area, Connecticut, USA. We examined how environmental factors related to colonization patterns of invasive and non-invasive introduced species over time, and whether these patterns were consistent with the proposed mechanisms. Non-invasive introduced species declined rapidly with forest development, while many invasive plants persisted or even increased over time as the forest aged. Colonization was focused in areas that were unforested when the study began, although this declined with time. Dispersal distance, soil conditions, and initial land cover played important roles in patterns of colonization, while the effect of shading was less clear. There was some evidence for each mechanism, but the relative importance of each mechanism was species-dependent, making generalizations about how invasive plants invade forests di�cult. We found that land use history impacted invasion more strongly than forest age, but over time even mature forests were slowly being invaded by some species. Thus, invasive species management may be required even in mature forests.


Introduction
Closed-canopy forests have long been considered resistant to invasive plants due to shading and competition with native trees (Simberloff et al. 2002), the long-lived nature of many forest trees (Von Holle et al. 2003), and because invasive species often have disturbance-adapted traits.(Rejmánek and Richardson 1996).Indeed, surveys of introduced species presence and abundance have shown fewer introduced plant species in undisturbed forests both in temperate (Von Holle and Motzkin 2007;Chytrý et al. 2008;Rejmánek et al. 2013) and tropical forests (Waddell et al. 2020) compared to other plant communities.However, it is increasingly clear that invasions of closed-canopy forests are occurring, and that some invasive species are shade-tolerant, and thus able to invade even closed-canopy forests (Martin et al. 2009).While there is now widespread recognition that some closed-canopy forests are being invaded, it is unclear if all forests are equally invasible and what factors may in uence the degree of invasion in different forests.
Forests may be more susceptible to invasion earlier in successional development (Holmes and Matlack 2019).Although patterns and mechanisms may differ for introduced species as a whole compared to shade-tolerant invasive species (Martin et al. 2009), numerous chronosequence studies from the northeastern USA have shown that younger forests have much greater frequency and/or abundance of both introduced species generally (Matlack and Schaub 2011; Holmes and Matlack 2019) and invasive species speci cally (Lundgren et al. 2004; Flory and Clay 2006; Mosher et al. 2009).A regional scale analysis of factors in uencing the distribution of invasive species also showed that invasives declined with forest age, although at this scale factors such as mean annual temperature and landscape openness were much better predictors (Golivets et al. 2019).Studies have found similar patterns of lower frequency and/or abundance of introduced species in older regenerating forests in California (Dudney et al. 2021), Puerto Rico (Pascarella et al. 2000), and the southeastern USA (Wang et al. 2012).This repeated pattern of fewer introduced and invasive species in older forests could support the idea that older forests are more resistant to invasion.However, given that these studies are a snapshot of different-aged forests at a speci c time, it is not possible to tell whether young forests have more invasive plants because they are at a different stage of development or because of confounding variables, such as the time period when they established or the land use history prior to forest regeneration.In fact, we do not have a good understanding of how invasive plant populations develop through succession in developing forests (Holmes and Matlack 2019), in part due to a relative paucity of long-term studies that follow the same forest over time.Some long-term studies have looked at general patterns of invasion over time in forests.In the most detailed study to date, Meiners et. al (2002) found that introduced species abundance generally declined over the rst 40 years following abandonment of old elds in New Jersey.At the same sites, Rosa multi ora, a common invasive shrub increased for 30 years, but then started to decline for the next 14 years (Banasiak and Meiners 2009).In contrast, (Huebner 2020) found that both mature (>80 years) and young (10-15 years) forests in West Virginia showed an increase in invasive richness over a 16 year period, although the mature forests generally had lower richness than the young forests.Similarly, Rogers et al. (2008) found that richness and abundance of introduced species increased in forest stands in Wisconsin that were resampled after a 50-year period, although this study did not report on the successional stage of the forest stands.
Several mechanisms have been proposed that could lead to a pattern of greater numbers of introduced non-invasive or invasive species in younger forests than older forests.Each of these mechanisms has distinct implications for the future of invasion in these forests, and thus understanding the prevalence of different mechanisms is critical for informing management and conservation decisions.

