Transporter gene family evolution in ectomycorrhizal fungi in relation to mineral weathering capabilities

King, Katharine; Fransson, Petra; Finlay, Roger; Sánchez-García, Marisol

doi:10.5194/bg-22-8047-2025

Articles | Volume 22, issue 24

https://doi.org/10.5194/bg-22-8047-2025

© Author(s) 2025. This work is distributed under
the Creative Commons Attribution 4.0 License.

https://doi.org/10.5194/bg-22-8047-2025

© Author(s) 2025. This work is distributed under
the Creative Commons Attribution 4.0 License.

Articles | Volume 22, issue 24

Research article

|

17 Dec 2025

Research article |

| 17 Dec 2025

Transporter gene family evolution in ectomycorrhizal fungi in relation to mineral weathering capabilities

Katharine King, Petra Fransson, Roger Finlay, and Marisol Sánchez-García

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Final revised paper (published on 17 Dec 2025)
Supplement to the final revised paper
Preprint (discussion started on 17 Jan 2025)

Interactive discussion

Status: closed

RC1:
'Supplemental information not found for egusphere-2024-3930', Anonymous Referee #1, 07 Feb 2025

I tried to find supplemental information to review this manuscript, but there is no DOI at https://doi.org/10.6084/m9.figshare.28016633. Please notify authors to fix this so I can review all materials in a timely manner.

Citation: https://doi.org/10.5194/egusphere-2024-3930-RC1
- EC1:
  'Reply on RC1', Mark Lever, 07 Feb 2025
  
  Thank you for pointing that out. I just heard from the authors: The SI can be found at this link: https://figshare.com/s/bdf230f68420e2ed9404
  
  Citation: https://doi.org/10.5194/egusphere-2024-3930-EC1
  - RC2: 'Reply on EC1', Anonymous Referee #2, 19 Feb 2025
    
    The authors investigate how base cation transporter gene families are important for mineral weathering processing by fungi. The authors use different approaches, but the different parts could be brought together more organically. Overall, this is a very interesting idea, but the rationale is not well articulated and the different parts are not connected. I think that the article could be published but not in its current form. Broadly, it would be helpful to have a clearer motivation stated and some way of bringing together the different components. I have some general suggestions:
    Please add at the beginning of the introduction some information about mineral weathering and perhaps a definition.
    I'm wondering if it would be helpful to have a diagram of the soil and the movement of the nutrients through weathering and fungal uptake in the introduction. Perhaps including Mg and the elements in Table 1 to bring the different parts of the paper together that feel a bit dispersive.
    It is unclear overall what is the logic of the questions. For example, why did the authors look into the fungal mating-type pheromone? Justify the biology? Explain why is the expansion in pheromone receoptors suggestive of adaptation to an environment?
    For hypothesis 2, does the base cation uptake simulated in experimental conditions by the mineral additions?
    Are hypotheses 3 and 4 fundamentally the same? Or is there something specific about Suillus that the authors are trying to test?
    If it's just Suillus, how can we know if it's just lineage-specific? How can you exclude the evolution at the root? Or explain more clearly what the analysis is showing within Suillus.
    Add the goal/context for the bioinformatics section (2.1) to understand the questions those methods will help address.
    Provide more context for looking into Mg transporter family.
    Why is there no figure with the whole Agaricomycotina tree?
    I have a few specific suggestions:
    Figure 1 - what is the input matrix to calculate the PCAs? counts of genes? Perhaps a multipanel figure with also the phylogeny might help explain the analysis?
    Figure 2 - add branch lengths. The reason for this tree is to show the taxa that are tested in the lab? Since the bioinformatics section talks about a Agaricomycotina tree, I am puzzled by the selection of these taxa and what the purpose of this tree is.
    Figure 3 - highlight panels with significant slopes and mention statistical test in caption.
    Figure 4 - highlight different species on the figure with boxes around the names or labels. Report significance and statistical tests used in the caption.
    L18 list species of saprotrophic fungi
    
    L397-401 sentence is incredibly long
    
    L417 onwards, is there a way of summarising the information on significance so that it's not so difficult to read through? perhaps a table?
    
