). In urban areas, novel ecosystems can consist primarily of species that are adapted to or 146 persist under urban-associated stresses (e.g., urban heat, air, soil, light, and noise pollution).

147 Introduced (e.g., exotic/non-native) -including invasive species -are disproportionately 148 represented in urban species pools (Avolio and others 2015; Pregitzer and others 2019). Invasive 149 species tend to thrive in nutrient-rich soils, often escape natural enemies, and are frequently 150 quick to establish in unmanaged lands (Ehrenfeld 2003 175 Phoenix, AZ). We tested whether woody plant community composition in interstitial sites 176 differed from that in natural reference sites and whether soil properties were related to those 177 differences. Woody community composition was measured to identify whether plant 178 assemblages included combinations of introduced (e.g., non-native/exotic) and native species 179 that differed from assemblages in natural reference sites. We measured basic soil properties 180 (moisture, bulk density), soil microbial biomass C and N content, basal respiration, inorganic N 181 pools, potential net N mineralization and nitrification, and denitrification potential and visually 182 inspected soil profiles to ensure that there were no major anthropogenic differences between 183 reference and interstitial sites such as compaction, profile disturbance, or fertilization, and to 184 explore effects of novel plant communities on soil processes. We aimed to answer two questions:

185 1) How does woody plant community composition differ between interstitial and natural 186 reference sites? 2) Are these differences in vegetation associated with variation in soil C and N 187 cycling processes? We hypothesized that: 1) Woody plant community composition in interstitial 188 sites would differ from that in reference sites, with higher proportion of introduced species and 189 2) soil N cycling would be altered in interstitial sites, with higher N pools and rates of N cycling 190 processes in the sites with plant communities most distinct from those in reference sites. Results S1), and there were no consistent differences in 399 soil moisture or bulk density between interstitial and reference sites (data not presented).

Across cities, at least 61% of the total variance in soil parameters was explained by the 401 first two components (Dim1 and Dim2) in a principal components analysis (Fig. 3; Table S4).

402 On average, the first principal component (Dim1) explained 44.6% and the second principal 403 component (Dim2) explained 26.3%. of the variation in interstitial and reference soils. Nitrogen 404 cycle variables were the most strongly loaded on the two principal component axes, especially 405 Dim1. NO 3 -and NH 4 + strongly loaded on Dim1 and Dim2 in every city, except for Miami (Fig. 406 3; Table S4). Potential net nitrification and denitrification potential were strongly loaded on 407 Dim1 or Dim2 in every city except for Los Angeles (Fig. 3; Table S4). Potential net nitrification, 408 microbial biomass N and total inorganic N had strong loadings with both Dim1 and Dim2 in 409 every city. Carbon cycle variables were rarely significantly loaded on either Dim1 or Dim2 410 although organic matter and bulk density were strongly loaded on Dim2 in Miami (Fig. 3; Table

). Centroids of reference and interstitial sites did not overlap, except in Los Angeles and 412 Miami. Variation among sites was noticeable in many cities, with some interstitial and reference 413 sites having strong association with particular soil variables, while in other cities, site variation 414 was not strongly associated with soil variables (Fig. 3).

Across cities, in a canonical correlation analysis (CCA) of woody plant community and 416 soil variables, the proportion of variance explained by the first CCA axis was at least 12%, and 417 the second axis explained at least 10% of the variation across both interstitial and reference sites 418 (Fig. 4; Table S5). Variance explained was higher (> 30%) in the driest cities, Los Angeles and 419 Phoenix, that had many fewer species present (Fig. 4; Table S5). The proportion of variance in 420 woody plant community composition explained by soil variables was at least 62%, except in S5). While the number of soil variables that influenced community 422 composition varied per city, some variables were consistent across cities (Fig. 4; Table S5). For 423 example, soil NO 3 -, NH 4 +, and organic matter contents were related to woody plant community 424 composition in both interstitial and reference sites in all cities except Phoenix (Fig. 4; Table S5).

