Sequence-based abundance, diversity, community

composition and activity of arbuscular mycorrhizal fungi

vary with phosphorus and altitude in Andean Solanum
tuberosum fields.

Jessica Duchicela1, Jose Luis Hernández1, Eduardo Furrazola2, Ricardo Herrera-

Peraza2†
1
Departamento de Ciencias de la Vida, Laboratorio de Botánica, Universidad de las Fuerzas
Armadas ESPE, Av. Gral. Rumiñahui s ⁄ n, P.O.BOX. 171101, Sangolquí, Ecuador
2
Instituto de Ecología y Sistemática, CITMA, La Habana 19, C.P. 11900. La Habana, Cuba
[email protected]

Abstract. Potato is a culturally and economically important crop worldwide,

with particular importance in Andean regions of South America. Solanum tu-
berosum (potato) has been shown to form a relationship with arbuscular mycor-
rhizal fungi. Nonetheless, current agricultural practices disregard this association
and historical practices, such as substantial nutrient addition, compromise the
fungi activity. Here, we study the impacts of edaphic factors and altitude on my-
corrhizal activity and sequence-based abundance, diversity and community com-
position in the Andean region of South America. We do this first through field-
work in the Ecuadorian Andes, and then through re-analysis of an Andean-wide
sequencing effort by Senés-Guerrero (2016). We find that phosphorus and alti-
tude were the dominant drivers of arbuscular mycorrhizal fungal activity (Ecua-
dorian Andes); soil phosphorus was the most important predictor of the sequence-
based metrics of diversity (Andean-wide sequence data). Our results bring novel
insights to the environmental factors structuring AMF communities in the Andes,
suggesting that managing phosphorus concentration may be an important avenue
to harness the plant-mycorrhizal symbiosis in potato agriculture.

Keywords: Arbuscular mycorrhizal fungi (AMF), diversity, Solanum tu-

berosum, phosphorus.

1 Introduction

Arbuscular mycorrhizal fungi (AMF) are the most widespread symbionts of plant roots
in terrestrial habitats [1]. These ancient, obligate root symbionts contribute to the
2

transport of nutrients and water to their host plants (Augé 2001, Smith & Read, 2008),
pathogen resistance (Azcon-Aguilar & Barea, 1996; Jung et al., 2012), and the for-
mation and stability of soil aggregates (Duchicela, 2013). As the need of sustainable
agricultural practices increases, AMF are key to improving crop productivity and eco-
system function as well as other non-nutritional benefits (Delavaux et al., 2017).

Studies have shown that AMF abundance, diversity and functioning are af-
fected by differences in management practices in agroecosystems. High nitrogen and
phosphorus inputs are related to a decrease in host plant resource allocation to AMF
and therefore have a negative impact on AMF biodiversity (Verbruggen et al., 2012).
In contrast, plant intercropping systems can have a positive impact on AMF; for exam-
ple, Bainard et al., (2011) found that these systems support a more abundant and diverse
AMF community. Therefore, it is relevant to understand how agricultural management
practices influence AMF, especially when associated with crops of global importance,
such as potato.

Potato (Solanum tuberosum) is a widespread crop of global importance (Ortiz

& Mares, 2017). It is the fourth most consumed crop in the world, worth more than a
$5.5 billion industry (FAO, 2008; FAO 2012). It was domesticated between 8,000 to
10,000 years ago from a wild species native to the Andes. The Andean region is an arid
region between 3,000 to 4,500 m above sea level characterized by cold temperatures,
saline soils, and high solar radiation (Spooner et al., 2005). Potato cultivation usually
involves high inputs of fertilizers and pesticides (Wu et al., 2013). Previous studies
have reported greater AMF root colonization levels under organic than conventional
farming, by indigenous AMF (Loit. et al., 2018). The knowledge of AM fungi colo-
nizing potato roots in field conditions under common agricultural practices is still
scarce, in particular in the Andean region where AMF is expected to improve the plant
resistance to the extreme environmental conditions. Therefore, it is pertinent to under-
stand how agricultural management and environmental conditions in the Andes affects
AMF function, diversity and community composition.