    L447 italicise luteus
    
    Citation: https://doi.org/10.5194/egusphere-2024-3930-RC2
    
    AC2: 'Reply on RC2', Katharine King, 08 Apr 2025
    
    Here are our responses to reviewer 2's comments
    
    Citation: https://doi.org/10.5194/egusphere-2024-3930-AC2
RC3:
'Comment on egusphere-2024-3930', Anonymous Referee #1, 11 Mar 2025

Using bioinformatic and culture-based approaches, this study examined the evolution of base cation transporter gene families in ectomycorrhizal fungi and their relationship to mineral uptake into mycelial biomass. Evolutionary expansions in copy numbers of these genes were linked to increased uptake of base cations into biomass. Overall this is an interesting and important study that covers a considerable amount of work. However, the presentation of the hypotheses, the results, the figures, and the overall writing of the manuscript need quite a bit of restructuring and simplification. I recommend accepting this manuscript for publication with major revisions.
General comments
How are hypotheses 1 and 4 different? It makes sense to combine them: “Greater base cation uptake is dependent on base cation transporter gene family copy numbers that result from evolutionary expansions”. This would require some reorganization of the results and discussion, but would them clearer and more concise.
Seventy-three supplemental figures is far too many. The authors should condense the expansion/contraction information from supplemental tree figures S3-S17 (which all appear to use the same phylogenomic tree) into one figure with a heatmap next to the tree, since they all represent the same idea. Likewise with figures S18-S56 for the partial dataset. The total number of gene family expansions/contractions shown in Fig 2 could also be included in this figure. This can be done in anvio by running anvi-import-misc-data for each tip of the tree.
The gene family evolution results are basically presented as a list, and could be made much more concise. I suspect that condensing and synthesizing gene expansion/deletion data from supplemental figures would lead to a broader-scale presentation of results that reads more naturally. Same idea for the mycelial cation correlation results.
Overall, the manuscript is very long. Significant sections require editing or removal to be made more concise or clear–more is not always better. I’ve given some examples below.
Introduction
Rewrite run-on or unclear sentences in lines 52-55, 57-60, and condense section from lines 80-91.
Methods
Line 220: I assume you meant micrometers instead of mm.
Line 246: How was biomass harvested? A brief description or citation would be sufficient.
Commas are needed to provide clarity on lines 160, 192, 279, and elsewhere in the manuscript. A period is missing in line 265.
Table 1: Why not simply specify the absolute concentrations of each mineral in each medium, rather than as a relative increase from the minimum medium?
Results/Figures/Supplementals
Line 300: Replace “There are” with “In the clade Agaricomycotina, we identified…”
Line 305: Assume you meant MFS instead
Figure 1a: Curious to know if you observe a separation between the non-Suillaceae, non-Russulaceae ECM and the non-ECM if you plotted on one of the other primary axes. Do all ECM fungi have different transporter gene composition from non-ECM fungi?
Similar comment for the correlations (Figures S58-S68), but do something like plot the R2 values to condense these into a single figure.
Fig. 2: Include in the legend that + refers to an expansion and – a contraction (I assume)
Fig. 3: Why show (or even mention, line 408) all the non-significant correlations? It distracts from the correlations you did find.
Line 444: Specify statistical test and results. Was this an ANOVA?
Discussion
Line 535: This point would be made more strongly if presented after synthesizing the base cation uptake results.
Line 540: How exactly might the choice or number of taxa influence the numbers of gene families found to have significant expansions/contractions, and what consequence might this have for interpreting these results? Please cite an example if possible.
Line 580: Add a reference to a figure here.
Awkward/unclear phrasing: lines 582-584
Line 601: Mention that this is hypothesis 2 (here, instead of in the next sentence that only references previous studies).
Line 612: Mention that this is hypothesis 3, since hypotheses were numbered in the abstract.
Lines 628, 632: Add references to figures here.
Line 624: Mention that this is hypothesis 4
Lines 625-634 belong in the results, not the discussion.
Line 634: typo?
Lines 649-652: run-on sentence