Biotic Resistance
First, increasing biotic resistance as forest succession progresses may cause younger forests to have greater numbers of introduced species.Biotic resistance indicates the ability of the resident community to resist invasion (Levine et al. 2004) and could increase as forests develop because of increasing richness of native species, increasing canopy cover and shading, or increased competition for limited resources in late succession.
The biotic resistance hypothesis is often focused on the diversity of native plant communities (often measured using species richness), which has long been proposed to reduce invasion by introduced species (Elton 1958).This hypothesis has been extensively studied and is clearly scale and context dependent (Traveset and Richardson 2020;Gioria et al. 2023).Generally, this pattern has been found to be more common at small spatial scales (Fridley et al. 2007) and when controlling for other factors (Beaury et al 2020).Given that species richness commonly increases at least through the early and middle stages of succession (Anderson 2007), this mechanism may allow older forests to have greater resistance to invasion.Because invasion may also impact native richness, measuring native richness prior to invasion can provide a clearer understanding about how native diversity impacts invasion than a snapshot of native and introduced diversity at a single time (Ernst et al. 2022).
In forests, tree biomass or canopy cover may play a bigger role than diversity in biotic resistance by reducing available light.As the canopy closes and shading increases, colonization by shade-intolerant species that dominate the introduced species pool is likely to decline (Meiners et al. 2002;Martin et al. 2009).Competition for resources, especially light, is expected to increase through succession (Walker and Chapin III 1987) which could also lead to a decline in introduced species as a forest ages.If increasing biotic resistance with succession is driving patterns of invasion, we would expect introduced species to be negatively associated with canopy cover and/or tree basal area.In a regional analysis of forests in the eastern USA, tree biomass was negatively related to both richness and cover of invasive species, even while tree richness had a positive relationship with degree of invasion (Iannone et al. 2015).This pattern has been found at more local scales as well.In Montana, Jang et al. (2021) found that 23 years after thinning and burning, introduced forb cover and richness and introduced graminoid cover were negatively related to tree basal area.In old elds in New Jersey, declines in introduced species over the rst few decades of forest development was negatively related to the increases in woody cover associated with canopy closure (Meiners et al. 2002).There is some evidence, however, that this mechanism may not be as strong when focusing on shade-tolerant invasive species (Martin et al. 2009).For example, even with the overall abundance of introduced species declining, Meiners et al. (2002) noted increases in individual shade-tolerant invasive species after 40 years of forest succession.
If biotic resistance is the primary driver of invasion patterns through succession, we would expect declining invasion over time, especially during the early stages of forest development (Fig 1).Thus, the pattern of greater invasive abundance in younger than in older forest stands in chronosequence studies (e.g.Lundgren et al. 2004;Mosher et al. 2009) would accurately represent a trajectory of declining invasion through succession.

Window of Opportunity
The timing of when forest development begins may be more important than actual forest age in creating a pattern of greater introduced species abundance in younger forests.There is a "window of opportunity" for colonization early in forest development (Hobbs 2000) that may interact with the timing of invasion (Degasperis and Motzkin 2007).Thus, areas that are still open when an invasive species arrives in an area may be colonized and then the species may persist, while older forests that were already developed when the species arrived may be resistant to invasion.For example, land use after introduction was the best single predictor of Berberis thunbergii presence in Massachusetts forests (Degasperis and Motzkin 2007), indicating that invasive species distributions in a forested landscape may re ect timing of past land use relative to introduction of the species.The intensity of land use and how much it initially opened up niches for colonization may also play a critical role, with higher intensity disturbances, even decades in the past, leading to increased current abundance of invasives (Holmes et al. 2021).This mechanism would show a trend of greater invasion in younger forests across a landscape, but for individual forests over time it would show an initial increase followed by persistence in the already-invaded sites (Fig 1).Older forests that developed prior to the arrival of the invasives would have persistently low invasion.