Considering the importance of this crop in the region, the aim of this study is
to gain an understanding of the impact of different agricultural management on envi-
ronmental conditions and ultimately, effects on AMF activity and sequence-based
abundance, diversity and community composition in the Andean region. First, we ex-
amine the impact of agricultural management and conditions on AMF activity in select
potato Ecuadorian Andean plots (Section 1: Mycorrhizal Activity in the Ecuadorian
Andes). Next, to broaden the scope of our research, we use published sequence data to
examine the relationship of AMF abundance, diversity and community composition
with edaphic factors and altitude across the Andean region, including Bolivia, Ecuador,
and Perú (Section 2: Mycorrhizal Diversity in the Andes). Collectively, our field and
molecular analysis results will facilitate the understanding of mycorrhizal ecology in
potato fields and inform better approaches for employing this symbiosis to increase the
sustainability of agroecosystems.
 3

2 Methods

2.1 Mycorrhizal Activity in the Ecuadorian Andes

Study sites. The study was conducted in 15 fields across the provinces of Cañar, Coto-
paxi, and Carchi, in the Andean region of Ecuador. Site selection was based on a range
of different regimes of phosphorus concentration (Table S.1). Soil samples of 500 cc
each were taken from each plot (pooled replicates) from each fertilization management
system described above. Five sub samples for each treatment were taken using a stand-
ard soil probe (2 cm x 30 cm) from each 5 distinct sampling plot within each of replicate
field, for a total of 15 samples. The original samples were pooled across treatment rep-
licates. Collected soil samples were air-dried for the characterization of arbuscular my-
corrhizae (described in 2.1.2 below) and for analysis of soil pH (1:2.5, w:v), organic
matter (OM, %), total nitrogen (N, %), available phosphorus P (mg kg-1), changeable
elements (Ca, Mg, K extractable with 1 mol/L ammonium acetate, expressed in mg kg-
1), and other elements (S, Fe, Zn, Cu, Mn and B (in ppm)). All soil analyses were
carried out at the National Institute of Agriculture and Livestock Production Research
(Instituto Nacional de Investigaciones Agropecuarias; INIAP), Experimental Extension
Sta. Catalina, Quito, Ecuador, following standard laboratory procedures.

AM fungus activity and diversity. AM fungus activity was quantified for external my-
celium biomass, internal mycelium biomass, percent mycorrhizal colonization and
rootlets dry weight according to Herrera et al. (2004); in addition, number of spores and
number of morphological species were determined. AM fungal spores were extracted
using the sucrose gradient centrifugation method described in Gerdemann and Nicolson
(1963) as described by Herrera et al. (2004). Spores were then visually quantified as
number of spores and number of morphological species identified to genus based on
AMF spore phenotypes described by Schüßler & Walker (2010) and others (Morton &
Msiska, 2010; Redecker et al., 2013).

2.2 Mycorrhizal sequence-based metrics in the Andean potato fields.

To further explore the impacts of nutrient concentrations on Solanum tuberosum (po-
tato) in the Andean region of South America in general, we used data presented in
Senés-Guerrero 6 (2016). This study targeted Solanum tuberosum root-associated AMF
though 454 sequencing in fields across Peru, Ecuador, and Bolivia. Representative se-
quences of the operational taxonomic units (OTUs) are deposited at the Sequence Read
Archive at the NCBI with accession number PRJNA242351. All bioinformatical
analysis is described in Senés-Guerrero (2016). We explored this data to understand
what edaphic properties and altitude control arbuscular mycorrhizal AMF abundance
(richness) and diversity in this important Andean crop. Shannon (Shannon, 1948) and
Simpson diversity (Simpson, 1949) were calculated using the vegan package in R
(Oksanen et al., 2013). Nucleotide diversity (Nei & Wen-Hsiung, 1979) was calculated
using the DnaSP v6 program (Librado & Rosas, 2009). We used the pez package
(Pearse et al., 2015) in R to determine phylogenetic species richness (PSR) values
4

(Helmus et al., 2007). This metric incorporates phylogenetic distance using a phyloge-
netic tree. In our phylogenetic tree construction, we included the Kruger LSU (large
subunit) database (Krüger et 124 al., 2012), as well as two outgroups, including one
ascomycete and one basidiomycete (Exophiala spinifera, Rhodotorula hordea), in addi-
tion to the target sequences (Senés-Guerrero and Schüßler, 2016), to more robustly pre-
dict phylogenetic relationships. We then used the resulting tree to predict PSR. Here,
we base branch length, used to calculate PSR, on substitution per base after running up
to 1000 iterations using RAxML (Stamatakis, 2006) after MUSCLE (Edgar, 2004)
alignment in MEGA 7 (Kumar et al., 2016). All metrics were calculated using R version
3.4.0.

