Saltmarsh Rhizosphere Fungal Communities Vary by Sediment Type and Dominant Plant Species Cover in Nova Scotia, Canada

Environmental Microbiology Reports (2021) 13(4), 458–463 doi:10.1111/1758-2229.12904

Brief Report

Saltmarsh rhizosphere fungal communities vary by sediment type and dominant plant species cover in Nova Scotia, Canada

Tyler W. d’Entremont, 1 Zoë Migicovsky,2 sediment carbon sequestration (Boesch and Juan C. López-Gutiérrez1 and Allison K. Walker 1* Turner, 1984). The term ‘blue carbon’ refers to carbon 1Department of Biology, Acadia University, Wolfville, stored in saltmarsh sinks and is greater, per unit area, Nova Scotia, Canada. than the carbon sinks of terrestrial counterparts (McLeod 2Department of Plant, Food, and Environmental et al., 2011). Despite large carbon stores, saltmarshes Sciences, Faculty of Agriculture, Dalhousie University, are declining globally, with minimal efforts targeting their Truro, Nova Scotia, Canada. conservation in Atlantic Canada (McLeod et al., 2011). Saltmarsh ecosystems in Atlantic Canada are dominated by two Sporobolus species in a zonal distribution. Summary Sporobolus alterniflorus (Loisel.) (Poaceae), formerly We surveyed Spartina saltmarsh sediment rhizo- Spartina alterniflora (smooth cordgrass), grows at the sphere fungal communities at three saltmarshes and tidal interface, and Sporobolus pumilus (Roth) two timepoints in coastal Nova Scotia. Based on ITS2 (Poaceae), formerly Spartina patens (saltmeadow cord- Illumina miSeq rDNA data and multivariate analysis, grass), dominates the high marsh (Bertness, 1991). neither sediment zone nor collection period corre- These grasses are adapted to growth in hypersaline sedi- lated with fungal ASV richness, but collection site ment and periodic tidal inundation. They excrete large did. However, Shannon diversity indicated that sedi- amounts of salt to regulate homeostasis, but it remains ment zone played a significant role in fungal diver- unknown how the stressors affect rhizosphere microbial sity. For unweighted and weighted UniFrac distance, communities in saltmarsh sediments. Abiotic factors such site was the major factor driving beta-diversity, with as pH, water content and nutrient loads all influence the sediment zone and collection period having smaller fungal community present at a site (Kendrick, 2017), roles. Sediment type and saltmarsh plant species which may affect coastal plant restoration success. may play important roles in structuring rhizosphere Assessing the sediment fungal diversity in different fungal assemblages, here dominated by ascomy- saltmarshes that are resistant to tidal erosion, such as cetes. To our knowledge, our study is the first to those in megatidal environments, may indicate which assess fungal sediment communities in saltmarshes species are key to a healthy ecosystem. in Atlantic Canada using metabarcoding. It provides To our knowledge, no eDNA studies of fungal assem- a biodiversity analysis of sediment fungi in a poorly blages in saltmarsh sediments have been done in Atlan- studied but highly important ecosystem and points to tic Canada. Saltmarsh sediment fungal communities their roles in nutrient cycling, blue carbon, coastal have been studied in southeastern Louisiana, USA, stability and coastal restoration. Our work will inform focusing on rhizosphere community shifts after the 2010 ongoing saltmarsh restoration in Atlantic Canada. Deepwater Horizon oil spill (Lumibao et al., 2018). Fungi associated with North American saltmarsh Sporobolus Introduction include arbuscular mycorrhizal symbiont (AMF) Funneliformis geosporum (Wilde et al., 2009; d’En- Saltmarshes are critical to sustainability of intertidal eco- tremont et al., 2018), the primary shoot decomposers systems; due to their highly productive vegetation and such as Phaeosphaeria spartinicola (Buchan et al., 2002; Walker and Campbell, 2010), and Fusarium pathogens of Sporobolus, F. palustre and F. incarnatum- Received 1 May, 2020; accepted 9 November, 2020. *For corre- spondence. E-mail [email protected]; Tel. 1-902-585-1333; equiseti (Elmer and Marra, 2011). Interestingly, a culture- Fax 1-902-585-1059 based study from New Brunswick, Canada saltmarsh