Historical Legacies
A third mechanism that could lead to greater abundance of introduced species in younger forests is the long-term effects of past land use on soil and other environmental variables in a developing forest (Holmes et al. 2021).Past land use, especially agriculture, can alter soil characteristics for decades or even a century after abandonment (Verheyen et al. 1999;Flinn and Marks 2007).Given that the distribution of invasive species in forests is related to patterns in these soil characteristics (e.g.soil richness, McDonald et al. 2008), forests may be more invasible as long as these legacies of past land use persist.Although there is considerable variation among studies, these legacies may decline over time and after 60+ years may be indistinguishable from much older forests (Holmes and Matlack 2017).If these post-disturbance legacies play an important role in invasion, then we might expect to see a decline in introduced species several decades to a century after forest establishment as these legacies diminish (Fig 1).Sites without historical land use causing these soil legacies would have persistently low levels of invasion.We would also expect to see a strong relationship between invasive species' distribution and soil characteristics both spatially and over time.

Invasion Debt
All three of the previous mechanisms may contribute to the general pattern that younger forests are more invaded than older ones.However, they may also help mask a mechanism that could actually lead to increased invasion as forests age.Relative to more disturbed habitat types, it is clear that forest invasions are slower (Martin et al. 2009) and may develop after a signi cant time lag either because of the longer-lived nature of many forest species or because of more limited propagule pressure in undisturbed forests (Essl et al. 2012).For example, bird dispersal into undisturbed forests may be limited (Holmes et al. 2021).Species that are able to disperse into undisturbed forests may establish at low levels but then "sit and wait" until small-scale disturbances allow increased establishment (Greenberg et al. 2001).Given the increasing prevalence of invasive species surrounding many forests, propagule pressure is likely increasing over time.Thus, the relative lack of invasion in older forests may only be an indication of a delayed "invasion debt" rather than a resistance to invasion per se (Essl et al. 2012).Multiple studies in Europe have shown a pattern of increased introduced species abundance in forests nearer to invasion sources, suggesting that propagule pressure may play a key role in forest invasions (Essl et al. 2012;Wagner et al. 2021).Similarly, a number of studies have shown increased invasions near the edges of forests (e.g., Yates et al. 2004;Riitters et al. 2018).However, invasion near forest edges may be due to environmental differences in forest edge habitat rather than dispersal distance, and thus may not be indicative of invasion debt.It is also unclear if the importance of dispersal distance changes as a forest ages.If an invasion debt is a driving mechanism, then we would expect to see a gradual increase in invasive species over time in forests of all different initial ages, with distance from potential seed sources being more important than forest age (Fig 1).
Disentangling these mechanisms requires a long-term dataset with forests that initiated at different times.Such a dataset was begun in the 1950s in the Bolleswood Natural Area (BNA) of the Connecticut College Arboretum when permanent plots were sampled in abandoned elds, transitional forest, and mature forest (Niering and Goodwin 1962).These plots have been resampled every 10 years for the past 70 years which allows an exploration of the patterns and mechanisms of introduced plant spread in developing forests of different ages and land use histories.
We use this long-term dataset to address three major questions.

Study Area
The Bolleswood Natural Area is a 23.1 ha unmanaged tract in the Connecticut College Arboretum in New London and Waterford, Connecticut, USA.The western end of the BNA was used for agriculture up until the natural area was created in 1951.Other areas had previously been cleared for agriculture but abandoned earlier.The eastern portion of the BNA contains rocky ridges and a ravine and was likely never completely cleared, but had been subject to localized res and windthrow in the decades prior to 1952 (Niering and Goodwin 1962).The natural area is bordered by a residential neighborhood to the west, a powerline corridor to the south and a road to the north, all of which are potential sources of invasive species.Forests in BNA are dominated by Quercus spp., Tsuga canadensis (L.) Carr.and Fagus grandifolia Ehrh., with Tsuga canadensis increasing in abundance until 1992 followed by a steep decline after the invasion of Adelges tsugae (hemlock wooly adelgid) in the late 1980s (Small et al. 2005).Since its establishment, the BNA has been allowed to develop without management or human disturbance, with the exception of surrounding it with a deer exclusion fence in 1989 to reduce herbivory (Buchanan et al. 2016).