Citation: https://doi.org/10.5194/egusphere-2024-3930-RC3
- AC1: 'Reply on RC3', Katharine King, 08 Apr 2025
  
  Here are our responses to reviewer 1's comments
  
  Citation: https://doi.org/10.5194/egusphere-2024-3930-AC1
AC3: 'Comment on egusphere-2024-3930', Katharine King, 08 Apr 2025

Here we attach the figures (Fig. 1, 3, 4, 5, S3, S4, S7 and S8) which have been add/condensed/improved in accordance with the reviewer's suggestions.
The figure legends are as follows:
Figure 1 – In boreal forest podzols, ECM fungi, in symbiosis with the fine roots of host trees (left), can exude LMWOAs, siderophores, free radicals and protons which can weather minerals via their extraradical hyphae, and mobilise base cations and P (right). Both the exudation of weathering agents, and the uptake of base cations and P occurs through transporter proteins.
Figure 3 - Summary of copy number changes in significantly expanding and contracting transporter families (TFs) across the partial family dataset. The number of TFs expanding and contracting at each node of the phylogenomic tree (not to scale) are indicated with a positive (+) number for expanding families or a negative (-) number for contracting families. The heatmaps show changes in gene copy number of significantly expanding and contracting TFs, with contractions in blue and expansions in red at each node (left heatmap) and tip (right heatmap). Significant expansions and contractions are indicated with an asterisk (*). The heatmap for internal nodes (left) has gene copy number changes ranging from -22 to +49, and dashed red lines indicating which node corresponds to with row of the heatmap. The heatmap for tips (right) has gene copy number changes ranging from -48 to +101. For both heatmaps, base cation TFs are positioned to the left and separated from other TFs which are positioned to the right.
Figure S3 - Summary of copy number changes in significantly expanding and contracting transporter families (TFs) across the full family dataset. The number of TFs expanding and contracting at each node of the phylogenomic tree (not to scale) are indicated with a positive (+) number for expanding families or a negative (-) number for contracting families. The heatmaps show changes in gene copy number of significantly expanding and contracting TFs, with contractions in blue and expansions in red at each node (left heatmap) and tip (right heatmap). Significant expansions and contractions are indicated with an asterisk (*). The heatmap for internal nodes (left) has gene copy number changes ranging from -13 to +14, and dashed red lines indicating which node corresponds to with row of the heatmap. The heatmap for tips (right) has gene copy number changes ranging from -10 to +61. For both heatmaps, base cation TFs are positioned to the left and separated from other TFs which are positioned to the right.
Figure S4 - Summary of copy number changes in significantly expanding and contracting transporter families (TFs) across the partial subfamily dataset. The number of TFs expanding and contracting at each node of the phylogenomic tree (not to scale) are indicated with a positive (+) number for expanding families or a negative (-) number for contracting families. The heatmaps show changes in gene copy number of significantly expanding and contracting TFs, with contractions in blue and expansions in red at each node (left heatmap) and tip (right heatmap). Significant expansions and contractions are indicated with an asterisk (*). The heatmap for internal nodes (left) has gene copy number changes ranging from -8 to +14, and dashed red lines indicating which node corresponds to with row of the heatmap. The heatmap for tips (right) has gene copy number changes ranging from -21 to +24. For both heatmaps, base cation TFs are positioned to the left and separated from other TFs which are positioned to the right.
Figure S7 - Ratios of elemental concentration between gabbro and limited treatments for each fungal isolate. Panels show (a) iron (Fe), (b) potassium (K), (c) magnesium (Mg), (d) sodium )Na), and (e) phosphorus (P). Isolates include Suillus bovinus (Sb1-4), S. luteus (Sl1-3), S. variegatus (Sv1-3), S. granulatus (Sg1-3), S. grevillei (Sg4), Piloderma byssinum (Pb), P.aff fallax (Pf), P. olivaceum (Po), P. sphaerosporum (Ps), P. sp. 1, Pi. sp. 2, Coniophora puteana (Cp), and Serpula lacrymans (Sl). Data points represent the mean ration between gabbro and limited for each isolate, with error bars indicating the 95 % confidence interval.
Figure S8 - Ratios of elemental concentration between granite and limited treatments for each fungal isolate. Panels show (a) iron (Fe), (b) potassium (K), (c) magnesium (Mg), (d) sodium )Na), and (e) phosphorus (P). Isolates include Suillus bovinus (Sb1-4), S. luteus (Sl1-3), S. variegatus (Sv1-3), S. granulatus (Sg1-3), S. grevillei (Sg4), Piloderma byssinum (Pb), P.aff fallax (Pf), P. olivaceum (Po), P. sphaerosporum (Ps), P. sp. 1, P. sp. 2, Coniophora puteana (Cp), and Serpula lacrymans (Sl). Data points represent the mean ration between granite and limited for each isolate, with error bars indicating the 95 % confidence interval.