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd Saltmarsh rhizosphere fungal communities 459 sediment using buried wooden baits recovered a different Our study provides the first analysis of sediment fungal fungal community, dominated by ascomycete fungi eDNA data from Atlantic Canada saltmarshes. Known (Mansfield and Bärlocher, 1993). Our study addresses a terrestrial, amphibious and marine fungi were detected in knowledge gap and will inform saltmarsh restoration sediments at all saltmarsh sites. This may result from fungi practices throughout Atlantic Canada to combat current in root tissues or spore deposition from wind, freshwater habitat degradation and loss due to anthropogenic inputs or ocean currents. Some fungi detected are unlikely destruction and natural stressors. to be metabolically active in these ecosystems, although With this ITS2 rDNA metabarcoding study we: we did not test for this. Ascomycete fungi were the most (i) compared the rhizosphere sediment fungal diversity at common fungi found, which correlates with data from two time points within three different saltmarshes border- saltmarshes of Rhode Island, USA (Mohamed and ing the megatidal Minas Basin, Nova Scotia to test the Martiny, 2011) and Louisiana, USA (Lumibao et al., 2018). fi hypothesis that fungal communities are site dependent Unclassi ed fungi were also detected, having no DNA and (ii) determined whether saltmarsh plant zonation sequence matches in the publicly available UNITE data- (sediment zone) influences total sediment fungal base (version 7.0)(Kõljalg et al., 2005); tidal wetland sedi- diversity. ment is a source of novel fungal diversity. It is worth noting that ITS metabarcoding can miss some fungal classifica- tions due to missing reference sequences in public data- Results and discussion bases (Heeger et al., 2019). Fungi identified in this study such as Lulworthia Saltmarsh rhizosphere fungal community composition sp. Phaeosphaeria halima, Phaeosphaeria spartinicola, 3Taxonomy was assigned using the UNITE version 7.0 Scheffersomyces spartinae,andFunneliformis geosporum database (Kõljalg et al., 2005) and relative abundance are known from other saltmarshes in the USA and Canada was assessed using QIIME2 (Bolyen et al., 2019) for (Filip and Alberts, 1993; Walker and Campbell, 2010; 65 saltmarsh sediment samples. Saltmarsh rhizosphere Kurtzman et al., 2011; d’Entremont et al., 2018). fungal assemblages, as revealed by ITS2 metabarcoding, Phaeosphaeria halima and P. spartinicola have both been showed differences by site and sediment zone, as well as isolated from the leaves of S. alterniflorus and play much unclassified diversity (Fig. 1, Appendix S1). Number important roles in the decomposition of Sporobolus litter of reads per sample are given in Appendix S2, and rare- and nutrient cycling in saltmarsh habitats (Filip and faction curves are provided in Appendix S3. Of the fungi Alberts, 1993; Buchan et al., 2002; Walker and able to be classified, ascomycetes dominated the sedi- Campbell, 2010). Scheffersomyces spartinae is only ment fungal communities at all sites and sediment zones, known from aquatic environments and it is unknown including obligate marine genus Lulworthia. Some fungal whether a true interaction between Sporobolus species plant pathogens, such as Gaeumannomyces graminis, and S. spartinae exists. Occurrence may be from suspen- were more abundant at some sites and sediment zones sion in the water column (Kurtzman et al., 2011). than others. One salt-tolerant arbuscular mycorrhizal fun- Funneliformis geosporum forms a mutualistic relationship gus (AMF), Funneliformis geosporum, was common to all with both Sporobolus alterniflorus and Sporobolus pumilus sites. Three other AMF species (a Claroideoglomus sp., in Nova Scotia saltmarshes (d’Entremont et al., 2018). Archaeospora trappei,andRhizophagus irregularis)were Funneliformis geosporum is an arbuscular mycorrhizal fun- also detected. It is important to note that eDNA-based gus that colonizes Sporobolus roots, although the strength assessments of sediment fungal diversity can include of the interaction is different for S. pumilus and detections of dormant or nonliving fungi. Sequences S. alterniflorus; the former is more extensively colonized obtained may be from fungal hyphae, spores, or other fun- than the latter. gal reproductive structures; future detection of fungal RNA from coastal sediments (metatranscriptomics) is needed to Species richness among Minas Basin saltmarsh sites determine which fungi are metabolically active in these and sediment zones habitats (Amend et al., 2019). Diversity of saltmarsh sediment fungal communities in We estimated alpha diversity using observed ASV rich- megatidal environments such as the Bay of Fundy, ness for samples taken from three locations, two Canada, have been poorly studied compared with terres- collection periods, and two sediment zones (Fig. 2, trial soil fungal communities. Both provide critical ecosys- Appendix S1). Considering all three variables, the total tem services such as decomposition, nutrient cycling and number of samples was insufficient to test all possible symbioses forming the basis of most food webs and are interactions and therefore we only performed pairwise essential for nutrient turnover and ecosystem sustainabil- comparisons for location, collection period, and sediment ity (Dighton, 2016). zone independently. Using a Kruskal–Wallis test, we