2.3 Statistical analysis.

For both the Ecuadorian field data (Mycorrhizal activity in the Ecuadorian Andes) and
the Andean sequencing data reanalyzed from Senés-Guerrero (Mycorrhizal sequence-
based 7 metrics in the Andes), we constructed (generalized) linear regression models
to assess the impacts of edaphic variables and altitude on (1) mycorrhizal activity,
including external mycelium biomass, internal mycelium biomass, mycorrhizal
colonization percentage, rootlets dry weight (RDW), number of spores and number of
morphological species, and (2) richness (OTU, Genera and Species, and phylogenetic
species richness (PSR)) and diversity (Shannon and Simpson diversity indices, and nu-
cleotide diversity (Pi)). The relationship between the soil variables and the richness and
diversity of AMF was assessed using generalized linear models using the GLM
function in the built-in stats package in R. For Senés-Guerrero, we additionally con-
structed a Permutational multivariate analysis of variance (PERMANOVA) to test
effects of the soil and environmental factors on the AM fungal community composition
using the adonis2 function in the vegan package in R (Oksanen et al., 2013).

Mycorrhizal activity in the Ecuadorian Andes potato fields

To investigate the impacts of soil nutrient enrichments and other environmental varia-
bles in our field data we constructed simple and multiple linear regression models. After
transforming and scaling of variables (see Table S.2), we performed variable selection
using PCA (principal 151 components analysis) (see Figure S.1) and correlation be-
tween independent variables to avoid multicollinearity between predictor variables
within the multivariate model. The response variables were those described in the
Methods section. We ran separate multiple linear regression models for each response
variable and the four uncorrelated independent nutrient variables. We then ran simple
linear regression models for each response variable and each environmental variable to
understand the individual impacts of variables.

Mycorrhizal sequence-based metrics in the Andes

We used generalized linear models (GLMs) to test whether environmental variables

explained differences in AMF OTUs, genera and species. For determination of genera
 5

and species, please see Senés-Guerrero (2016). After transforming and scaling of vari-
ables (see Table 162 S.3. in the Supplementary material), we performed variable selec-
tion to avoid multicollinearity between predictor variables within the multivariate
model. Because there was a significant correlation between all variables of interest (al-
titude, nitrogen, phosphorus, pH and mean temperature), no multivariate model could
be assessed. Therefore, we ran separate simple linear regressions for each predictor
with each response variable, using sequence number as a covariate in all models and
assuming Gaussian distribution of the response variables. The response variables were
Shannon and Simpson diversity and richness (OTU, genera, and species), OTU nucle-
otide diversity and OTU phylogenetic species richness (PSR). We included a random
effect to properly account for structured variation present in this study (1|county:prov-
ince:cultivar). All previously described statistical analysis were carried out in R version
3.4.0 (Team, 2017). In order to investigate the environmental variables that predict
community composition, we ran separate PERMANOVAs with the OTU table, the spe-
cies table, and the genera table. Before using the tables, we included only samples
where a minimum of 5 OTUs, species or genera were found; we also only included
samples where a minimum of 1000 sequences were recovered, using rrarify from the R
package vegan (Oksanen et al., 2013). We then constructed PERMANOVAs with 999
permutations using adonis2 in vegan. We included phosphorus, nitrogen, pH, and alti-
tude as environmental predictors. In addition, to account for the structured variation
present in this study, we included a blocking variable using the strata option
(1|county:province:cultivar:sample).