425 NO 3 -and community composition were significant in Boston (p = 0.033) and Miami (p = 0.004), 426 while organic matter content was statistically significant in Baltimore (p = 0.019), and Miami (p 427 = 0.005, Fig. 4; Table S5). Furthermore, the CCA models were only statistically significant in 428 Baltimore (p = 0.044) and Miami (p = 0.021), and marginally significant in Boston (p = 0.064; 429 Fig. 4; Table S5). S3). Even in cities that did not show clear differences along the 449 NMDS axes (e.g., Minneapolis-St. Paul), there was evidence for clustering among the interstitial 450 sites indicating that interstitial sites were more similar to each other than reference sites. As we 451 discuss below, the differences between reference and interstitial sites were likely the result of a 452 greater proportion of introduced species and higher species richness in interstitial sites, 453 especially in the sapling layers (Table 1).

Our careful selection of sites allowed us to assess how changes in plant community 455 composition affect soil N cycling, which is important for a variety of ecosystem services (e.g., 456 primary productivity). In our study sites, there were no noticeable anthropogenic effects on soils, 457 e.g., compaction, profile disturbance, or fertilization. Therefore, we were able to examine how 458 differences in plant community composition between interstitial and reference sites were 459 associated with variation in N availability. This finding is shown by separation of interstitial and 460 reference sites along PCA axes of soil characteristics in Baltimore, Boston, Los Angeles, Miami, 461 and Minneapolis-St. Paul in the PCA, and by relationships between N pools (NO 3 -, NH 4 + , TIN, 462 and microbial biomass N) and woody vegetation composition in the CCA. It is important to note 463 that there were no systematic differences in N availability between interstitial and reference sites, 464 and no evidence that interstitial sites had artificially elevated N availability based on soil 465 taxonomy (Table S1). Closely matching the soil series allowed us to avoid differences in soil 466 moisture retention and having the sites interspersed across the region avoid local pollution (e.g.,

Comments to the Author:

Thank you for submitting your paper "Woody plant-soil relationships in urban interstitial spaces have implications for future forests within and beyond urban areas" to Ecosystems. We now received two reviews and both reviewers see the relevance and the potential in the study conducted.

However, both reviewers also raise a series of concerns, and based on my own reading, I must add another one: In the title, the highlights and throughout the manuscript you put a lot of weight on the relevance of your results for forests within and beyond urban areas. However, I do not see how your specific results contribute to this finding. The existing literature alone allows to draw these conclusions, but to which specific aspect do your results contribute? I strongly suggest being more precise and specific regarding the implications you refer to throughout the text.

We have addressed the concerns regarding the relevance of our results to forests within and beyond urban areas. At the beginning of the manuscript, we clarify that in this study, we asked the question "if analysis of forests that have spontaneously assembled in urban interstitial spaces provide insight into how global environmental change will affect the forests of the future." We ask if the complex mix of anthropogenic factors affecting these spaces (altered climate and atmospheric chemistry, altered disturbance regimes, altered species pool) are analogous to factors playing out across the globe at lower intensity. If so, the novel communities that assemble in these spaces may provide a glimpse of the forests that may become widespread across the world. The fact that we observed the formation of novel communities in these spaces in six cities across a very broad range of climates, suggests that our results do have relevance for forests beyond urban areas.

We note that Reviewer #2 appreciates this relevance, "it is of high relevance to better understand novel ecosystems as they develop in described urban and peri-urban areas. We will face novel ecosystems across the globe and therefore it is important to learn on how and where to manage in order to maintain ecosystem services, biodiversity and climate resilience."

On some occasions, there seems to be a tension between the desire to draw more general conclusion and the heterogeneity of site conditions, landscape level context, land use legacies etc. in your sites selected. At least this is my impression when I read for example on Line 576 about a lack of difference in C dynamics between interstitial and reference sites, but just five lines further down that your results support the idea that the quantity of C in urban ecosystems can be significant compared to native ecosystems. Reviewer 1 similarly refers to aspects where a more precise wording might help to avoid such misunderstandings. The reader has to be very clear regarding which insights arise from your results and what statements are based on the literature -this does not seem to be always the case now.