Citation: https://doi.org/10.5194/egusphere-2024-3930-AC3

Peer review completion

AR – Author's response | RR – Referee report | ED – Editor decision | EF – Editorial file upload

ED: Publish subject to minor revisions (review by editor) (19 Apr 2025) by Mark Lever

ED: Reconsider after major revisions (25 Apr 2025) by Mark Lever

AR by Katharine King on behalf of the Authors (28 Apr 2025) Author's response Author's tracked changes Manuscript

EF by Polina Shvedko (28 Apr 2025) Supplement

ED: Referee Nomination & Report Request started (12 Jun 2025) by Mark Lever

RR by Anonymous Referee #1 (18 Jun 2025)

Suggestions for revision or reasons for rejection

Overall, this revised version reads considerably better, particularly in the introduction, discussion, and updated figures. I have a few (mostly minor) suggestions that I believe should be addressed before publication. Here they are organized by section:

Abstract
In my opinion, 475 words is way too long for an abstract. Try reducing it to around 300 words, highlighting only the major takeaways from this study.
Lines 19-20: Put the results after the hypotheses.

Intro
Lines 57-59: Awkward wording.
Overall, the additions/changes (including the addition of Figure 1) provide a clearer background for the reader.

Methods
Please describe the significance threshold used for determining a significant expansion or contraction in copy number for a gene family.
For most of the bioinformatics analyses, it is not specified whether DNA or amino acids were used. Please clarify these.

Results and figures
Fig. S1: Not every branch has a bootstrap value. For these, was no value assigned, or was it not significant enough? A minor detail, but worth clarifying briefly.
Paragraph beginning at line 350: There are several mentions of “most frequent transporter gene families”. I assume these are the gene families that gained/lost the most copies? Please clarify.
Add 2+ or + superscripts to Mg and other cations, they are omitted in several places.
Table 2 legend: Pearson correlation coefficient (r) should be lowercase to distinguish it from R2 used in a linear regression.

Discussion
The interpretation of MAT-PR expansions in several Suillus sp enhancing sexual reproduction makes sense and is a great addition to the discussion.
More genes, more cation uptake- this makes sense. However, is there evidence for specific variants of certain genes leading to more efficient uptake? Possibly an interesting point to add.
Line 535: typo
Lines 594-599: this sentence should be split up
LInes 617-617: This last sentence leaves me a bit confused. Don’t the results of figure 4 suggest that increased copy numbers of MgtE correlate with increased cation uptake in Piloderma and other species as well?