3 Results

3.1. Mycorrhizal Activity in the Ecuadorian Andes. In our analysis of Ecuadorian

mycorrhizal activity data, we found that phosphorus and altitude are the most important
predictors. In our multivariate model, we found that phosphorus significantly predicts
the number of spores. With greater soil phosphorus, there was a significantly lower
number of spores (Fig 1.A, Table S.4., p = 0.01). In our simple linear model, we found
that altitude was also an important predictor of several variables (Fig 1 D - F, Table
S.5), including percent mycorrhizal colonization, number of spores and rootlets dry
weight. An increase in altitude was correlated with a higher number of spores (p =
0.035) and percent of mycorrhizal colonization (p = 0.021), while it was correlated with
a lower rootlets dry weight (p = 0.008). All other results are presented in Table S 4-5.
6

Fig. 1. Regression plots for Mycorrhizal Functioning: A) Number of spores response to phos-
phorus concentration; B) Species Simpson diversity response to phosphorus concentration; C)
Genera Simpson diversity response to phosphorus concentration; D) Number of spores re-
sponse to Altitude; E) Mycorrhizal colonization response to Altitude & F) Rootlets dry weight
response to Altitude.

3.2. Mycorrhizal Diversity in the Andes. In our analysis of Andean-wide sequences

from Senés-Guerrero (2016), soil phosphorus concentration was the most important
predictor. Specifically, we found that phosphorus was a significant predictor of Simp-
son diversity from species and genera data. In our simple generalized linear models, we
found that an increase in phosphorus was correlated with a decrease in Simpson diver-
sity (Table S.7; Species p = 0.032; Genera p = 0.003). All results are presented in Table
S 6-8.

When examining community composition, the number of significant predictors de-

creased from OTUs to species to genera. Our results showed (PERMANOVA) that for
OTU data, all variables tested were significant predictors for community composition.
For species data, only phosphorus and altitude remained significant (Table S.8; P p =
0.042; altitude p = 0.032) in predicting community composition. Finally, for genera
data, no variables were significant. We found no significant results using Pi and PSR
as response variable in our simple generalized linear model.

4 Discussion

This study addressed how soil factors and altitude influence AMF activity and
sequence- based abundance, diversity and composition in Solanum tuberosum within
Andean Region agroecosystems. Specifically, we choose potato plots that represent a
gradient of phosphorus fertilization in the region. This allowed us to detect the influence
 7

of agriculture on soil mutualistic symbionts through changes in soil nutrient load. Over-
all, we found that phosphorus and altitude were important in determining AMF activity
in Ecuador field data and sequence- based abundance, diversity and composition in the
Andean region. An increase in phosphorus broadly led to lower AMF activity and di-
versity, while increasing altitude led to greater AMF abundance and sequence-based
abundance, diversity and composition.

Generally, an increase in phosphorus led to a decrease of number of AMF spores and

decrease of diversity (genera and species data Simpson diversity index). This agrees
with the general principle in mycorrhizal ecology that an ecosystem with greater phos-
phorus reduces plant reliance on AMF (Koide & Elliott, 1989). This is already generally
known, but here, we show it is important in terms of potato agriculture across the An-
dean region of South America.

Nonetheless, our results are supported by a study by Verbruggen et al. (2012). These
authors found in a corn potato intercropping system that management practices impact
AMF abundance and community composition through changes in phosphorus. Altitude
is also a good predictor of AMF activity. Increasing altitude leads to increased AMF
activity, including number of spores and percent mycorrhizal colonization. We also
found that rootlets dry weight is negatively correlated with altitude. This shows that
there is less investment in rootlets is decreasing with altitude, as plants may rely more
on AMF. This is congruent with our finding that at higher altitude, there is a greater
number of AMF spores and root colonization. Although altitude is an important predic-
tor here, it is important to acknowledge the drawbacks of altitude as a predictor. Alti-
tude encompasses many predictors (temperature, precipitation) and so does not mech-
anistically help us explain what is driving AMF abundance and diversity.

Although both phylogenetic species richness (PSR) and nucleotide diversity (Pi) have
recently been used to describe sequence-based richness, we find no significant relation-
ships between these metrics and edaphic or altitudinal factors. PSR is thought to be a
better measure of richness as opposed to simple OTU richness because it takes phylo-
genetic relationships in a community into account. Pi measures nucleotide diversity
(nucleotide variation). The lack of results we see using nucleotide diversity metrics may
be due to sample size. For nucleotide diversity, due to non-independence of samples,
we could only assess Pi at 12 levels of P and 12 levels of altitude. Our PERMANOVA
results that show a decreasing significance of variables with broader definition of tax-
onomic groups (from OTUs to species to genera) may also result from a decreasing n.
With broader classification, the n available to the model decrease, potentially decreas-
ing any impact variables may have on the community composition.

