Supplementary information

Author response:
We sincerely thank the editor and both reviewers for their time and thoughtful feedback on our manuscript. We have addressed several of the concerns in the responses below and are currently working on additional analyses to further strengthen the study. These results will be incorporated into the final version of the research paper.
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Reviewer #1 (Public review):
Summary:
The authors investigated the population structure of the invasive weed Lantana camara from 36 localities in India using 19,008 genome-wide SNPs obtained through ddRAD sequencing.
Strengths: 
The manuscript is well-written, the analyses are sound, and the figures are of great quality.
Weaknesses:
The narrative almost completely ignores the fact that this plant is popular in horticultural trade and the different color morphs that form genetic populations are most likely the result of artificial selection by humans for certain colors for trade, and not the result of natural selfing. Although it may be possible that the genetic clustering of color morphs is maintained in the wild through selfing, there is no evidence in this study to support that. The high levels of homozygosity are more likely explained as a result of artificial selection in horticulture and relatively recent introductions in India. Therefore, the claim of the title that "the population structure.. is shaped by its mating system" is in part moot, because any population structure is in large part shaped by the mating system of the organism, but further misleading because it is much more likely artificial selection that caused the patterns observed.
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The reviewer raises the possibility that the observed genetic patterns may have originated through the selection of different varieties by the horticultural industry. While it is plausible that artificial selection can lead to the formation of distinct morphs, the presence of a strong structure between them in the wild populations cannot be explained just based on selection. In the wild, different flower colour variants frequently occur in close physical proximity and should, in principle, allow for cross-fertilization. Over time, this gene flow would be expected to erode any genetic structure shaped solely by past selection. However, our results show no evidence of such a breakdown in structure. Despite co-occurring in immediate proximity, the flower colour variants maintain distinct genetic identities. This suggests the presence of a barrier to gene flow, likely maintained by the species' mating system. Moreover, the presence of many of these flower colour morphs in the native range—as documented through observations on platforms like iNaturalist—suggests that these variants may have a natural origin rather than being solely products of horticultural selection.
While it is plausible that horticultural breeding involved efforts to generate new varieties through crossing—resulting in the emergence of some of the observed morphs—even if this were the case, the dynamics of a self-fertilizing species would still lead to rapid genetic structuring. Following hybridization, just a few generations of selfing are sufficient to produce inbred lines, which can then maintain distinct genetic identities. As discussed in our manuscript, such inbred lines could be associated with specific flower colour morphs and persist through predominant self-fertilization. This mechanism provides a compelling explanation for the strong genetic structure observed among co-occurring flower colour variants in the wild.
While a recent bottleneck may have increased inbreeding, the strong and consistent genetic structuring we observe within populations is more indicative of predominant self-fertilization. To further validate this, we conducted a bagging experiment on Lantana camara inflorescences to exclude insect-mediated cross-pollination. The results showed no significant difference in seed set between bagged and open-pollinated flowers, supporting the conclusion that L. camara is primarily self-fertilizing in India.
As the reviewer rightly points out, the mating system of a species plays a crucial role in shaping patterns of genetic structure. However, in many natural populations, structuring patterns are often influenced by a combination of factors such as selection, barriers to gene flow, and genetic drift. In some cases, the mating system exerts a more prominent influence at the microgeographic level, while in others, it can shape genetic structure at broader spatial scales. What is particularly interesting in our study is that - the mating system appears to shape genetic structure at a subcontinental scale. Despite the species having undergone other evolutionary forces—such as a genetic bottleneck and expansion due to its invasive nature—the mating system exerts a more pronounced effect on the observed genetic patterns, and the influence of the mating system is remarkably strong, resulting in a clear and consistent genetic structure across populations.
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Reviewer #2 (Public review):
Summary:
The authors performed a series of population genetic analyses in Lantana camara using 19,008 genome-wide SNPs data from 359 individuals in India. They found a clear population structure that did not show a geographical pattern, and that flower color was rather associated with population structure. Excess of homozygosity indicates a high selfing rate, which may lead to fixation of alleles in local populations and explain the presence of population structure without a clear geographic pattern. The authors also performed a forward simulation analysis, theoretically confirming that selfing promotes fixation of alleles (higher Fst) and reduction in genetic diversity (lower heterozygosity).
Strengths:
Biological invasion is a critical driver of biodiversity loss, and it is important to understand how invasive species adapt to novel environments despite limited genetic diversity (genetic paradox of biological invasion). Lantana camara is one of the hundred most invasive species in the world (IUCN 2000), and the authors collected 359 plants from a wide geographical range in India, where L. camara has invaded. The scale of the dataset and the importance of the target species are the strengths of the present study.
Weaknesses:
One of the most critical weaknesses of this study would be that the output modelling analysis is largely qualitative, which cannot be directly comparable to the empirical data. The main findings of the SLiM-based simulation were that selfing promotes the fixation of alleles and the reduction of genetic diversity. These are theoretically well-reported knowledge, and such findings themselves are not novel, although it may have become interesting these findings are quantitatively integrated with their empirical findings in the studied species. In that sense, a coalescent-based analysis such as an Approximate Bayesian Computation method (e.g. DIY-ABC) utilizing their SNPs data would be more interesting. For example, by ABC-based methods, authors can infer the split time between subpopulations identified in this study. If such split time is older than the recorded invasion date, the result supports the scenario that multiple introductions may have contributed to the population structure of this species. In the current form of the manuscript, multiple introductions were implicated but not formally tested.
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Through our SLiM simulations, we aimed to demonstrate that a pattern of strong genetic structure within a location—similar to what we observed in Lantana camara—can arise under a predominantly self-fertilizing mating system. These simulations were not parameterized using species-specific data from Lantana but were intended as a conceptual demonstration of the plausibility of such patterns under selfing using SNP data. While the theoretical consequences of self-fertilisation have been widely discussed, relatively few studies have directly modelled these patterns using SNP data. Our SLiM simulations contribute to this gap and support the notion that the observed genetic structuring in Lantana may indeed result from predominant self-fertilisation.
We thank the reviewer for the suggestion regarding the use of simulations based on genomic data from Lantana and for explaining the importance of it. We are currently conducting demographic simulations using genomic data from Lantana to estimate divergence times between the different flower colour variants. We believe this analysis will offer deeper insights and provide further clarity on the points raised by the reviewers.
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I also have several concerns regarding the authors' population genetic analyses. First, the authors removed SNPs that were not in Hardy-Weinberg equilibrium (HWE), but the studied populations would not satisfy the assumption of HWE, i.e., random mating, because of a high level of inbreeding. Thus, the first screening of the SNPs would be biased strongly, which may have led to spurious outputs in a series of downstream analyses.
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Hardy-Weinberg Equilibrium (HWE) filtering is a commonly used step in SNP filtering analysis to exclude loci potentially under selection, thereby enriching for neutral variants and minimizing bias in downstream analyses. To ensure that our results are not influenced by selection-driven SNPs, we conducted the analysis both with and without applying the HWE filter. Notably, the number of SNPs retained did not drop significantly after filtering, and the overall patterns observed remained consistent across both approaches.
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Second, in the genetic simulation, it is not clear how a set of parameters such as mutation rate, recombination rate, and growth rate were determined and how they are appropriate. Importantly, while authors assume the selfing rate in the simulation, selfing can also strongly influence the effective mutation rate (e.g. Nordborg &amp; Donnelly 1997 Genetics, Nordborg 2000 Genetics). It is not clear how this effect is incorporated in the simulation.
</blockquote>
The aim of the SLiM simulation was to demonstrate that the extreme genetic structuring observed in Lantana camara can plausibly arise in natural systems under predominant self-fertilization. For the simulation, we used mutation and recombination rates estimated for Arabidopsis thaliana, as these parameters are currently unknown for Lantana. The details of this will be added in the revised version, and thanks to the reviewer for pointing this out. While we acknowledge that this simulation does not provide an exact representation of the species' evolutionary history, the goal of the simulation was not to produce precise estimates but rather to illustrate the feasibility of such strong genetic structuring resulting from self-fertilization alone. The impact of the selfing on the mutation rate is not incorporated in the simulations now. We will look into the details of this.
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Third, while the authors argue the association between flower color and population structure, their statistical associations were not formally tested.
</blockquote>
We recognize that one of the key improvements needed for the manuscript is to provide experimental evidence supporting self-fertilization. To address this, we conducted a bagging experiment on Lantana camara inflorescences to prevent insect visitation and eliminate insect-mediated cross-fertilization. The results showed no significant difference in seed set between bagged and open-pollinated inflorescences, indicating that Lantana is predominantly self-fertilizing in India. This finding is consistent with our genetic data and will be included in the revised version of the manuscript.
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Also, it is not mentioned how flower color polymorphisms are defined. Could it be possible to distinguish many flower color morphs shown in Figure 1b objectively? I am concerned particularly because the authors also mentioned that flower color may change temporally and that a single inflorescence can have flowers of different colors (L160).
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The different flower colour variants are visually distinguishable. Our classification of these variants is not based on the colour of individual flowers at a single time point, but rather on the overall colour change pattern across the inflorescence over time. In other words, the temporal aspect of colour change has been considered in our grouping. For example, in the “yellow-pink” variant, flowers begin as yellow when young and gradually turn pink as they age. Importantly, variants that follow this pattern do not transition to an orange type at any stage, which distinguishes them from other colour types. The varieties that don't change colours are named based on the single flower colour like “orange”.

