Host Phylogenetic Relatedness and Soil Nutrients Shape Ectomycorrhizal Community Composition in Native and Exotic Pine Plantations

Round 1

Reviewer 1 Report

A well written and well executed study of exotic pines and their ectomycorrhizal fungi.  My only concern would be that the results and conclusions are based on quite young pine stands (27 years old) which undoubtedly do not yet have the full EMF diversity of mature forests.  So differences among native and exotic tree species may become more evident further into the rotation age.  Is there any literature from Asia that might have documented the typical alpha or gamma diversity of EMF in mature Pinus forests for comparison? (e.g. Kranabetter et al. 2018. Diversity and Distributions 24: 755-764)

Perhaps this topic is under consideration for future studies (line 381-386) but there is considerable interest in how host genetics affect ectomycorrhizal communities and overall fitness.  Some literature (see below) suggests quite fine-tuned local adaptation (e.g, Gehring et al. 2017, Kranabetter et al. 2015) while others suggest the opposite (Gundale et al. 2014) so the science is not settled.  Given the added pressures of ongoing climate change, the ability of exotic trees to thrive with native EMF will be crucial. 

Gehring, C.A., Sthultz, C.M., Fores-Rentería, L., Whipple, A.V., and Thitham, T.G. 2017. Tree genetics defines fungal partner communities that may confer drought tolerance. PNAS 114(42):11169-11174.

Gundale MJ, Kardol P, Nilsson M-C, Nilsson U, Lucas RW, Wardle DA. 2014. Interactions with soil biota shift from negative to positive when a tree species is moved outside its native range. New Phytologist 202: 415–421.

Kranabetter, J.M., Stoehr, M., and O’Neill, G.A. 2015. Ectomycorrhizal fungal maladaptation and growth reductions associated with assisted migration of Douglas-fir. New Phytol. 206(3):1135-1144.

Pickles, B.J., Twieg, B.D., O’Neill, G.A., Mohn, W.W., and Simard, S.W. 2015. Local adaptation in migrated interior Douglas-fir seedlings is mediated by ectomycorrhizas and other soil factors. New Phytol. 207(3):858-871.

Rúa MA, Lamit LJ, Gehring C, Antunes PM, Hoeksema JD, Zabinski C, Karst J, Burns C, Woods MJ. 2018. Accounting for local adaptation in ectomycorrhizas: a call to track geographical origin of plants, fungi, and soils in experiments. Mycorrhiza 28: 187–195

Some further comments:

Line 118. One time extractions of NH4 and NO3 are generally not credible as indices of N availability (see Binkley and Hart 1989. The components of nitrogen availability assessements in forest soils. Adv. Soil Sci. 10: 57-112).  C:N ratios would be more defendable as a general index of N supply.

Line 122. The way this is written it seems the sporocarp survey was part of the community analysis?  The only reference i see to it again is in the DNA extraction for a ‘mock community’ (line 138).

Line 214. See comment above, a total of only 43 EMF species suggests an early seral EMF community, many of which may be host generalists because that ensures better survival in recently disturbed forests requiring wide spore dispersal. 

Line 300. Native host range has emerged as a possible factor as well, see Karst J, Burns C, Cale JA, Antunes PM, Woods M, Lamit LJ, Hoeksema JD, Zabinski C, Gehring CA, La Flèche M, Rúa MA. 2018. Tree species with limited geographical ranges show extreme responses to ectomycorrhizas. Global Ecology and Biogeography 27: 839-848.

Line 302. Confusing, which pine plantations are being referred to here?

Line 308-314. Which makes perfect sense for a young pine plantation, and may not apply at all to a mature pine forest

Line 356. ?! fertilizers have an enormous effect on EMF, at least in the short-term.  Please add any details on stand management relevant to this in the Site descriptions – line 95.