As farmers look to more sustainable, but also profitable ways to farm potatoes, arbus-
cular mycorrhizal fungi (AMF) should be considered. It has been argued that part of
the solution to more sustainable agriculture can be found belowground in the rhizo-
sphere (Gewin, 2010; Rillig et al. 2019) by manipulating soil microbes which improve
8

the root function and support important soil properties. AMF help in nutrient acquisi-
tion, reducing the need for fertilization, but also provide additional benefits to the crop
plant as well as soil conservation, promotion sustainability in the long-term economic
benefits to the farmer.

5 Conclusion

Here, we have shown that changes in soil nutrients lead to important change in both
activity and diversity of AMF of potato fields in the Andes within Ecuador, Bolivia,
and Peru. Overall, we show that, across the Andean region, phosphorus is the main
driver of both arbuscular mycorrhizal fungal activity and diversity in potato fields. In
order to reap the benefits of this ubiquitous symbiosis, we encourage farmers to con-
sider the impacts of increasing phosphorus addition on their crops. Namely, this addi-
tional phosphorus is likely to lead to the reduction of AMF abundance and diversity in
their fields, along with the reduction of benefits particularly in the Andean region. Fi-
nally, we hope these results support more research into sustainable agriculture that aims
to promote naturally occurring microbes to the crop and farmers’ benefit.

6 Acknowledgements

We thank the KU Center for Research Computing for their ongoing bioinformatics sup-
port throughout this project. We are grateful to Carolina Senés-Guerrero for sharing her
data with us. Thanks to Camille Delavaux, Andrew Mehring for statistical support and
Francisco Flores for bioinformatic analysis assistance.

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7 Supplementary Information

Fig. S.1. PCA for independent variables (Altitude, pH, Organic mater, N, P, K, Ca, Mg, S, Fe,
Zn, Cu, Mn & B )from Mycorrhizal activity in the Ecuadorian Andes data, independent varia-
bles where standardized using scale function from the base package i
 Table S.1. Agricultural plots studied at provinces of Cañar, Cotopaxi, and Carchi.

Code Province Alti- P (mg. External Internal my- Rootlets Percentage of Number Species num-
tude(masl kg-1) mycelium celium bio- dry weight mycorrhizal col- of spores ber
) biomass mass (RDW) onization (MC)
M1r1 Cañar – Sizho 3074 85 53.36 89.39 4.91 60.60 11668 15
M1r2 Cañar – Cooperativa San 3074 12 10
Rafael 19.12 169.27 10.45 63.54 13231
M1r3 Cañar – Cooperativa San 3320 14 12
Rafael 28.62 103.68 7.68 49.07 16666
M2r1 Cañar – Antenas de Bueran 3600 33 61.74 41.77 4.11 41.51 32606 18
M2r2 Cañar – Antenas de Bueran 3600 24 84.00 60.49 3.44 73.58 43089 17
M2r3 Cañar – Antenas de Bueran 3600 15 23.87 57.67 4.52 63.11 31134 15
M3r1 Cotopaxi – Pagtag 4570 71 64.02 15.96 1.07 82.82 18285 15
M3r2 Cotopaxi – Yacubamba 3470 63 42.71 33.37 2.82 75.42 22493 15
M3r3 Cotopaxi – Romerillos 3520 44 93.74 111.79 4.57 78.78 8598 14
M4r1 Carchi – San Luis 2750 84 34.52 47.66 6.56 50.00 7816 14
M4r2 Carchi – Loma del Centro 3270 30 13
9 67.62 60.85 8.90 47.09 6423
M4r3 Carchi – El Tambo 3010 66 106.70 57.48 11.68 44.52 16943 18
M5r1 Carchi – San Luis 2750 77 105.26 49.53 8.45 48.84 4790 9
M5r2 Carchi – Loma del Centro 3270 21 14
0 68.25 94.12 5.14 50.96 7191
M5r3 Carchi – San Pedro de 3000 22 8
Dacha 0 44.94 73.18 3.97 64.29 3218
 Table S.2. Variable transformation - Mycorrhizal activity in the Ecuadorian Andes. all the pre-
dicted variables where standardized using scale function from the base package in R.

