Mikania micrantha genome provides insights into the molecular mechanism of rapid growth.

Mikania micrantha is one of the top 100 worst invasive species that can cause serious damage to natural ecosystems and substantial economic losses. Here, we present its 1.79 Gb chromosome-scale reference genome. Half of the genome is composed of long terminal repeat retrotransposons, 80% of which have been derived from a significant expansion in the past one million years. We identify a whole genome duplication event and recent segmental duplications, which may be responsible for its rapid environmental adaptation. Additionally, we show that M. micrantha achieves higher photosynthetic capacity by CO2 absorption at night to supplement the carbon fixation during the day, as well as enhanced stem photosynthesis efficiency. Furthermore, the metabolites of M. micrantha can increase the availability of nitrogen by enriching the microbes that participate in nitrogen cycling pathways. These findings collectively provide insights into the rapid growth and invasive adaptation.

d) On a more subjective note, do you feel that the paper will influence thinking in the field? The paper will influence thinking in the field (see also part b)

Conclusion
The manuscript should be accepted, but only with major revisions (see also part b and c).
Reviewer #2 (Remarks to the Author): Liu et al. report a chromosome scale genome of the rapid growing plant Mikania micrantha and analyze genomic features associated with its high photosynthetic rate and fast growth. Gene duplicates retained from its whole genome and segmental duplications are enriched in functions related to growth and photosynthesis, reflecting adaptive evolution to fast growth. The authors provide several lines of evidence supporting CAM photosynthesis activity in M. micrantha, and when combined with stem photosynthesis, may help boost growth. The authors also identified candidate biosynthesis genes for the allelochemicals STLs and propose a model where STLs promote soil nitrogen cycling. I have some minor concerns that should be addressed to strengthen this manuscript.
1. Line 118. The authors state the sharp Ks peak < 0.2 correlates to recent segmental duplications that span ~23% of the M. micrantha genome. Based on the Kmer peak in Supplemental Figure 2, it seems like this genome is highly heterozygotic. These duplicated regions may represent haplotype specific sequences that were not filtered out using falcon-unzip from the assembly. The amount of haplotype-specific sequence identified and filtered out of the assembly is not discussed in the methods or the results, but this would be useful to provide in a supplemental table.
2. Plant genomes contain multiple copies of core CAM pathway genes, and most are involved in housekeeping processes and metabolic functions unrelated to CAM. The genes shown in the heatmap of figure 3b may not be the copies functioning in CAM. For instance, PEPC is usually expressed and transcribed during the day time in CAM plants, and post-translationally activated through phosphorylation by PEPCK at night. The three PEPC genes shown in Figure 3b have high nocturnal but low diurnal expression, contrasting what has been shown in other CAM lineages. A single subfamily of PEPC has been recruited independently in all C4 and CAM lineages and a phylogenetic tree of PEPC genes may help the authors resolve which copies are functioning in CAM. Based on the results, it seems like only two timepoints were collected for surveying CAM gene expression, but six are shown in the heatmap in Figure 3b. Is this heatmap just showing independent replicates?
Reviewer #3 (Remarks to the Author): This manuscript deals with the genome of M. micrantha, an invasive species of the Asteraceae family that is characterized by a very rapid growth. The authors describe the assembly and annotation, they perform a comparative genomics study with other Asteraceae species, and date the expansion of LTRretrotransposons. Then they investigate the genomic basis of rapid growth and production of allelochemicals in M. micrantha.
The manuscript is generally well written, although some sentences are long and some jargon is used (generalist readers may not know what "allelochemical" means). The main text nevertheless lacks some basic information on some methods (e.g., a network is built but it is not clear in the text whether this is a metabolic or co-expression network), or there is a reference to a control without explicit stating of what is this control. There is also a lack to the references to the proper sections of the methods or supplementary material (e.g. L235-236, there is no indication as where the methods that were used to build the network are described).
The genome assembly and annotation metrics are good, but unless I am wrong, the authors did not provide accession number for their release (from the detailed description of data availability only for raw data, which is not enough). An increasing number of plant genome are "published" without actually releasing the genome assembly, which I find really concerning, because the ressource are not really "provided" to the community.
Concerning the general genome analysis performed in the manuscript, the comparative genomics analysis in Astereaceae shows that M. micrantha, which belongs to the Heliantheae tribe, shares a whole-genome-duplication with sunflower. However, this not a scoop, the results presented here are very similar to previous findings (Badouin et al 2017, but also Barker et al 2008, who already shown that the whole-genome duplication of sunflower occurred at the basis the Heliantheae tribe). The analysis of LTR-retrotransposons is rather superficial and does not bring very interesting information.