Data Collection
In 1952, four parallel permanent transects were established in the BNA (Fig. 2).The transects are 122 m apart and range from 265 to 439 m long.Each transect is 6 m wide and made up of two rows of contiguous 3x3m plots (Niering and Goodwin 1962).The western ends of three of the transects were offset to avoid eld edges.For this study, we focused on the upland areas (without standing water during the growing season; 750 of the 890 total plots).
All plots have been surveyed every ten years beginning in 1952.In each survey year, the presence of each species of vascular plant was recorded in each of the plots.In addition, the diameter at breast height (DBH) of all trees was recorded.Trees < 2.5 cm DBH were categorized as either < 1 cm or > 1 cm DBH.In 2012 and 2022, we photographed the canopy from the center of each plot using a Sigma SD14 camera with a 4.5 mm circular sheye lens at ~ 1 m height.In these same years we also collected a soil sample from each plot.We collected soils from the top 10 cm below the O horizon from three locations in each plot and then mixed them to produce one sample per plot.
All species were categorized as native, introduced non-invasive, or invasive based on Dreyer et al. ( 2014), which follows the o cial Connecticut list of invasive species.Using the species presence data, we determined which plots were colonized for the rst time by a given invasive species in each survey year (newly colonized plots).For each year, we de ned plots that had never been previously colonized by a given species as uncolonized plots for that species.
For all sample years, we determined ve characteristics of each upland plot.Initial land cover was based on conditions at the start of the survey (open = recently abandoned open elds, transitional = shrublands or thickets that had previously been used for agriculture but had been abandoned in previous decades, and forest = closed-canopy forest) (Niering and Goodwin 1962).We used total basal area (in m 2 per hectare) as a measure of tree density and light availability.Because the 3x3m plots are too small to adequately represent the tree density surrounding a point, we calculated total basal area for 6x15m sections containing ten plots (75 total sections) and then assigned that basal area to each plot in the section.Species richness was measured as the number of species present in the plot in the previous survey.We calculated distance to the edge of the natural area for each plot using ArcMap 10.8.2.For each species, we also used ArcMap 10.8.2 to calculate the distance from the nearest plot in the previous survey where the target species occurred.
In 2012 and 2022 we also measured canopy openness and seven soil variables.We calculated canopy openness as the percentage of open sky in the hemispherical photos using Gap Light Analyzer (Canham et al. 1999).We calculated percent soil moisture (percent of dry weight) by weighing soils immediately on return to the lab and then drying them at 105°C for 24 hours.Soils were collected following at least 24 hours of dry conditions.We calculated soil organic matter using loss on ignition test at 500° C for 12 hours.For pH and soil nutrient analysis, we mixed soils from the 10 plots in a section and sent them for analysis to the Cornell Nutrient Analysis Laboratory in 2012 and the University of Connecticut Soil Nutrient Analysis Laboratory in 2022.We assigned each plot the value of pH, nitrate nitrogen, phosphorus, calcium, and potassium for its section.

Statistical Analysis
To assess invasion over time, we calculated the average number of introduced non-invasive and invasive species per plot in each of the three initial land cover types in each survey year.We also calculated the number of plots where each invasive species occurred in each survey year and the total number of plots where at least one invasive species was present.We determined the proportion of newly colonized plots (for all species combined) in each survey year that occurred in each of the initial land cover classes.
We used linear regression to assess changes in the characteristics of newly colonized plots over time.For each species we used time since the beginning of the study as the predictor variable for each of the four response variables separately (distance to nearest individual, distance to edge, basal area and species richness).
We then compared each of the four variables between newly colonized plots and uncolonized plots for each species in each year in which at least 10 plots were newly colonized (N = 15).Colonization for most species was focused on the western half of the natural area.We were interested in the differences in colonized and uncolonized plots within the range in which colonization might occur, rather than larger scale spatial patterns that might correlate with distance from the seed source.Thus, we limited the uncolonized plots used in this analysis to those within the colonization range.For each species in each year we noted the maximum distance that a newly colonized plot was from a plot containing that species in the previous survey and removed all uncolonized plots beyond this maximum distance from analysis.Because Lonicera morrowii Gray colonization occurred in a very limited area of the natural area and in only one initial land cover class, it was not included in further analyses.
For each species and year, we used t-tests to compare the mean values for each variable (distance to nearest individual, distance to edge, basal area, and species richness) between newly colonized and uncolonized plots.Canopy openness and the seven soil variables were only available for species spreading to ten or more new plots in 2012 or 2022 (N = 4).For each variable, signi cance of comparisons was adjusted for multiple tests using the Holm-Bonferroni procedure.For species richness we compared the mean values for total species richness and for native and introduced (invasive and noninvasive together) richness separately.
To understand the relative importance of the variables, we used relative weights analysis using the rwa package in R (Tonidandel and LeBreton 2011; Chan 2022).Relative weights analysis addresses multicollinearity among variables in a multiple regression model by separating the total variance into weights that re ect the relative contributions of each predictor variable (Tonidandel and LeBreton 2011).We conducted this analysis using multiple logistic regression models for each species in each year with colonization status as the response variable and six