Transfor- p-value from Shapiro

Variable
mation test

External mycelium biomass p-value = 0.5233

Endophyte mycomass p-value = 0.1775

Rootlets dry weight p-value = 0.5652

Percentage of mycorrhizal colonization p-value = 0.2084

Number of spores log10(Num- p-value = 0.9681

ber of spores)

Fungal spore phenotypes p-value = 0.2645

Table S.3. Variable transformation -Mycorrhizal Diversity in the Andes. lambda value was cal-
culated using Tukey’s ladder of powers function (transformTukey) from the rcompanion pack-
age and all the predicted variables where standardized using scale function from the base pack-
age in R

Variable Transformation p-value from Shapiro test

OTU richness log10(OTU richness) p-value = 0.4204

OTU Shannon diversity p-value = 0.08985

OTU Simpson diversity (OTU Simpson diversity) ^lambda p-value = 0.001068

Phylogenetic species Richness (PSR) ^ lambda p-value = 0.671

Genera richness (Genera richness) ^ lambda) p-value = 0.001247

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Genera Shannon diversity (Genera Shannon diversity) ^ lambda p-value = 0.05453

Genera Simpson diversity (Genera Simpson diversity) ^ lambda p-value = 0.009

Phylogenetic species richness (PSR) ^ lambda p-value = 0.6614

Species richness p-value = 0.2943

Species Shannon diversity (Species Shannon diversity) ^ lambda p-value = 0.0168

Species Simpson diversity (Species Simpson diversity) ^ lambda p-value = 0.0009369

Phylogenetic species richness (PSR) ^ lambda p-value = 0.7125

Table S.4. Mycorrhizal Activity in the Ecuadorian Andes: significance (p-value) and estimate
results for multivariate regression models

Multivariate linear regression model (Yi = β0 + β1 × P + β2 × K+ β3 × Ca + β3 × Cu)

Environ- External Internal my- Rootlets Percentage of Number Species

mental Var- mycelium bi- celium biomass dry weight mycorrhizal colo- of spores number

iables omass (RDW) nization (MC)

P p-value 0.960 0.892 0.6511 0.2587 0.0101 * 0.158

Esti- 1.442e-02 -4.694e-02 1.316e-01 -3.393e-01 -7.087e- -

mate 01 4.199e-01

K p-value 0.532 0.330 0.0554 . 0.0654 . 0.7939 0.569

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Esti- -1.862e-01 -3.503e-01 -6.206e-01 5.948e-01 -6.095e- -

mate 02 1.645e-01

Ca p-value 0.233 0.773 0.9864 0.7861 0.8580 0.437

Esti- -3.733e-01 1.034e-01 -5.098e-03 -8.186e-02 -4.262e- -

mate 02 2.311e-01

Cu p-value 0.125 0.995 0.1222 0.1397 0.1137 0.131

Esti- 4.647e-01 -2.226e-03 4.661e-01 -4.446e-01 -3.796e- -

mate 01 4.428e-01

Table S.5. Mycorrhizal Activity in the Ecuadorian Andes: Significance (p-value) and estimate
results for simple linear regression models.

Table S.6. Generalized linear models predicting OUT. genera and species Richness. Significant
predictors are in bold.

Table S.7. Generalized linear models predicting Shannon and Simpson diversity calculated for
OTUs. species and genera. Significant predictors are in bold.
Table S.8. Generalized linear models predicting Phylogenetic Species Richness each calcu-
lated for OTUs. species and genera. Significant predictors are in bold.
Table S.9. PERMANOVA results from Senés-Guerrero for (a) the operational taxonomic unit
OTU table. (b) the Species table and (c) Genus table. Each analysis included all variables on the
table. Significant predictors are in bold.

*Table S.5 to S.9 were located in an external file due to pages limitation for the
submission of only one pdf file.
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Fig. S2. Linear regression models for Nucleotide diversity and a) Soil phosphorus concentra-
tion levels (p= 0.88); b) Altitude levels(p=0.940). No significant p-value