The investigation of the genomic bases of specific life-history traits is more interesting. The authors find an enrichment of photosynthesis-related genes, but I am concerned that such genes are often left out in genome assemblies and that the enrichment seen here may be very dependent on methods that were used to assemble the different genomes in the dataset. The investigation of allelochemicals production is very interesting, especially because of the soil metagenomic experiments, but I must admit that I am not a specialist of soil biology and metabolism and that I lack the skills to evaluate this part. The defoliating experiment is also interesting but the manuscript should state if defoliated growth is common in M. micrantha, otherwise one does not see the revelance of the experiment. Introduction -paragraph L42: add a comma to cut this long sentence "to the canopy, and blocking the sunlight" -paragraph2: very clear -paragraph3: L57: of "the" invasive species: suppress "the" L61: use past tense ("we generateD" ... "and performED") L63: use plural: "genetic processes and molecular mechanisms" Results Section 1 LTR-RT -paragraph1 assembly how did the authors validate the correspondance between heterozygote fragments. How was genome size evaluated? What is the mean length of subreads? -Paragraph 2 annotation: L85-86: not very informative, the different "functional" databases provide very different levels of evidence. What databases were used? Paragraph 3 LTR L89: "high coverage and continuity": no information on how many gaps remain. The "continuous" aspect is not well accounted for. L90-91: "which play multiple role...": this part of the sentence is not informative. Be more precise or remove.
L96-99: no information on the method that was used to estimated the age of LTR-RT expansion.
Section2 WGD: problem with the title "improve ecological adaptation" (no evidence for that). Please tone down. L108-111: missing R in "orthologous" I do not understand the difference between the two parts of the sentence, separated by 'as well'. I do not understand what "syntenic genes" are: synteny is studied at the level of multiple genes. L117: "with Ks values less than 0.2": Do you mean "lower than 0.2"? Sentence not clear "the enrichment of 127 synteny blocks": enrichment in what? L123-127: sentence too long that is difficult to read, cut it in to.
-Section 2 metabolism. Again, a very affirmative title. Is the level of evidence sufficient? Paragraph 2 L152-155: the important information is whether all genes in the CAM pathway are present in the genome, not the raw number of candidate genes. Is this the case, or are there missing genes? Paragraph 3 L160: diel? L179-L181: tone down the conclusion, this is suggested but not the definitive evidence. Section 4: paragraph 1: L188-L189: why this choice of plants. Are they particularly representative? paragraph 2: defoliating experiment L211-212: "on the basis of contrasted expression pattern": uncessary piece of sentence.
Section allelopathic effects: L230-231: again, define allelopathic for the generalist reader. L231: point at the end of the sentence, not comma. L235: "genome-scale network": do you mean a metabolic network? Co-expression network? This must be stated.
L243: Define LC-MS? L244: of five genes: of the five genes L244-246: sentence not clear. What do you mean by "was demonstrated in this study"? L246-248: not clear either. What is the "five genes interaction network?" Overall, paragraph 2 of this section needs to be re-written in a clearer way, removing redundancy and highlighting the conclusions L276: a, not "an" at the end of the sentence. L279: use "increased", not "improved" What is proof that the increased CO2 content comes from the microorganisms and not from the plants? Why add the STLs to soil near a monoculture rather than to bare soil alone? It is not clear in the text whether the five STLs were added together or separately. paragraph 2: L285-287: what is the control? Overall, the interest of this experiment should be better justified.
paragraph soil metagenomic L302-305 sentence not correct, delete "the abundance" (no ok with "is significantly enriched") L319-320: not clear, what do you mean by "able to prevent the loss of available nitrogen". L322: "was all" > were L322: "these evidences" > these evidence (no S) All this message on the effect of STL in interesting, but there is no evidence of the allelopathic effect of these STL. Relevant with the message? Discussion: L327-330: not informative L330-332: WGD not a scoop, already known that it was shared by most Heliantheae. L333-334: redundant with the results section. L334: double T L335 with "a" published genome L336: upper case in Asterids L336-337: how? L344-L345: "most than ..." only if the set of other species chosen in the stem photosynthesis experiment was representative. It is not possible to judge it because this choice was not justified. L348-349: what do you mean by "transferring enzymes from C4 to C3 plants"? L352-353: this sentence has no verb. remove the ", which "? L356: "could not only promoted" > "could not only promote". What is dissimilatory? L360-364: is this biosynthesis pathway for STL different than in other plants? If not it is not "new".