Fig. 1. Average relative abundance of fungal taxa collected at each saltmarsh zone, at each site, as determined by ITS2 metabarcoding. Each label represents the lowest possible classiﬁcation for each read.

Fig. 2. Fungal alpha diversity, as determined using ITS2 metabarcoding, at saltmarshes in Kingsport, Windsor and Wolfville, Nova Scotia show- ing the difference in observed ASV richness between sites and at different sediment zones, separated by dominant vegetation type, Sporobolus alterniﬂorus or Sporobolus pumilus.

© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology Reports, 13, 458–463 Saltmarsh rhizosphere fungal communities 461 found that ASV richness did not differ significantly by col- resulting interactions for both unweighted and weighted lection period (P = 0.22) or sediment zone (P = 0.31). UniFrac distance matrices using an Adonis test (Table 1). However, ASV richness in Minas Basin saltmarsh sedi- For both unweighted and weighted UniFrac, ‘site’ ment was site specific(P = 0.03). Pairwise comparisons explained the variation. The unweighted UniFrac PCoA between sites indicated that richness at the Windsor site plot had strong separation along PC1 between Kingsport was significantly higher than the Kingsport site and the other sites, with weaker separation between sites (Benjamini and Hochberg adjusted P = 0.04), with neither for the weighted UniFrac, which is supported by a smaller site differing significantly from the Wolfville site. Kingsport R2. Sediment zone and collection period also explained a had the lowest species diversity in both sediment zones significant amount of variation in beta diversity, as compared with other sites, which may indicate that assessed using unweighted and weighted UniFrac dis- coarse, sandy sediment is less favourable for intertidal tance matrices. This supports previous findings in a Rhode fungi than the clay-rich sediments of Wolfville and Island saltmarsh study that plants may create different Windsor saltmarshes. niches for sediment fungi (Mohamed and Martiny, 2011). Using Shannon diversity, we assessed alpha diversity Principal coordinate analysis indicated that sediment for the same metrics as above. Using a Kruskal–Wallis fungal communities at all sites differed, but Kingsport test, we found Shannon diversity did not differ for collec- was most dissimilar compared with both Wolfville and tion period (P = 0.75) or site (P = 0.39). Shannon diver- Windsor. Sediment characteristics at saltmarsh sites may sity of saltmarsh zones differed between S. alterniflora structure the composition of rhizosphere fungal and S. pumilus (P = 0.02). For Wolfville and Windsor, the communities. S. alterniflorus sediment zone had higher sediment fun- Unweighted UniFrac distance assessed community dif- gal species richness, which may be due to the frequency ferences using presence or absence, without accounting of tidal inundation. Kingsport had the opposite trend with for abundance. As an exploratory study, we were primar- the S. pumilus sediment having higher species richness. ily interested in whether rhizosphere fungal species com- Differences in sediment porosity and salinity may contrib- position was similar at our three saltmarsh study sites, ute to this phenomenon but are untested (Mohamed and not fungal abundance, although we also ran weighted Martiny, 2011). UniFrac distance that showed similar trends. We also wanted to identify symbiotic fungi for potential use in restoration practices in Atlantic Canada. By determining common saltmarsh sediment fungi in Nova Scotia, our Comparison of species composition in saltmarshes of study provides a preliminary understanding of which spe- the Minas Basin cies may be essential for cold water saltmarsh ecosys- We performed a principal coordinate analysis (PCoA) tem function. using unweighted UniFrac distance, which measures read presence or absence, and weighted UniFrac dis- Conclusions tance, which includes relative abundance of reads with the aforementioned (Fig. 3). We estimated the amount of We provide the first record of saltmarsh sediment fungal variance (R2) explained by each variable and the diversity from Atlantic Canada based on eDNA data and