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RR by Anonymous Referee #3 (18 Aug 2025)

Suggestions for revision or reasons for rejection

General comments

In their work, King et al. investigated mineral weathering by ECM fungi from both an evolutionary and an experimental perspective. They conducted a bioinformatic analysis focusing on expanding and contracting genes families, with particular attention to on base cation transporter gene families and the genera Suillus and Piloderma. They also grew several fungal species in a setup which allowed them to observe and quantify weathering capabilities of each species. Their final aim was to combine the bioinformatic analyses with growth assays, thereby testing for a direct connection between base cation uptake transporters and weathering.

I found the paper somewhat difficult to read and understand. I am not a specialist in phylogeny or bioinformatics, and ECM-related mineral weathering is also outside my main expertise. Still, I think some of the difficulty comes from the way the aims and outcomes are described. On close reading, it seemed to me that the authors could not directly link individual transporter genes to function. This is absolutely fine – negative or inconclusive results are part of the scientific process – but I think it should be stated more openly. Otherwise, the reader invests a lot of effort and may in the end feel a bit “tricked”.

Beyond that, the manuscript feels rather long, and several sections cold be condensed, as they seem to stray from the main topic. I provide more concrete suggestions in the specific comments below.

Specific comments and suggestions:

18: Since the fungi were always grown in pure culture, it may not be necessary to repeat “growing in pure culture” every time.
60 – 75: This section could be shortened. It describes the effects of C and N availability on weathering, which doesn’t seem like the main focus of your experiment.
73 – 75: It’s unclear what exactly needs to be understood better. If you specify – for example, it should be temporal variations, interspecific or intraspecific differences, somethis else… - the paragraph would have a clearer conclusion.
84: The extra sentence about LMWOA may not be necessary, as this is not the focus of your experiments.
94: Mentioning the two additional mineral-weathering species seems unnecessary since they are not included later in analyses or discussion.
100: Suggestion for clarity and flow: “can take place at multiple stages, from genomic to post-translational. On the genomic level, one mechanism is …”
101: Here CNV is mentioned for the first time. A short clarification would help: (for example “CNV refers to differences in gene copy number within a species (among individuals or populations). This does not equal long-term gene family expansion, which is the evolutionary outcome of repeated duplication events”). Adding this distinction would make the text easier to follow.
111 – 115: The sentences here are unclear. Is K⁺ transporter regulation the only case identified so far? For the second sentence, a shorter version would improve flow: “Yet, no studies have addressed the evolution of these transporter genes in the context of ECM-mediated mineral weathering.”
120: Again, repetitions of “pure culture” can be avoided.
163: What are the three levels of criteria? If they matter, please list them briefly; if not, the remark can be removed.
164: Numbers (e.g. 173) are clearer than words (“one hundred and seventy-three”).
170: Suggested rewrite to avoid repetition: “Relationships between transporter gene family copy numbers and fungal taxa were analysed and visualised using principal component analysis (PCA) on count data (Tab. S3) with CANOCO 5.10 (Microcomputer Power, Ithaca, NY, USA; Braak and Smilauer, 2012).”
179: A short explanation of “birth” and “death” in CAFE5 would help (less knowledgeable) readers: e.g. “Due to the birth–death model (duplication rate – loss rate) employed by CAFE5…”
199: Please clarify what the four levels of criteria are, or if they don’t matter, the remark can be removed.
200: A schematic of the three datasets, showing how they relate to each other and how they are used in analyses, would greatly help the reader.
219: Suggested edit: “Isolates of Suillus and another ECM genus, Piloderma, which inhabits both organic and mineral soil horizons, as well as two saprotrophic fungi, Coniophora puteana and Serpula lacrymans, were included…” (to avoid suggesting Suillus is not ECM).
242: Possible typo: should the fraction size be 63–2500 µm (sand particle size)? Also, since you later call it “sand,” clarify this earlier: “to retain a sand-sized fraction (63–2500 µm) to maximise surface area…”