Author Response

A well written and well executed study of exotic pines and their ectomycorrhizal fungi.  My only concern would be that the results and conclusions are based on quite young pine stands (27 years old) which undoubtedly do not yet have the full EMF diversity of mature forests.  So differences among native and exotic tree species may become more evident further into the rotation age.  Is there any literature from Asia that might have documented the typical alpha or gamma diversity of EMF in mature Pinus forests for comparison? (e.g. Kranabetter et al. 2018. Diversity and Distributions 24: 755-764)

Agreed, and thanks so much for the great suggestion. Due to the history of heavy anthropogenic disturbance in subtropical China in 1970s, most of recently established forests are either secondary regeneration or monoculture plantation (20~50 years). However, we indeed noticed that some other studies have investigated similar number of OTUs in young-aged masson pine forests (43 OTUs in Huang et al. 2014; and 47 OTUs in Huang et al. 2012), and also dominated by most of ECM generalists (e.g. Russula and Thelephoraceae). In a recent study by Chen et al., 2018, they found thousands of OTUs by Illumina sequencing in a ~50yr masson pine–broadleaf mixed forest. However, our data is not comparable with their results, since: a) they did not list a detail of ECM OTUs; b) they directly sampled the soil material including root hyphae, soil hyphae, dormant propagules or even fungal necromass, which was different from our in-growth bag sampling methods for active hyphae; c) OTU inflation may happen since they only applied 97% similarity thresholds for de novoclustering, which could overestimate the species richness (see Nguyen et al. 2015).

Chen L, Xiang W, Wu H, Ouyang S, Zhou B, Zeng Y, Chen Y, Kuzyakov Y. 2018. Tree species identity surpasses richness in affecting soil microbial richness and community composition in subtropical forests. Soil Biology and Biochemistry 130: 113-121.

Huang J, Nara K, Lian C, Zong K, Peng K, Xue S, Shen Z. 2012. Ectomycorrhizal fungal communityes associated with masson pine (Pinus massonianaLamb.) in Pb-Zn mine sites of central south China. Mycorrhiza 22:589-602.

Huang J, Nara K, Zong K, Wang J, Xue S, Peng K, Lian C. 2014. Ectomycorrhizal fungal community associated with masson pine (Pinus maasoniana) and white oak (Quercus fabri) in a manganese mining region in Hunan Province, China. Fungal Ecology 9: 1-10.

Nguyen NH, Smith D, Peay K, Kennedy P. 2015. Parsing ecological signal from noise in next generation amplicon sequencing. New Phytologist 205:1389-1393.

Perhaps this topic is under consideration for future studies (line 381-386) but there is considerable interest in how host genetics affect ectomycorrhizal communities and overall fitness.  Some literature (see below) suggests quite fine-tuned local adaptation (e.g, Gehring et al. 2017, Kranabetter et al. 2015) while others suggest the opposite (Gundale et al. 2014) so the science is not settled. Given the added pressures of ongoing climate change, the ability of exotic trees to thrive with native EMF will be crucial. 

Gehring, C.A., Sthultz, C.M., Fores-Rentería, L., Whipple, A.V., and Thitham, T.G. 2017. Tree genetics defines fungal partner communities that may confer drought tolerance. PNAS114(42):11169-11174.

Gundale MJ, Kardol P, Nilsson M-C, Nilsson U, Lucas RW, Wardle DA. 2014. Interactions with soil biota shift from negative to positive when a tree species is moved outside its native range. New Phytologist 202: 415–421.

Kranabetter, J.M., Stoehr, M., and O’Neill, G.A. 2015. Ectomycorrhizal fungal maladaptation and growth reductions associated with assisted migration of Douglas-fir. New Phytol. 206(3):1135-1144.

Pickles, B.J., Twieg, B.D., O’Neill, G.A., Mohn, W.W., and Simard, S.W. 2015. Local adaptation in migrated interior Douglas-fir seedlings is mediated by ectomycorrhizas and other soil factors. New Phytol. 207(3):858-871.

Rúa MA, Lamit LJ, Gehring C, Antunes PM, Hoeksema JD, Zabinski C, Karst J, Burns C, Woods MJ. 2018. Accounting for local adaptation in ectomycorrhizas: a call to track geographical origin of plants, fungi, and soils in experiments. Mycorrhiza 28: 187–195

Again, we really appreciate the great suggestions!

Some further comments:

Line 118. One time extractions of NH4 and NO3 are generally not credible as indices of N availability (see Binkley and Hart 1989. The components of nitrogen availability assessements in forest soils. Adv. Soil Sci. 10: 57-112).  C:N ratios would be more defendable as a general index of N supply.