Alexandre Fournier-Level

Detlef Weigel

eLife Assessment
The submission by Gopal and colleagues reports important findings describing the structure of genetic and colour variation in its native range for the globally invasive weed Lantana camara. Whilst the importance of the research question and the scale of the sampling is appreciated, the analysis, which is currently incomplete, requires further tests to support the claims made by the authors.

anonymous

Reviewer #1 (Public review):
Summary:
The authors investigated the population structure of the invasive weed Lantana camara from 36 localities in India using 19,008 genome-wide SNPs obtained through ddRAD sequencing.
Strengths: 
The manuscript is well-written, the analyses are sound, and the figures are of great quality.
Weaknesses:
The narrative almost completely ignores the fact that this plant is popular in horticultural trade and the different color morphs that form genetic populations are most likely the result of artificial selection by humans for certain colors for trade, and not the result of natural selfing. Although it may be possible that the genetic clustering of color morphs is maintained in the wild through selfing, there is no evidence in this study to support that. The high levels of homozygosity are more likely explained as a result of artificial selection in horticulture and relatively recent introductions in India. Therefore, the claim of the title that "the population structure.. is shaped by its mating system" is in part moot, because any population structure is in large part shaped by the mating system of the organism, but further misleading because it is much more likely artificial selection that caused the patterns observed.

Reviewer #2 (Public review):
Summary:
The authors performed a series of population genetic analyses in Lantana camara using 19,008 genome-wide SNPs data from 359 individuals in India. They found a clear population structure that did not show a geographical pattern, and that flower color was rather associated with population structure. Excess of homozygosity indicates a high selfing rate, which may lead to fixation of alleles in local populations and explain the presence of population structure without a clear geographic pattern. The authors also performed a forward simulation analysis, theoretically confirming that selfing promotes fixation of alleles (higher Fst) and reduction in genetic diversity (lower heterozygosity).
Strengths:
Biological invasion is a critical driver of biodiversity loss, and it is important to understand how invasive species adapt to novel environments despite limited genetic diversity (genetic paradox of biological invasion). Lantana camara is one of the hundred most invasive species in the world (IUCN 2000), and the authors collected 359 plants from a wide geographical range in India, where L. camara has invaded. The scale of the dataset and the importance of the target species are the strengths of the present study.
Weaknesses:
One of the most critical weaknesses of this study would be that the output modelling analysis is largely qualitative, which cannot be directly comparable to the empirical data. The main findings of the SLiM-based simulation were that selfing promotes the fixation of alleles and the reduction of genetic diversity. These are theoretically well-reported knowledge, and such findings themselves are not novel, although it may have become interesting these findings are quantitatively integrated with their empirical findings in the studied species. In that sense, a coalescent-based analysis such as an Approximate Bayesian Computation method (e.g. DIY-ABC) utilizing their SNPs data would be more interesting. For example, by ABC-based methods, authors can infer the split time between subpopulations identified in this study. If such split time is older than the recorded invasion date, the result supports the scenario that multiple introductions may have contributed to the population structure of this species. In the current form of the manuscript, multiple introductions were implicated but not formally tested.
I also have several concerns regarding the authors' population genetic analyses. First, the authors removed SNPs that were not in Hardy-Weinberg equilibrium (HWE), but the studied populations would not satisfy the assumption of HWE, i.e., random mating, because of a high level of inbreeding. Thus, the first screening of the SNPs would be biased strongly, which may have led to spurious outputs in a series of downstream analyses. Second, in the genetic simulation, it is not clear how a set of parameters such as mutation rate, recombination rate, and growth rate were determined and how they are appropriate. Importantly, while authors assume the selfing rate in the simulation, selfing can also strongly influence the effective mutation rate (e.g. Nordborg &amp; Donnelly 1997 Genetics, Nordborg 2000 Genetics). It is not clear how this effect is incorporated in the simulation. Third, while the authors argue the association between flower color and population structure, their statistical associations were not formally tested. Also, it is not mentioned how flower color polymorphisms are defined. Could it be possible to distinguish many flower color morphs shown in Figure 1b objectively? I am concerned particularly because the authors also mentioned that flower color may change temporally and that a single inflorescence can have flowers of different colors (L160).

The population structure of invasive Lantana camara is shaped by its mating system

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