Responses to comments from Reviewer 1
What is rather surprising, however, is that the STL are supposed to have a stimulatory effect on the soil microbiome and are considered as a "new biofertilizer to promote nutrient cycling". This would indeed be a novelty, but the authors do not explain by what mechanism these STL are supposed to mediate this nutrient cycling effect. This part needs a more convincing explanation and more discussion.
In previous researches, allelochemicals were mainly reported to inhibit microorganisms. However, some studies also showed that allelochemicals can stimulate soil microfauna and microorganisms. For example, the study on a serious invasive species, Fallopia japonica, which is greatly threating to Europe, found that the abundance of soil predator Acari could be increased by adding rhizome extract of According to the reviewer's comments, we performed two experiments to find more evidences that STLs promote nutrient cycling of soil.

Experiment 1:
Five STL (Dih, Anh, Deo, Sca, 3-epi) were adding to the bare soil to identify whether STL affects microorganisms and promote nutrient cycling. The concentration of NH4 + -N was increased when adding STL to the soil (Supplementary Table 11). Then, we selected the Dih soil, which was the highest among the five STL in the soil colonized by Mikania micrantha, for high-throughput sequencing. The 16S sequencing results showed that the relative abundance of Bradyrhizobium was significantly increased after adding Dih compound at genus levels (P-value = 0.002, two-sided t-tests, see Supplementary Figure   35). Bradyrhizobium belong to the nitrogen fixation bacteria and convert molecular nitrogen in the air into ammonia or related nitrogenous compounds in soil.

Experiment 2:
The density of cultured nitrogen-fixing bacteria was used to prove the effects of STL on microorganisms. Adding 1mL of 2 mg/L STLs (Dih, 3-epi, and mixture of five STL) and 0.5 mL cultured nitrogen-fixing bacteria respectively to 25mL of culture medium.
Meantime, adding STL (Dih, 3-epi, and mixture of five STL) respectively to culture medium, and adding nothing as the control. These samples were cultured in a shaker at 28℃ and 150 rpm for 7 days. Each treatment was replicated 5 times. After then, OD values of each treatment were measured at 600 wavelengths by ultraviolet spectrophotometer (UV-2450) to evaluated the density of azotobacter. We found that the colony-forming units (CFU) for nitrogen-fixing bacteria were significantly increased after adding STL to the culture medium (Figure a). These results showed that the STLs promote nitrogen-fixing bacteria growth. According to these results, STLs can increase concentration of NH4 + -N and promote nitrogen-fixing bacteria growth. However, we cannot explain the molecular mechanism of STL's influence on soil microorganisms exactly. Not to give the reader ambiguous conclusions, we changed the title of this section from "Mikania sesquiterpene lactones promote nutrient utilization by reinforcing the soil microbial functioning" to "Reinforced soil microbial functioning promotes its nutrient utilization". We modified the conclusion that STL promotes soil microbial enrichment and soil nutrient cycling, including Figure 6a

General considerations
1. Generally, the grammar and style should be checked in the main manuscript and the supporting material.
We agree with the reviewer's comment. The grammar and style have been revised in main manuscript and supporting material.