Results
Although correlations among variables were generally low (|r|<0.55),the initial land cover classes were different in many respects (Online Supplement S1).Initially open sites had consistently higher richness, although richness uctuated over time in all initial land covers.Basal area increased over time in all initial land cover classes but then decreased starting in 2002 in forests and 2022 in the transitional forests.
Basal area increased more rapidly in the initially open areas but remained slightly lower than the other land cover classes at the end of the study.Initially open sites were nearer to the edge of the natural area and had higher canopy openness, nitrate and calcium levels, and pH and lower soil moisture, organic matter, and potassium.The pattern for phosphorus varied by year (Online Supplement S1).
Non-invasive introduced species were initially much more abundant in the initially open areas compared to other land cover classes, but declined rapidly over time (Fig. 3).By 1992, very few non-invasive introduced species occurred anywhere on the transects.In contrast, invasive species increased dramatically over time in the initially open sites (Fig. 3).Invasive species were much less common in the initially forested sites but also increased over time, while invasive richness uctuated in the transition forest plots.Ten invasive species were found in the upland plots.One of these, Rumex acetosella L., was initially present primarily in the open areas and declined rapidly, disappearing completely from the transects by 1982.Fallopia japonica (Houtt.)R. Decr.and Rubus phoenicolasius Maxim.rst appeared on the transects in 2012 and 2022 respectively and occurred in no more than four plots.The other seven species were analyzed in more detail.The frequency of these invasive species over time varied by species (Fig. 4).Several species (Lonicera japonica Thunb., Lonicera morrowii Gray, and Celastrus orbiculatus Thunb.)initially increased in abundance and then declined.Berberis thunbergii DC. and Rosa multi ora Thunb.ex Murr.increased and then leveled off while Ligustrum vulgare L. and Euonymus alatus (Thunb.)Sieb.continued to increase.For all species, colonization was concentrated in areas that were open in 1952, even after 70 years, with Lonicera morrowii colonization completely restricted to these areas.However, the proportion of the new occurrences that occurred in areas forested in 1952 has increased over time.Prior to 2002, no more than 7% of the new occurrences for all species were in the areas forested at the start of the study.This increased to 12% in 2002, 14% in 2012 and 30% in 2022.The number of newly colonized plots peaked in 1982, but new plots continue to be colonized in every survey since (Online Supplement S2).
The mean distance of newly colonized plots from individuals of the same species in the previous survey was always less than 40m, and usually less than 20m (Fig. 5).The maximum distance ranged from 21.7m for R. multi ora in 1992 to 256.1m for Lonicera japonica in 1982.Even when removing uncolonized plots farther away than the maximum colonized distance from the analysis, colonized plots were closer to plots with previously existing individuals than uncolonized plots for most species and years (Fig. 5).The mean distance declined over time for R. multi ora but not for any other species (Table 1).Colonized plots were closer to the edge of the natural area for all species and years except for Ligustrum vulgare in 1992 (Fig. 6).When considering colonization across all years, only C. orbiculatus colonized plots increasingly distant from the edge over time (Table 1).Ligustrum vulgare, Lonicera japonica, and R. multi ora actually colonized plots closer to the edge over time.This pattern appears to be driven by years when only few plots were colonized (Supplement S3).A few plots distant from the edge were initially colonized by Ligustrum vulgare and R. multi ora in the early stages of the study, while the bulk of later invasion in the years with enough data for complete analysis were very near the edge (Fig. 6).For three of the species, the maximum distance from the edge did increase over time -but only a few individuals colonized at distances greater than 200 m from the edge (Online Supplement S3).
In most years and for most species, colonization occurred in plots with higher species richness in the previous survey (Fig. 7).This pattern was most obvious for introduced species richness, but four of the six analyzed species colonized areas with higher native richness in at least one year (see Online Supplement S4 for full statistical analysis).There were no obvious trends over time, except for Lonicera japonica, which colonized plots with lower richness over time (Table 1).
In at least one survey year for each species except B. thunbergii, colonized plots had lower basal area than uncolonized plots (Fig. 8).However, this pattern was not consistent.C. orbiculatus colonized plots with lower basal area initially but not later on, while Ligustrum vulgare and E. alatus showed the opposite pattern.All of the species except E. alatus and Ligustrum vulgare colonized plots with higher basal area over time as the overall basal area in the forest increased (Table 1).Canopy openness in 2012 and 2022 did not show a similar pattern (Fig. 9).Only for E. alatus in 2022 was there a difference in canopy openness between colonized and uncolonized plots and the uncolonized plots had a higher canopy openness.
Soil variables did differ between uncolonized and colonized plots in 2012 and 2022 (Fig. 9).Soil pH was higher and potassium lower in colonized plots for all species.For some species, soil moisture and organic matter were lower, and nitrate and calcium were higher in colonized plots.Phosphorus was lower in colonized plots only for Ligustrum vulgare in 2012.
When including all of the explanatory variables, the importance of each variable changed by species and year with few obvious patterns (Fig. 10).Having the vegetation be open in 1952 was the most important variable in many cases (as high as 82% for R. multi ora in 2012).Basal area generally had low relative weights, while the importance of the other factors varied among species and year.