As described in detail below, we have clarified the specific places where this tension arises in our revised manuscript. We have eliminated the sentence that created confusion in (former) Line 576.

I do think that the topic addressed is indeed of high relevance and also of high interest to the readership of our journal. However, in its present form, the manuscript lacks specificity regarding the insights generated based on results and how these insights specifically translate into what kind of implications for future forest management. I therefore suggest that you revise and resubmit your manuscript based on the recommendations of the reviewers and the suggestions regarding sharpening the message outlined above.

Thank you for considering our manuscript. We are glad to hear the topic is relevant and useful to readers of ECOSYSTEMS. We have revised the manuscript according to the comments suggested, and more specifically addressed the implications for future forest management.

General comments:

The study by Mejia et al examines woody plant species composition and richness with respect to soil C and N properties across six contrasting urban areas in the United States. These comparative types of studies are very important as they highlight where ecological theory becomes limited to different ecosystems. I think the data sets are robust and should absolutely be published.

However, the content of the study needs revision as outlined in my following general and specific comments:

The introduction needs to be revised. First, the authors need to emphasize how this is novel with respect to their previous works, in particular, the "Trammell, T.L., Pataki, D.E., Pouyat, R.V., Groffman, P.M., Rosier, C., Bettez, N., Cavender-Bares, J., Grove, M.J., Hall, S.J., Heffernan, J. and Hobbie, S.E., 2020. Urban soil carbon and nitrogen converge at a continental scale. Ecological Monographs, 90(2), p.e01401." has very similar conclusions using much of the same data. Moreover, the Trammell paper was not cited in the introduction and should be as it is incorporating many of the same themes and concepts.

Second, there has been extensive work on C and N cycling in urban systems and this work does not approach it in a quantitative fashion. I understand the need to advance theory, but there needs to be quantitative data to describe sizes of storage and fluxes and the current introduction is a disservice to those authors and to readers.

A major focus of our revision has been to clarify that the soil data were used for two very different purposes than the analyses in Trammell and others (2020) and Ryan and others (2022). Given that the focus of our study was what we can learn from the vegetation communities that spontaneously assemble in urban interstitial spaces, our soils analysis was focused on 1) ensuring that there were no major anthropogenic differences between reference and interstitial sites such as compaction, profile disturbance, or fertilization, and 2) to explore effects of novel plant communities on soil processes. A major focus of our revision was to clarify these objectives. We have also included references to the Trammell and others (2020) and Ryan and others (2022) papers and clarified that these were focused on evaluating homogenization and changes in soil pools and processes rather than relationships with vegetation communities.

The methods were written very well. I only had a specific comment on nondimensionalizing the data used in the PCA.

My first issue with the content of the manuscript is what seems to be either an inconsistency or cherrypicking regarding composition vs richness. For example, the abstract states "We observed marked differences in woody plant community composition between interstitial and reference sites in most metropolitan regions." but the first line of the results state "Across cities, there was no consistent difference in mean woody plant species richness between reference and interstitial sites ( composition and touted as an important response variable but here in the results and discussion it seems to have been forgotten/buried.

We have clarified in the Methods section (lines 600 -602) that species composition is "the identity of species present in a community." We had already defined species richness as "the overall number of species," in a community (lines 344-345). In the Cubino and others (2019) study the focus was on the role of non-native species in biotic homogenization in residential yards and addressed both species richness and composition. Our focus here was not on homogenization, but rather to explore effects of novel plant communities (composition) in interstitial (i.e., unmanaged) spaces.

My second issue is that soil C and N are discussed and framed irrespective to soil moisture and pollution. Soil moisture controls the growth of woody plants in southern California (shrubs and invasive grasses dominate the dry areas). Soil moisture retention data should be available from NRCS to compare soil data within each city. Also, that could help eliminate any potential aquic conditions creating outliers in temperate forests. N pollution sin several of the cities are dominant factors, particularly LA which has had massive N fluxes of N deposition exceeds 10 kg ha-1 yr-1 well into the 1990s (N pollution during acid rain peaked around 12 kg ha-1 yr-1 in NY for example). These are two very important processes not covered in the manuscript as needed.