Part: "Metabolic pathway and genetic basis of Mikania sesquiterpene lactones"
2. l. 230: "Sesquiterpene lactones (STLs), which are mainly derived from the precursor germacrene A acid (GAA), are well known for their allelopathic effects and are characteristic of the family Asteraceae." => End sentence with a dot. Cite references for these statements.
We have cited references for these statements in revised version.
3. l. 230: "The biosynthetic pathways of STLs related to synthetases including HaG8H, LsCOS, and Ih8H from sunflower, Lactuca sativa and Inula hupehensis have been elucidated 32-34" Like this the sentence makes no sense. => keep references but rephrase: "Several enzymes acting downstream of GAA have been elucidated recently, such as HaH8H, LsCOS and Ih8H, that lead to the formation of different STL with distinctive stereochemistry32-34." We agree with the reviewer's comment. The sentence has been revised. 4. l.234: "However, the biosynthetic pathways of STLs in Mikania remain unknown." Here some information is missing: At this point the reader must be informed about the common stereochemistry of all STL detected in this study to understand why the authors chose to look for the Ih8H homolog. Include the following sentence: "As all the detected STL had a 7,8-trans lactone, we were investigating for a homolog of Ih8H, an enzyme capable of synthesizing 7,8-trans inunolide, the putative precursor of these Mikania micrantha STL." This part has been revised to "However, the biosynthetic pathways of STLs in Mikania remain unknown. Lactone ring formation in STLs is dependent upon the (C6 or C8) position and stereoselective hydroxylations of GAA, which can result in STLs with four unique configurations (12, 6α-, 12, 6β-, 12, 8α-, and 12, 8β-olides derivatives of GAA)     Table S8)." Several things have to be corrected here: 1. Fig 5a has a poor resolution and is as such meaningless, as the labeling can not be read.
The resolution of Figure 5a have modified in the revised manuscript.
2. Fig 5b: y-axis label has to be corrected.
We used the standard curve of inunolide to calculate the content of inunolide in fresh tissues. Thus, y-axis label of Figure 5b have been corrected.
Eupatolide is a C12,6α lactone, which have been changed in Figure 5c of revised manuscript.

Fig 5e
: resolution and illustrative graphics must be improved.
We have corrected those in the revised manuscript.  We isolated and identified five STLs in M. micrantha, the purity was analyzed by HPLC and 1 H NMR (Figure f).

Responses to comments from Reviewer 2
1. Line 118. The authors state the sharp Ks peak < 0.2 correlates to recent segmental duplications that span ~23% of the M. micrantha genome. Based on the Kmer peak in Supplemental Figure 2, it seems like this genome is highly heterozygotic. These duplicated regions may represent haplotype specific sequences that were not filtered out using falcon-unzip from the assembly.
The amount of haplotype-specific sequence identified and filtered out of the assembly is not discussed in the methods or the results, but this would be useful to provide in a supplemental table.
We agree with the reviewer's comment. We added the methods of genome assembly and haplotype identification in supplemental Note 1. "First, the PacBio reads were used to assemble the M. micrantha genome by the software canu-1.6, and 7,523 contigs were generated with a total length of 2.16 Gb. Based on the distribution of k-mer frequencies, the estimated genome size of M. micrantha is 1.87 Gb. Then, we used the following methods to filter the heterozygous sequences: Firstly, we used the genome sequence to align to itself by MUMmer3.23, and filtered out the heterozygous segments with more than 70% coverage and 70% identity. Secondly, the PacBio reads were re-assembled by falcon/falcon-unzip, which generated 1,684 Mb primary reference sequence and 352 Mb alternative heterozygous haplotype sequence (haplotig). Then, we aligned the 352 Mb alternative heterozygous haplotype sequence (haplotig) to the canu assembled contigs, to further filter the heterozygous sequences in the canu reference sequences. The We agree with the reviewer's comment that most of multiple copies of core CAM pathway genes are involved in housekeeping processes and metabolic functions unrelated to CAM. And there was also reports that PEPC was activated by phosphorylation to catalyse the primary assimilation of CO2 in Crassulacean acid metabolism plants at night. The expression patterns of three PEPC genes shown in The three PEPC genes in pineapple were used to align to the protein sequences with e-value cutoff of 1 × 10 −5 in P. equestris, A. shenzhenica, D. officinale and M. micrantha, respectively. Based on BLASTP analysis, totally eleven candidate PEPC genes were identified in P. equestris (2), A. shenzhenica (2), D. officinale (2) and M. micrantha genome (5) (identify > 60% and coverage > 60%). The phylogenetic tree showed the relationships of the putative PEPC genes in these CAM plants (Figure k). In phylogenetic tree, all the putative PEPC genes were clustered into two clades. Two M. micrantha