351-372: These paragraphs are difficult to follow due to switching between the main dataset and subsets. It may be clearer to structure them by transporter gene family, describing and summarizing trends for each.
418: Suggested edit for clarity: “The phylogeny showed the latter: several clades of MgtE genes are present in each taxon.”
423: Suggested rewrite: “Of the 25 base cation transporter gene families that were significantly expanding or contracting, 11 also had copy numbers significantly correlated with base cation uptake.”
426: The relevance of reporting all 28 significant correlations is not clear. Many (16/28) correlations are under control conditions (which don’t directly prove weathering), several are negative, and for some TCDB IDs it is unclear how they relate to the reported ion transport. See also my comments on Table 2.
443: Questions regarding Table 2 (as I was not able to find a confirmation of these):
• Are 2.A.3 or 2.A.39 involved in Ca transport?
• Is 3.A.1 involved in Mg and Na transport?
• Are 3.A.3 or 3.A.3.2 involved in Mg transport
463: Isolate IDs are mislabeled (for example, S. bovinus are labeled as Sb4, Sb16, Sb25 and Sb36 but described as Sb1-Sb4 in the caption; same goes for all other isolate IDs)
477: Reporting here doesn’t fully match Figure 5. The figure shows significant ratios for 10/14 Suillus and 5/6 Piloderma isolates, but this is not stated. Also, C. puteana is omitted, though its isolate also showed a significant ratio.
479: Results do not mention that C. puteana had a high gabbro:limited Ca ratio. Since this species is part of your saprotrophic comparison, this seems important to highlight.
General note on discussion:
Overall, many conclusions seem only loosely supported by the results.
520: The 28 significant correlations are, as far as I can say, not sufficient to support Hypothesis 1 (see earlier comment for line 426).
561-565: For S. bovinus, no expansions were found in base cation transporter families, yet significantly higher Ca, Mg, and Fe were observed in gabbro versus limited treatment. This appears to contradict Hypothesis 1. The statement that this supports previous findings seems unrelated.
568-577: S. luteus has no significant expansions yet shows significantly higher Ca and Fe uptake in gabbro, which again contradicts Hypothesis 1. However, this is presented as supporting Hypothesis 2. The paragraph goes on by referring to other publication’s results and concludes by saying “these findings align with our first hypothesis”, which seems inconsistent.
579-583: S. variegatus shows expansions in MgtE and Na-related genes but actually takes up Ca and Fe. This also seems inconsistent with Hypothesis 1, yet the paragraph mainly cites other studies.
586: Mention of S. lakei, S. cothurnatus, and S. cf. subluteus feels disconnected from the rest of the discussion.
594: The phrase “not all transporter gene families associated with a particular base cation showed a significant positive correlation” seems misleading; in fact, only a small minority did.
607-617: This section is hard to follow. The hypothesis was that Suillus species would show greater base cation uptake than Piloderma. Only Ca showed this pattern, so the claim that Suillus has stronger weathering ability seems based only on Ca. The final sentence (“if we broaden our hypothesis…”) comes across as uncertain—almost as if the authors themselves are not sure whether Suillus performs better.

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ED: Reconsider after major revisions (27 Aug 2025) by Mark Lever

AR by Katharine King on behalf of the Authors (30 Oct 2025) Author's response Author's tracked changes Manuscript

EF by Katja Gänger (30 Oct 2025) Supplement

ED: Referee Nomination & Report Request started (07 Nov 2025) by Mark Lever

RR by Anonymous Referee #1 (10 Nov 2025)

ED: Publish as is (11 Nov 2025) by Mark Lever

AR by Katharine King on behalf of the Authors (19 Nov 2025) Manuscript

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Short summary

Symbiotic ectomycorrhizal (ECM) fungi have an important role in mineral weathering and base cation uptake in boreal forests. We characterised base cation transporter diversity and their evolution in ECM fungi in relation to weathering capabilities, and quantified base cation uptake from pulverised rock. Base cation transporter gene families evolved rapidly in the genus Suillus, with gene copy numbers correlating with base cation uptake in some cases, suggesting links to weathering capabilities.