We have clarified in the methods section (lines 501 -509) that the selection criteria for the interstitial sites "included sites with natural soil profiles similar in texture and landscape position to those in the reference areas, without signs of anthropogenic soil disturbance. In some cities, e.g., Minneapolis St.-Paul this required locating sites on different soil parent materials. Unmanaged patches that fit these criteria were located within the same region as the refence sites, either on the edge of the city, at the interface with suburban residential land, or within public parklands or woodlands (for more detailed description, see Padullés Cubino and others 2020; Lerman and others 2021). Soil taxonomy was identified using USDA Natural Resource Conservation Service (NRCS) maps for each native reference and interstitial site in each city (Table S1)." Closely matching the soil series allowed us to avoid differences in soil moisture retention and having the sites interspersed across the region avoid local pollution gradients. However, we have highlighted the importance of these gradients, especially in Los Angeles (lines 804 -806).

The title should be revised. Having "urban interstitial spaces" and "urban areas" in the title seems redundant.

We have revised to: "Woody plant-soil relationships in interstitial spaces have implications for future forests within and beyond urban areas."

For author contributions, authors should use CRediT to really specify their intellectual contributions. The second batch of authors contributed equally through what kind of comments? Making sure their names are spelled correctly?

We have extensively revised the author statement to more clearly specify the contributions that people made based on CRediT descriptions and the journal guidelines. This paper is the product of a research group that has been working together since 2010 so there are people who contributed to the study and experimental design as well as relative newcomers who were more involved in interpretation of this specific data. This is an active group; there were multiple iterations of comments and suggested edits on the early drafts.

Bullet 2: either 'N' or 'nitrogen'. Abstract:

The authors should consider being more quantitative, as the only number in their abstract is 'six' for the number of sites.

While we have extensively revised the Abstract to clarify several key points (as described above), we have not made it more quantitative as suggested here. Most of our results are from multivariate analyses that are not particularly conducive to simple quantitative statements.

Line 341: One of my least favorite aspects of ecology, p values. Please remember it is significant or it is not. Marginally significant defeats the purpose of a priori statistical tests and shows post-hoc significance hunting. Instead, focus on the R2 explanatory power which is huge for Los Angeles.

We have clarified (lines 680 -681) that 'there were strong, but not statistically significant compositional differences between interstitial and reference sites in Los Angeles (r2 = 0.47, p = 0.10, respectively; Table S2)."

Line 370 and 371: Is "Strongly loaded" the correct terminology? From the methods it is unclear if the data were normalized/nondimensionalized to their standard deviation and thus would have an oversized effect on the eigenvalues and eigenvectors. Please either clarify the methods or re-do the PCA with nondimensionalized data.

We clarified in the methods sections (lines 627 -628) that the data was normalized/nondimensionalized to their standard deviation before conducting the PCA. This enabled us to use the loadings to determine the importance of the factors (i.e., strong or weak contribution to the components).

Line 384: The high variability seems like soil macro-and micro-topography were not properly controlled during sampling in LA and PHX. Such is the way of field sampling.

We have clarified (line 515) that "two soil cores up to 30 cm depth were collected at random locations along transects at each site." Indeed, some of the variability that we observed may be due to soil macroand micro-topography as the reviewer suggests.

Line 399: Why not mention the lack of differences in richness?

We have clarified (lines 481 -497) in the discussion that the focus of our analysis is species composition, which we define as "the identify of species present in a community." We do talk about species diversity (richness), later in the discussion, but our focus is composition.

Discussion:

Line 410: The authors should really consider nitrogen in the context of precipitation limitations and N pollution in their overview as it is misleading to draw a link directly between plants and soil N without those two major factors being stated immediately.

We have extensively revised this "overview" section as well as the description of the objectives and experimental approaches to our soil and nitrogen work. Of particular importance here is that having the sites interspersed across the region avoids local pollution gradients. Line 476: I believe the major deforestation of New England in the 1800s-1900s was far more fragmentation than current parcels. Just look at the stone walls!