Agreed, and thanks for the suggestion. In our study, soil samples were collected at the same time as the hyphal bags as an indicator of the soil conditions experienced by the ECM community at that time. This approach has been widely used in previous studies of ECM communities (see Cox et al. 2010, Corrales et al. 2017).

Line 122. The way this is written it seems the sporocarp survey was part of the community analysis?  The only reference i see to it again is in the DNA extraction for a ‘mock community’ (line 138).

We did not include these taxa in the community analysis. Unfortunately, some of the edible species (e,g. Lactarius and Russula) are occasionally collected by local farmers. Therefore, the abundance data for sporocarps cannot reflect the real situation. Instead, we have adjusted the text to more clearly articulate the use of this collection (lines 141- 146).

Line 214. See comment above, a total of only 43 EMF species suggests an early seral EMF community, many of which may be host generalists because that ensures better survival in recently disturbed forests requiring wide spore dispersal. 

We agree that many of the ECM taxa are generalists, and commented to this effect in the original manuscript (see lines 231-233, 310, 314-316) 

Line 300. Native host range has emerged as a possible factor as well, see Karst J, Burns C, Cale JA, Antunes PM, Woods M, Lamit LJ, Hoeksema JD, Zabinski C, Gehring CA, La Flèche M, Rúa MA. 2018. Tree species with limited geographical ranges show extreme responses to ectomycorrhizas. Global Ecology and Biogeography 27: 839-848.

Agreed, and thanks for the suggestion. We have noted this possibility on line 321 of the revised manuscript, and added the reference to the citations.

Line 302. Confusing, which pine plantations are being referred to here?

This should be ‘exotic pine plantations’, and has been updated (line 323-325 in revised manuscript). 

Line 308-314. Which makes perfect sense for a young pine plantation, and may not apply at all to a mature pine forest

Thanks for the suggestion. We agree that a future investigation is needed to address this possibility.

Line 356. ?! fertilizers have an enormous effect on EMF, at least in the short-term.  Please add any details on stand management relevant to this in the Site descriptions – line 95.

To the best of our knowledge, fertilization was not applied to the plantations.  In addition, we did not apply any fertilizer in both study sites in the last years. Top reduce the confusion, we deleted this sentence and changed the citation #77.

Reviewer 2 Report

The article “Host Phylogenetic Relatedness and Soil Nutrients Shape Ectomycorrhizal Community Composition in  Native and Exotic Pine Plantations” is  based on well-designed study.

My only remark is to the fig. 2 and fig. 4. To the fig. 2,  I would suggest to add as a legend the names of fungal genera, to my mind it would improve readability.a

As for fig. 4, I do not think the size of point which reflects “the richness of each sample” is the best issue of depiction, perhaps different texture of the points  would be a better issue.

Author Response

The article “Host Phylogenetic Relatedness and Soil Nutrients Shape Ectomycorrhizal Community Composition in Native and Exotic Pine Plantations” is  based on well-designed study. 

My only remark is to the fig. 2 and fig. 4. To the fig. 2,  I would suggest to add as a legend the names of fungal genera, to my mind it would improve readability.a 

We have added color legend for each ECM genus. Please see the updated version.

As for fig. 4, I do not think the size of point which reflects “the richness of each sample” is the best issue of depiction, perhaps different texture of the points would be a better issue. 

We have kept the original version of Figure 4 because richness is a continuous parameter for each sample. 

Reviewer 3 Report

This is a well designed study and a well written article that was a pleasure to read.  It shows the type of interesting relationships that can be learned about ectomycorrhizal fungi and their hosts through the use of high through-put sequencing and bioinformatics analysis.  The introduction provided a good background for understanding the problem.  The Material and Methods were clear and highly detailed.  The data was well described and illustrated in the Results section.  There were no redundancies among the figures and tables - all need to be included in the paper.  The conclusion related the results to those from other studies. 

Author Response

This is a well designed study and a well written article that was a pleasure to read.  It shows the type of interesting relationships that can be learned about ectomycorrhizal fungi and their hosts through the use of high through-put sequencing and bioinformatics analysis.  The introduction provided a good background for understanding the problem.  The Material and Methods were clear and highly detailed.  The data was well described and illustrated in the Results section.  There were no redundancies among the figures and tables - all need to be included in the paper.  The conclusion related the results to those from other studies. 

Thank you!