Responses to comments from Reviewer 3
The manuscript is generally well written, although some sentences are long and some jargon is used (generalist readers may not know what "allelochemical" means).
Thanks for the reviewer's suggestion. The defines of Allelochemical and allelopathic have been added in the introduction part.
The main text nevertheless lacks some basic information on some methods (e.g., a network is built but it is not clear in the text whether this is a metabolic or coexpression network), We constructed a co-expression network to speculate STL proposed biosynthesis pathway by transcriptome data. We have made corresponding modifications in the main text.
or there is a reference to a control without explicit stating of what is this control.
We have explained it in the revised version.
There is also a lack to the references to the proper sections of the methods or supplementary material (e.g. L235-236, there is no indication as where the methods that were used to build the network are described).
We changed the sentence from "We reconstructed a genome-scale network for M. micrantha and focused on the metabolic pathways involved in inunolide synthesis" to "We reconstructed a genome-scale co-expression network for M. micrantha and focused on the metabolic pathways involved in inunolide synthesis". In the transcriptome data analysis of materials and methods part, we described the method of co-expression network analysis.
"Co-expression network analysis was performed by WGCNA package using all transcriptomes. WGCNA network construction and module detection was identified using an unsigned type of topological overlap matrix (TOM) with a soft-thresholding power β of 28 (R 2 > 0.9)".
The genome assembly and annotation metrics are good, but unless I am wrong, the authors did not provide accession number for their release (from the detailed description of data availability only for raw data, which is not enough). An increasing number of plant genome are "published" without actually releasing the genome assembly, which I find really concerning, because the resource are not really "provided" to the community.
We agree with the reviewer's suggestion. When the manuscript is submitted, the genome sequence and gene annotation results had been uploaded to NCBI. We have shown the accession number in the maintext.
The defoliating experiment is also interesting but the manuscript should state if defoliated growth is common in M. micrantha, otherwise one does not see the revelance of the experiment.
Defoliation is a common phenomenon in the whole life history of plants, such as insect bite, drought and other conditions can lead to plant leaf wilting, litter and so on. In this study, the stem photosynthetic capacity of Mikania micrantha, other vines and associated plants under normal conditions was measured. It was found that the stem of Mikania micrantha has a certain photosynthetic capacity, which is much higher than that of the control plant. In order to further study whether the stem photosynthetic capacity of  We have added the define of allelochemicals in the introduction part.
Introduction -paragraph L42: add a comma to cut this long sentence "to the canopy, and blocking the sunlight" We have added a comma to cut this sentence. -paragraph3: L57: of "the" invasive species: suppress "the" We have delete "the" in this sentence in revised version.
L61: use past tense ("we generateD" ... "and performED") According to the comment, we have corrected the sentence in revised version.
L63: use plural: "genetic processes and molecular mechanisms" According to the comment, we have corrected the sentence in revised version.

Section 1 LTR-RT
-paragraph1 assembly how did the authors validate the correspondance between heterozygote fragments. How was genome size evaluated? What is the mean length of subreads?
For the heterozygote fragments, we added the methods of genome assembly and haplotype identification in supplemental Note 1. "First, the PacBio reads were used to assemble the M. micrantha genome by the software canu-1.6, and 7,523 contigs were generated with a total length of 2.16 Gb. Based on the distribution of k-mer frequencies, the estimated genome size of M. micrantha is 1.87 Gb. Then, we used the following methods to filter the heterozygous sequences: Firstly, we used the genome sequence to align to itself by MUMmer3.23, and filtered out the heterozygous segments with more than 70% coverage and 70% identity. Secondly, the PacBio reads were re-assembled by falcon/falcon-unzip, which generated We agree with the reviewer's comment. The sentence has been change from "A total of 42,804 (92%) coding proteins were annotated by functional databases (Supplementary   Table S5)" to "A total of 42,804 (92%) coding proteins were annotated by functional databases (Supplementary Table S5), including eggNOG, KEGG, Interpro and uniprot".