Discussion
Non-invasive introduced species and invasive species generally had opposite abundance patterns as forests developed over time.Non-invasive introduced species and one disturbance-adapted invasive species, Rumex acetosella, occurred almost exclusively in the open sites and rapidly declined as forests developed on these sites.While patterns varied somewhat among the rest of the invasive species, colonization was generally concentrated in the formerly open sites and increased over time, particularly between 1972 and 2002.Areas initially forested showed the lowest level of invasion, but invasion has increased in these areas in the past 30 years.
Shading and competition, as measured by basal area, canopy cover, and existing species richness, played a somewhat ambiguous role in colonization patterns.We found some evidence that colonization in a given year was more common in areas with lower basal area, but when multiple explanatory variables were considered, basal area explained only a small proportion of the total variation.In addition, most invasive species colonized areas with increasing basal area over time, suggesting that basal area by itself may not be the driving factor.Basal area was previously shown to have little association with sitelevel invasion patterns in this region (Howard et al. 2004), although it has been associated with invasion elsewhere (Jang et al. 2021).We also did not nd evidence that colonization was greater in areas with greater canopy openness, which is a more direct measure of shading.Likewise, species richness was usually higher in colonized vs non-colonized plots.Some but not all of this pattern is due to the richness of pre-existing introduced species.The lack of a clear role of shading and competition for colonization patterns of species in this study may not be surprising given that several of these species have been shown to tolerate shaded conditions (Greenberg et al. 2001;Martin et al. 2009;Driscoll et al. 2016).For species such as these, canopy closure and forest density may not be the primary driver of invasion patterns in forests.We only focused on invasive species' presence, however, so light availability, such as canopy gaps, may play a greater role in these species' growth rates and abundance within plots (Greenberg et al. 2001;Driscoll et al. 2016).
Dispersal clearly plays a role in patterns of colonization.Newly colonized plots were consistently closer to the edge of the natural area and to previously existing individuals of the same species than uncolonized plots.The average newly colonized plot was within about 100m of the edge of the natural area for all species and years and only for C. orbiculatus did this distance increase over time.We did see an increase in the maximum distance from the edge for three of the species over time, but the other three never occurred more than 130m from the edge.Thus, while invasive species are more common near the edge as found in previous studies (Yates et al. 2004;Riitters et al. 2018), there is moderate evidence that the species continue to move farther into the natural area.This evidence could be limited because many of the colonizing individuals are still small and are not reproductive, so more time may be necessary for this pattern to become more apparent for all species.Although it has been hypothesized that invasive species will continue to move into more remote forests over time (Essl et al. 2012), this may be a very long-term process in undisturbed forests.
Soil conditions were related to colonization patterns, with pH, nitrate, and calcium generally higher in colonized plots and soil moisture, organic matter, potassium, and to some extent phosphorus lower in colonized plots.Nitrate and calcium have previously been shown to be positively associated with invasion in forests in the northeastern USA, while the patterns for potassium and phosphorus have been less consistent (Howard et al. 2004;Degasperis and Motzkin 2007).Because these variables were only measured recently, and thus were not included in the multivariate analysis, it is unclear how important they are relative to other variables.
Initial land cover continued to be an important predictor of invasion throughout all years.Most new occurrences were in areas that were initially open-even decades after canopy closure.However, the other measured variables are connected to initial land cover, with the initially open areas having lower basal area, being closer to both the edge of the natural area and the initially invaded plots, and having higher richness and distinct soil conditions.Nevertheless, initial openness was often the most important variable even with these other variables included in the analysis.Thus, initial land cover may be an important proxy for several variables that together in uence colonization patterns.It is also critical to note that over the past 20 years, colonization into the initially forested, and thus oldest, areas has increased.