We have clarified (lines 922 -924) that "temperate forests of New England have experienced increased fragmentation over recent decades" to avoid confusion with earlier periods of more intensive fragmentation.

Line 517: Finally soil moisture constraints on N are mentioned. Soil moisture constraints need to be a more prominent feature in describing the C and N data as soils in LA and PHX are moisture constrained for most of the year while C and N data were determined using incubated soils which are artificial conditions in the dry areas for most of the year.

We have a paragraph (lines 974 -977) that discusses how "comparison of interstitial and reference sites in our most arid cities (Phoenix and Los Angeles) produced an interesting contrast to mesic cities." We note in the methods (lines 712 -714) that this comparison is not affected by differences in soil moisture between the interstitial and reference sites, none of which received water additions. We also note that there were no water additions to our incubations for C and N cycle processes.

Line 536: Soils and their moisture retention control plants success outside of the udic soil moisture regime (and even to an extent in udic regimes where aquic conditions dominate). This basic feature of soil moisture controlling plant richness and composition seems lost on the authors' discussion.

As noted above, we do spend some time talking about the importance of soil moisture effects on plants and soils, and how this varies between the mesic and arid cities. However, these effects do not affect our comparison of interstitial and reference sites, which were chosen to avoid differences in inherent soil characteristics. Detailed soil classifications are presented in Table S1. This topic is extremely important for the provisioning of future ecosystem services. It is of high relevance to better understand novel ecosystems as they develop in described urban and peri-urban areas. The study is well presented and touches an important interface of forest dynamics considering soil dynamics. We will face novel ecosystems across the globe and therefore it is important to learn on how and where to manage in order to maintain ecosystem services, biodiversity and climate resilience. I do support the publication of this paper. However, it would be beneficial to increase the perspective a bit as there is quite a bit of experience across the world. There are some basic papers that might improve the manuscript especially in the light of a legal framework on introducing tree species. In the light of controlling and managing ecosystems in the future. e.g. Brundu et al. 2020 in Neobiota Global guidelines for the sustainable use of non-native trees to prevent tree invasions and mitigate their negative impacts.

Thank you for feedback and recognition of the contribution of our manuscript to understanding novel ecosystems. We agree that broadening our references would strengthen the paper and make the study more relevant to urban and peri-urban forests outside the U.S. We have added 23 additional sources, including Brundu et al. 2020.

What I miss and think it should be discussed is a stronger link to ecosystem services. Especially the provision of clean and filtered water in such environments seems to be a real challenge for now and the future. There are experiences with invasive woody species with regards to water consumption of introduced species from e.g. South Africa. There is lots of literature available for the Center of Invasion Biology in Stellenbosch (e.g. van Wilgen or Richardson). Also Australia has a broad expertise on this topic. The same accounts for biodiversity and the competition of non-native to native species. This is also only touched very briefly and might be expanded in a few sentences.

We expanded our discussion of ecosystem services, specifically the effects of invasive species on water consumption and soil conservation. We have also included the references recommended. Many thanks for these specific suggestions! In general, the reference list is quite focussed on US literature. This certainly makes sense as the study is tailored for US environments, however, a view across borders is reasonable as in other countries where non-natives (in the US) are native, management experiences for certain species might be available. This also accounts for invading processes. Also here, countries as New Zealand, Australia, South Africa and many countries in South America have vast experiences with such developments. There are also strong pubications from author groups from across the globe that might be considered as refs. (e.g. Brundu et al, 2020 Neobiota Global guidelines for the sustainable use of non-native trees to prevent tree invasions and mitigate their negative impacts; or Krumm and Vitkova, Introduced tree species -opportunities and challenges, EFI 2016. Also the groups of Pysek, Essl or Blackburn have important publications on basic principles on invasive processes.

As the definition of crucial terms, such as exotic, native, non-native is interpreted and perceived in different ways, it would be helpful to include a definition in the manuscript. It usually confuses readers as there are also varying definitions around.