Paragraph 3 LTR
L89: "high coverage and continuity": no information on how many gaps remain. The "continuous" aspect is not well accounted for.
There are only 1,599 (0.0089% of whole genome) gaps in M. micrantha reference genome sequences. This result has been added in Table 1 in revised version.
L90-91: "which play multiple role...": this part of the sentence is not informative. Be more precise or remove.
We remove this sentence "which play multiple roles in driving genome evolution in eukaryotes" in the revised version.
L96-99: no information on the method that was used to estimated the age of LTR-RT expansion.
We have added this sentence "The LTR insert time is estimated by LTR-retriever." to describe the method of LTR expansion time estimation in Materials and Methods.
Section2 WGD: problem with the title "improve ecological adaptation" (no evidence for that). Please tone down.
We agreed with reviewer's comment. The title has been change to "Genome-wide replication and subsequent fragment replication of Mikania micrantha may play an important role in ecological adaptation". L108-111: missing R in "orthologous" We have revised this error.
I do not understand the difference between the two parts of the sentence, separated by 'as well'. I do not understand what "syntenic genes" are: synteny is studied at the level of multiple genes.
The sentence has been changed to "To study the conservation of genomic structure, we identified the inter-species syntenic genes between M. micrantha and sunflower, artichoke, and grape, and intra-species syntenic genes within the M. micrantha, sunflower, lettuce, artichoke and coffee genomes, then calculated the Ks values of the syntenic fragments for orthologous pairs".
Yes, we changed this sentence from "less than 0.2" to "lower than 0.2".
Sentence not clear "the enrichment of 127 synteny blocks": enrichment in what?
We changed this sentence from "we identified the enrichment of 127 syntenic blocks" to "we identified 127 syntenic blocks".
L123-127: sentence too long that is difficult to read, cut it in to.
This sentence has been changed to "The WGT-1, WGD-2 and SD-3 duplicated genes were significantly enriched in the functions of catalytic and hydrolase activity, nitrogen compound metabolism, transcription factor, response to stimulus and chemical ( Figure 2d and Supplementary Figure 9-10). It demonstrated that extensive gene fractionation has occurred during the evolutionary history of the M. micrantha genome, which retained the genes that are essential for survival and lost other redundant genes." -Section 2 metabolism. Again, a very affirmative title. Is the level of evidence sufficient?
We think it's a good suggestion. The title has been changed from "Cooperation of the C3 and CAM photosynthesis pathways provides abundant carbohydrates for rapid growth" to "High photosynthesis of the leaves provides abundant carbohydrates for rapid growth".
Paragraph 2 L152-155: the important information is whether all genes in the CAM pathway are present in the genome, not the raw number of candidate genes. Is this the case, or are there missing genes?
All key genes in the CAM and C4 pathway were identified in Mikania micrantha genome (see Figure m). The sentence has been changed from "To investigate the diel enzyme activity patterns of the CAM pathway" to "To investigate the enzyme activity patterns of the CAM pathway in day and night".
L179-L181: tone down the conclusion, this is suggested but not the definitive evidence.
The sentence has been changed from "Together, these results showed that M.
micrantha can not only fix carbon dioxide by the C3 pathway in the daytime, but also obtain sufficient carbon dioxide though the CAM pathway at night." to "These results indicate that one of the reasons for the higher photosynthetic capacity of Mikania micrantha leaves is that CO2 may be absorbed at night by photosynthetic pathway similar to CAM and stored in vacuoles as organic acids to supplement the carbon fixation capacity during the day".
In addition, we added some discussion in revised version (see discussion part). We have removed this sentence "on the basis of contrasted expression pattern" from the revised version.
We have added the define of allelopathic in the Introduction part. "allelopathic effects is a biological phenomenon by which an organism produces one or more biochemicals that influence the growth, survival, and reproduction of other organisms." L231: point at the end of the sentence, not comma.
This sentence has been revised.
L235: "genome-scale network": do you mean a metabolic network? Co-expression network? This must be stated.
"genome-scale network" means Co-expression network. We changed the sentence from "We reconstructed a genome-scale network for M. micrantha" to "We reconstructed a co-expression network for M. micrantha".