Biotic resistance
It is clear that biotic resistance is a strong mechanism for reducing the number of non-invasive introduced species in these forests.These species were initially abundant in the open sites and rapidly declined as has been found previously (Meiners et al. 2002).Very few non-invasive introduced species occurred in closed-canopy forests.However, there was little evidence supporting biotic resistance for the invasive species other than Rumex acetosella.All of the invasive species found in closed-canopy forests were most common in the youngest forest (with initially open land cover), but these invasions largely occurred after canopy development started and generally did not decline over time.Although within a given year colonized plots often had lower basal area than uncolonized plots, most species colonized plots with increasing basal area over time, which is not consistent with the biotic resistance hypothesis.Thus, we lack good reason to believe that biotic resistance is the primary cause of older forests being less heavily invaded.However, because this study focused on presence of invasive species rather than abundance, we may not have captured the full effect of biotic resistance.Indeed, biotic resistance may reduce the abundance of invasive species while not fully preventing colonization (Levine et al. 2004).
Even within forested habitat, we found that invasion occurred in sites with higher existing species richness.In particular, initially open sites generally had higher richness and were also the most invaded.
However, since the analysis here focused on site-scale patterns, this study may re ect processes at a spatial scale more likely to show a positive relationship between species richness and invasion (Fridley et al. 2007).For diversity to convey resistance to invasion to older forests, richness would have to increase during succession and be greatest in the oldest forests, and invasive species would tend to colonize sites with lower richness.However, none of these were true in our study system.Thus, species richness is not a form of biotic resistance that explains why younger forests sites have more invasive species.

Window of opportunity
Some of the species, especially B. thunbergii and R. multi ora, showed patterns consistent with the window of opportunity model which predicts greater invasion in younger forest sites with an initial increase in invasion followed by persistence.Between 1972 and 1992, both species spread into the initially open area while the tree canopy was still developing.Since 1992 the species have persisted but not really expanded into older forests, even those in close proximity.Lonicera japonica showed a similar pattern although it has declined in frequency since its peak.This pattern is consistent with the pattern found for B. thunbergii by DeGasperis and Motzkin (2007) where the species was found primarily in areas where forest development occurred after the species was already present in the area.B. thunbergii has previously demonstrated the ability to persist even in closed-canopy forests once present (Mosher et al. 2009).In contrast, previous studies for R. multi ora have shown a pattern more consistent with biotic resistance, with the species most common in early successional forests (Mosher et al. 2009) and then declining in time as the forests develop (Banasiak and Meiners 2009).Our results for R. multi ora are more consistent with the window of opportunity and consistent with the nding that both B. thunbergii and R. multi ora are less dependent on treefall gaps for persisting in forests than other invasive species (Driscoll et al. 2016).For these species, the pattern of lower abundance in older forests found in previous chronosequence studies may be due to younger forests being invaded in their early stages while older forests were not, instead of indicating that the species will actually become less common as forests age.