Fig. 3. Principal coordinate analysis of saltmarsh sediment ITS2 fungal communities separated by collection site (Kingsport, Windsor and Wolf- ville, Nova Scotia) and sediment collection zone [Sporobolus alterniﬂorus—dominated (low marsh) or Sporobolus pumilus—dominated (high marsh)].

Table 1. Variation (R2) explained by each variable and interaction for Walker AK, Yarden O, Gladfelter AS. (2019) Fungi in the unweighted and weighted UniFrac beta diversity distance matrices. marine environment: open questions and unsolved prob- lems. MBio 10: e01189–18. https://doi.org/10.1128/mBio. UniFrac UniFrac 01189-18. 2 2 Variable unweighted (R ) weighted (R ) Bertness, M.D. (1991) Zonation of Spartina patens and Site 0.14897*** 0.11500*** Spartina alterniflora in New England salt marsh. Ecology Sediment zone 0.04542*** 0.06846*** 72: 138–148. Collection period 0.02784** 0.02626* Boesch, D.F., and Turner, E.R. (1984) Dependence of fish- Site: sediment zone 0.03712** 0.07642*** ery species on salt marshes: the role of food and refuge. Site: collection period 0.03471* 0.02662 Estuaries 7: 460–468. Sediment zone: collection 0.01655 0.02162 period Bolyen, E., Rideout, J.R., Dillon, M.R., Bokulich, N.A., Site: sediment zone: 0.02711 0.03573 Abnet, C.C., al-Ghalith, G.A., et al. (2019) Reproducible, collection period interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37: 852–857. *P < 0.05; **P < 0.01; ***P < 0.001. Buchan, A., Newell, S.Y., Moreta, J.I.L., and Moran, M.A. (2002) Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern U.S. salt marsh. Microb Ecol 43: 329–340. demonstrate fungal community differences based on col- d’Entremont, T.W., Walker, A.K., and López-Gutiérrez, J.C. lection location and vegetation. Obligate marine as well (2018) Examining arbuscular mycorrhizal fungi in as terrestrial fungi were identified in this study, providing saltmarsh hay (Spartina patens) and smooth cordgrass an avenue for future study of ‘amphibious fungi’, to deter- (Spartina alterniflora) in the Minas Basin, Nova Scotia. mine which species may be metabolically active in these Northeastern Naturalist 25:72–86. productive and understudied megatidal environments. Dighton, J. (2016) Fungi in Ecosystem Processes. Boca Raton: CRC Press. Our data reveal a diversity of marine and terrestrial fungi Elmer, W.H., and Marra, R.E. (2011) New species of Fusar- present in rhizosphere sediment of valuable northern ium associated with dieback of Spartina alterniflora in coastal saltmarsh ecosystems. Fungal community com- Atlantic salt marshes. Mycologia 103: 806–819. position varied with sediment composition, plant zonation Filip, Z., and Alberts, J.J. (1993) Formation of humic-like and saltmarsh location. Some fungi such as the substances by fungi epiphytic on Spartina alterniflora. arbuscular mycorrhizal root symbiont of Sporobolus Estuaries 16: 385–390. (Spartina) plants, Funneliformis geosporum, may be Heeger, F., Wurzbacher, C., Bourne, E.C., Mazzoni, C.J., essential for saltmarsh ecosystem function and warrant and Monaghan, M.T. (2019) Combining the 5.8S and ITS2 to improve classification of fungi. Methods Ecol Evol 10: further investigation in the context of coastal restoration. 1702–1711. Our study supports coastal wetlands as sources of novel Mohamed, D.J., and Martiny, J.B. (2011) Patterns of fungal fungal biodiversity. diversity and composition along a salinity gradient. ISME J 5: 379–388. Kendrick, B. (2017) The Fifth Kingdom. Indianapolis: Hackett Acknowledgements Publishing Company. Kõljalg, U., Larsson, K.-H., Abarenkov, K., Nilsson, R.H., T.W. d’Entremont was supported by the Arthur Irving Acad- Alexander, I.J., Eberhardt, U., et al. (2005) UNITE: a data- emy for the Environment Master’s Student Scholarship in base providing web-based methods for the molecular Environmental Science. Z. Migicovsky was supported by identification of ectomycorrhizal fungi. New Phytol 166: National Science Foundation Plant Genome Research Pro- 1063–1068. gram 1546869. A. Walker gratefully acknowledges an Kurtzman, C.P., Fell, J.W., and Boekhout, T. (2011) The NSERC Discovery Grant (No. NSERC—2017-04325), and Yeasts, A Taxonomic Study. San Diego: Elsevier. an Arthur Irving Academy Research Grant to A. Walker, J. López-Gutiérrez and R. Browne. We thank G. M. Douglas Lumibao, C.Y., Formel, S., Elango, V., Pardue, J.H., (Dalhousie University), M. Mallory and R. Evans (Acadia Blum, M., and Van Bael, S.A. (2018) Persisting responses University), and J. Lundholm (Saint Mary’s University) and of salt marsh fungal communities to the deepwater horizon – anonymous reviewers for valuable comments on earlier ver- oil spill. Sci Total Environ 642: 904 913. fi sions of this manuscript, and the Dalhousie University Inte- Mans eld, S.D., and Bärlocher, F. (1993) Seasonal variation grated Microbiome Resource for Illumina sequencing. of fungal biomass in the sediment of a salt marsh in New Brunswick. Microb Ecol 26:37–45. Mcleod, E., Chmura, G.L., Bouillon, S., Salm, R., Björk, M., Duarte, C.M., et al. (2011) A blueprint for blue carbon: References toward an improved understanding of the role of vegetated