L243: Define LC-MS?
We changed the sentence from "we have developed an LC-MS method to determine the relative inunolide contents" to "we have developed a liquid chromatograph-mass spectrometer (LC-MS) method to determine the relative inunolide contents". This sentence has been revised from "Corresponding to the five genes interaction network, the candidate genes of STLs biosynthesis were detected" to "According to the co-expression network, the candidate genes of STLs biosynthesis were detected".
Overall, paragraph 2 of this section needs to be re-written in a clearer way, removing redundancy and highlighting the conclusions Thanks for reviewer's suggestion. We rewrote the paragraph to make it easier to understand.
This paragraph has been change into "STLs have been postulated to be derived from the precursor germacrene A acid (GAA L276: a, not "an" at the end of the sentence.
It has been revised. L279: use "increased", not "improved" It has been revised.
What is proof that the increased CO2 content comes from the microorganisms and not from the plants?
We collected the soil in the field, then sifted the soil through a sieve with 1mm diameter. Then, we add the STLs into the soil without any plants, and put it in a incubator.
So absolutely the increased CO2 content comes from the microorganism and not from the plants.
Why add the STLs to soil near a monoculture rather than to bare soil alone?
We think the soil near Mikania monoculture will be the nearest colony for Mikania in the near future during the period of the plant Mikania spreading. If the STLs could be the new weapon for Mikania colonizing, the soil near the monoculture would be influenced first.
The soil near the monoculture is the same as the bare soil because there are no plants growing in the soil near the Mikania monoculture. The picture we collected the soil is as follows: It is not clear in the text whether the five STLs were added together or separately.
We try to understand how STLs have an effect on the soil in the process of Mikania invading, and which one affected the most, so we added the five STLs into the soil separately, not together. We added this information in the text. Overall, the interest of this experiment should be better justified.
The control is the blank soil which no plants were planted. This sentence has been changed from "compared to that in the soil of its' two neighboring species and the control" to "compared to that in the soil of its' two neighboring species and the control which no plants were planted".
paragraph soil metagenomic L302-305 sentence not correct, delete "the abundance" (no ok with "is significantly enriched") We agreed with the reviewer's comment. We deleted the "the abundance of " in revised version.
L319-320: not clear, what do you mean by "able to prevent the loss of available nitrogen".
Because denitrifying bacteria are responsible for converting nitrate nitrogen into gaseous nitrogen oxides and consuming them, a reduction in denitrification genes means a reduction in the loss of available nitrate nitrogen.
We changed the sentence from "suggesting that the soil microbes of M. micrantha were able to prevent the loss of available nitrogen." to "suggesting that the soil microbes of M. micrantha were able to reduce the conversion of nitrate nitrogen into gaseous nitrogen oxides, thus preventing the loss of available nitrogen.".
L322: "was all" > were It has been revised.
L322: "these evidences" > these evidence (no S) It has been revised.
All this message on the effect of STL in interesting, but there is no evidence of the allelopathic effect of these STL. Relevant with the message?
The allelopathy of STL has been proved in previous studies (Shao et al., 2005;Huang et al., 2008;Piyasena et al., 2013). For example, Shao et al. reported that the sesquiterpenoids from M. micrantha inhibited both germination and seedling growth of tested species with deoxymikanolide possessing the strongest phytotoxicity. In a bioassay against lettuce (Lectuca sativa L.), deoxymikanolide reduced radicle elongation at low concentration (IC50 = 47 microg/ml); dihydromikanolide showed a weaker effect (IC50 = 96 microg/ml). Deoxymikanolide caused yellowish lesions at the root tips of lettuce at a concentration of 50 microg/ml, and a 250 microg/ml solution killed lettuce seedlings (Shao, 2005). Huang et al reported that deoxymikanolide and dihydromikanolide showed the inhibitory effect on seeding growth of B. parachinensis with I50 value at 1.08 and 0.88 mM. Deoxymikanolide and dihydromikanolide inhibited on shoot growth with the I50 values in the assay being 0.04 and 0.05 mM (Huang, 2008) In this manuscript, we provide possible synthesis pathways of STL and candidate genes.
"dissimilatory nitrogen reduction" should be "dissimilatory nitrate reduction to ammonium". L360-364: is this biosynthesis pathway for STL different than in other plants? If not it is not "new".
This biosynthesis pathway of STL was found in other species, but it is found in Mikania micrantha for the first time. We agreed with the reviewer's comment. We selected two chromosomes to display syntenic relationship between M. micrantha and sunflower. And Dot plot of syntenic orthologues in genome level was moved to supplementary. The total genes number of M. micrantha (51 genes) in the CAM pathway were more than that in pineapple (34 genes) and sunflower (37 genes). e) L443: add "proposed pathway", be careful on the conclusions.
Thanks for the reviewer's valuable suggestions. We have added the "proposed" in this sentence.