We have clarified in the introduction and methods our definition of introduced (non-native) species based on the USDA definition and offered more details on these definitions in the methods section. According to the USDA: Introduced species reproduce spontaneously in the wild without human help and tend to persist. Invasive species are (1) non-native (or alien) species to the ecosystem under consideration and We clarified in the methods that the threshold size for all vegetation measured was 1 cm diameter (line 514).

In my view, the first section "overview" would not be neccessary. It even confuses a bit and does not really support the readibility of the discussion. I would prefer to start right away with discussing the results and relationships. Adding a few sentsences on impacts on forest ecosystems would be valuable here. Lines 425ff Any other potential reasons for the observed differences? Or why are the differences that pronounced? Climatic changes? Land use changes? Invading processes, e.g. missing Mycorrhiza, missing antagonists etc?

We have eliminated the "overview" section and re-organized the Discussion section as suggested here. We now start by reminded the reader that our overarching question is "if analysis of forests that have spontaneously assembled in urban interstitial spaces provide insight into how global environmental change will affect the forests of the future." We then go on to discuss differences in plant communities and then discuss if local human alteration of soils has reduced the value of our sites as analogs for future environmental conditions. We then go on to discuss the effects of altered plant communities on soil processes and ecosystem services. Thank you for the suggestions, we have included these two references throughout the discussion (line 834, 848, and 1074).

Lines 437: Would it be an option that seeds are still rare from non-natives related to natives? This might then change soon? At least it could be a question of time and it might change. You mention this as a potential ecological time lag....there is this phase model from Blackburn et al. that might fit well. After the establishment phase of non-native species the bum and burst phase might follow.

We have revised this sentence to incorporate that introduced species may be going through some phase change as explained by the model developed by Blackburn This sentence has been revised (lines 1097 -1099) to clarify that soil conditions is also an important factor to consider in lands previously used for agriculture.

For the discussion, it might be worth discussing also management objectives of urban forest or tree managers. This might have a strong influence and must be considered anyway. Objectives to make cities greener must include non-native species that might become invasive with a time lag. The potential list that must consider urban environmental condidtions, narrow the list of species quite a bit. This is an interesting topic that is the focus of another paper in review by our research group. However, the focus of this paper is on what we can learn from spontaneously developing novel communities so we have not added discussion of management objectives for urban forests here. We have extensively revised the manuscript (previously ECOSYSTEMS MS# ECO-23-0052) in response to the thoughtful reviewer comments and hope and feel that we have produced a substantially changed and improved manuscript. We would like to request that this revised version of the manuscript be handled by the same subject matter editor (Matthias Bürgi) and the same reviewers. The comments from the editor and the reviewers were extremely useful in the revision process. The anonymous reviewers acknowledged that the topic is extremely important and of high relevance to better understand novel ecosystems and provisioning of future ecosystem services in urban areas and beyond.

In relatively unmanaged interstitial spaces (i.e., spontaneously forested areas surrounded by residential development), native and non-native vegetation have the potential to mix and assemble into new or novel communities. Our study examined differences in woody plant community composition between interstitial areas -at the residential-wildland interface -and natural reference areas in six cities in the continental U.S. (Baltimore, MD; Boston, MA; Los Angeles, CA; Miami, FL; Minneapolis-St. Paul, MN; and Phoenix, AZ). We also examined whether these differences in woody plant community composition in interstitial and reference areas are related to variation in soil C and N cycling processes. We found that there are marked differences in woody vegetation composition between interstitial and reference areas in six cities across the U.S. These differences are likely the result of a greater proportion of introduced species in interstitial sites, and variation in inherent N availability. These results increase our basic understanding of novel ecosystems that have emerged from transportation and spread of introduced species and have assembled without human intervention. The potential effects of these novel ecosystems are largely unknown, but they are of great concern due to growing urban expansion and land-use change. I hope that this topic is of interest to readers of Ecosystems. This manuscript has not been previously published and is not currently under consideration by another journal. There are no known conflicts of interest associated with this publication, and financial support provided for this study did not influence the results of the research. As the