Amend, A., Burgaud, G., Cunliffe, M., Edgcomb, V.P., coastal habitats in sequestering CO2. Front Ecol Environ Ettinger, C.L., Gutiérrez, M.H., Heitman J, Hom EFY, Ianiri 9: 552–560. G, Jones AC, Kagami M, Picard KT, Quandt CA, Walker, A.K., and Campbell, J. (2010) Marine fungal Raghukumar S, Riquelme M, Stajich J, Vargas-Muõiz J, diversity: a comparison of natural and created salt

marshes of the north-central Gulf of Mexico. Mycologia Appendix S1 2018 saltmarsh sampling sites located at: A) 102: 513–521. Wolfville (4505042.99"N, 6421029.73"W); B) Windsor Wilde, P., Manal, A., Stodden, M., Sieverding, E., (45005.35"N, 64807.13"W); and C) Kingsport, Nova Scotia, Hildebrandt, U., and Bothe, H. (2009) Biodiversity of Canada (459032.42"N, 6421036.13"W). arbuscular mycorrhizal fungi in roots and soils of two salt Appendix S2 Amplicon sequence variant frequency for – marshes. Environ Microbiol 11: 1548 1561. each of the saltmarsh sediment samples used in our study. Appendix S3 Rarefaction curves of all saltmarsh sediment Supporting Information samples used in our study. Shannon index, Faith phyloge- Additional Supporting Information may be found in the online netic diversity and observed OTUs were used to determine version of this article at the publisher’s web-site: adequate sampling depth.