Historical legacies
It is clear that historical legacies still persist and differentiate the areas with varying land use history even after 70 years forest development.Some soil nutrients remained twice as high in the initially open plots compared to initially forested plots in both 2012 and 2022.This is consistent with studies showing that soil legacies after agriculture can persist for as much as a century (Verheyen et al. 1999).It is possible that these differences are becoming less pronounced over time (as is the case with basal area), but because we do not have soil data from earlier surveys this is uncertain.Thus, the continued colonization in these young post-agricultural could be impacted by these historical legacies.Both Lonicera morrowii and C. orbiculatus have seen a steep decline in the number of colonized plots in the initially open sites in the past 20 years, which could be consistent with the historical legacies model if the legacies are in fact declining.Several other species, Ligustrum vulgare, R. multi ora, and B. thunbergii, are largely restricted to these younger sites, so even though they are not declining, their pattern of spread could be a result of the continuing biological legacies and it is possible that these species may decline in the future if these legacies fade over time.

Conclusion
We found some support for all four mechanisms for invasion into developing forests.It is likely that all are happening simultaneously but that their importance varies by species.Our results also clearly indicate that while developing forests may be resistant overall to introduced species (including disturbance-dependent invasive species), there is a set of shade-tolerant invasive species that can colonize forests even with closed canopies (Martin et al. 2009).Given the species-speci c and likely sitespeci c nature of these mechanisms, it will be di cult to generalize predictions.In particular, we recognize that this study represents invasion patterns at only a single site.Nevertheless, it is critical to recognize that all of these mechanisms may be happening simultaneously.
Long disturbed forests may be resistant, but not impervious, to invasion.Managers thus must recognize that despite the strong pattern found in many studies of younger forests being more invaded, monitoring and management of invasive species in older forests may be critical.The slower nature of these invasions, however, may offer some hope in successfully intervening to prevent widespread colonization.

Declarations Figures
Schematic

1 )
What are the patterns of spread of invasive and introduced non-invasive species in forests with different land use histories over time in the Bolleswood Natural Area? 2) To what extent can factors relating to canopy cover, propagule pressure, initial land cover and soil characteristics explain these patterns and how do these relationships change over time? 3) To what extent do these patterns in spread and associated factors support one or more of the mechanisms described above?
predictor variables: distance to nearest individual, distance to edge, basal area, species richness and two dummy variables representing the initial land cover (open = open elds in 1952; transition = shrubland, thicket or transitional forest in 1952).

Table 1
Results of linear regression of the effects of time on characteristics of newly colonized plots.Rows in bold indicate p < 0.05.
(Martin et al. 2009;Essl et al. et al. 2021)e history, particularly agriculture, can impact invasion patterns in forests in the northeastern USA(Holmes and Matlack 2019;Holmes et al. 2021).invasionoutside of the post-agricultural forest has been limited at this site, there are some initial indications of an ongoing invasion debt.A pattern of declining invasion with distance from seed sources is one indication of invasion debt(Essl et al. 2012) and as predicted, early colonization was restricted to plots nearer the edge.While the average distance of newly colonized plots from the edge only increased over time for C. orbiculatus, four of the seven species increased the maximum distance from the edge by more than 100m over the course of the study.Perhaps even more telling, over time the proportion of newly colonized plots in the initially forested habitat has increased, even though this is the oldest forest.In particular, E. alatus and C. orbiculatus have been spreading into this habitat, even while C. orbiculatus has been declining in the initially open sites.Thus, there is an indication that the lack of invasion throughout the study area was not due to resistance to invasion in older sites, but because of delayed dispersal to these areas.This is consistent with predictions of slow spread of invasive species into undisturbed forest, especially those that are more distant from seed sources(Martin et al. 2009;Essl et al. While Holmes et al. 2021critical to be able to disentangle these mechanisms and patterns, as forest age is often confounded with land-use history in chronosequence studies.In this study, we showed different patterns in colonization among forests with different land-use histories.As found repeatedly in other studies, older forests without a history of agriculture had fewer overall invasive species (Degasperis and Motzkin 2007; Holmes and Matlack 2019;Holmes et al. 2021).However, we also found that overall the abundance of invasive species increased over time in both the youngest and the oldest forests, suggesting that abundance of invasive species may not decline as forests age.In particular, in this study we found that both C. orbiculatus and E. alatus originally invaded the forests that had been open at the beginning of the study but then over the past few decades have increasingly shifted into older forest.
Our results suggest that while the rate of colonization in forests without a history of agriculture is slow(Martin etal.2009; Essl et al. 2012), at least some species are continuing to spread.These older, less