Molecular Ecology (2015) 24, 235–248 doi: 10.1111/mec.

13018

Relocation, high-latitude warming and host genetic

identity shape the foliar fungal microbiome of poplars
MIKLO
S BA

LINT,* LA
SZLO
BARTHA,† ROBERT B. O’ HARA,* MATTHEW S. OLSON,‡§
€ R G E N O T T E , * M A R K U S P F E N N I N G E R , * ¶ A M A N D A L . R O B E R T S O N , § * * P E T E R T I F F I N † † and
JU
IMKE SCHMITT*¶
*Biodiversity and Climate Research Centre, Senckenberg Gesellschaft fu€r Naturforschung, Senckenberganlage 25, 60325
Frankfurt, Germany, †Laboratory of Molecular Environmental Biology, Institute for Interdisciplinary Research in Bio-Nano
Sciences, Babe-Bolyai University, Treboniu Laurian 42, 400271 Cluj, Romania, ‡Department of Biological Sciences, Texas Tech
University, P.O. Box 43131, Lubbock, TX 79409-3131, USA, §Institute of Arctic Biology, University of Alaska Fairbanks, P.O.
Box 757000, Fairbanks, AK 99775, USA, ¶Institut fu €
€r Okologie, Evolution und Diversit€at, Goethe Universit€at, Max-von-Laue-
Str. 9, 60438 Frankfurt am Main, Germany, **Science Applications, U.S. Fish & Wildlife Service, 101 12th Avenue, Fairbanks,
AK 99701, USA, ††Department of Plant Biology, University of Minnesota, 1445 Gortner Avenue, St. Paul, MN 55108, USA

Abstract
Micro-organisms associated with plants and animals affect host fitness, shape commu-
nity structure and influence ecosystem properties. Climate change is expected to
influence microbial communities, but their reactions are not well understood. Host-
associated micro-organisms are influenced by the climate reactions of their hosts,
which may undergo range shifts due to climatic niche tracking, or may be actively
relocated to mitigate the effects of climate change. We used a common-garden experi-
ment and rDNA metabarcoding to examine the effect of host relocation and high-lati-
tude warming on the complex fungal endophytic microbiome associated with leaves of
an ecologically dominant boreal forest tree (Populus balsamifera L.). We also consid-
ered the potential effects of poplar genetic identity in defining the reactions of the mi-
crobiome to the treatments. The relocation of hosts to the north increased the diversity
of the microbiome and influenced its structure, with results indicating enemy release
from plausible pathogens. High-latitude warming decreased microbiome diversity in
comparison with natural northern conditions. The warming also caused structural
changes, which made the fungal communities distinct in comparison with both low-
latitude and high-latitude natural communities, and increased the abundance of plau-
sible pathogens. The reactions of the microbiome to relocation and warming were
strongly dependent on host genetic identity. This suggests that climate change effects
on host–microbiome systems may be mediated by the interaction of environmental fac-
tors and the population genetic processes of the hosts.
Keywords: climate change, community genetics, enemy release, metabarcoding, micro-organism
biogeography, plant–fungal interactions
Received 6 November 2014; accepted 19 November 2014

examples of such associations include mycorrhizae

Introduction
Micro-organisms associated with plants and animals
impact their host’s fitness and physiology. Long-known
Correspondence: Mikl
os B
alint, Fax: +49 69 7542 1855; E-mail:
miklos.balint@senckenberg.de and Imke Schmitt, Fax: +49 69 7542
1855; E-mail: imke.schmitt@senckenberg.de
(Remy et al. 1994), nitrogen-fixing bacteria of legume
roots (Long 1989), corals (Muscatine & Porter 1977), or
foliar endophytes (Redman et al. 2002). The complex
bacterial communities of the human gut influence the
host’s provision of energy and resistance against patho-
gen colonization (Dethlefsen et al. 2007). Plants rely on
complex bacterial or fungal consortia for protection

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LINT ET AL.
236 M . B A
against pathogens (Arnold et al. 2003; Mendes et al.
2011). These complex communities are so relevant for
host fitness and survival so that they have been termed
the host’s ‘second genome’ (Grice & Segre 2012).
The environment defines the composition of complex
plant-associated microbial communities. For example,
foliar endophytic fungal communities of a tropical tree
species are influenced by temperature and rainfall
(Zimmerman & Vitousek 2012). Ectomycorrhizal fungal
richness is defined by temperature and precipitation on
a global scale (Tedersoo et al. 2012). Given that climate
change will result in alterations in temperature and pre-
cipitation patterns (IPCC 2013), shifts in the distribution
of host species (Bellard et al. 2012), and in human-
assisted relocations of species of economic or conserva-
tion interest (Hoegh-Guldberg et al. 2008), it is likely
that climate change will reorganize plant-associated
microbiomes, just as it rearranges the communities of
macro-organisms (Walther et al. 2002).
The genetic identity of host specimens also influences
the microbiome, for example in the case of the root-
associated bacterial microbiome of Arabidopsis plants
(Bulgarelli et al. 2012; Lundberg et al. 2012), or the leaf-
associated fungal communities of poplar (Cordier et al.
2012; B
alint et al. 2013; Lamit et al. 2014). Living in close
association with a plant requires specialization to over-
come a sophisticated plant immune system (Dodds &
Rathjen 2010), and the genetically defined pathogen
resistance of plants (Bergelson & Purrington 1996) likely
impacts the microbiome. The microbiome may also be
structured along the interactions of host genetic proper-
ties with environmental factors. For example, both envi-
ronmental and host genetic factors control which
genotypes of a leaf-associated fungus may establish in
birch trees (Ahlholm et al. 2002). As climate change is
projected to influence the genetic composition of host
populations (either due to the loss of intraspecific
genetic diversity – B
alint et al. 2011 and Alsos et al.
2012 – or because the altered geographic distribution of
intraspecific lineages – Pauls et al. 2013), we can expect
that under future climates, plant microbiomes are struc-
tured by the hosts’ environment, by the hosts’ genetic
properties and by the impact of climate change on the
population genetic processes of the hosts.
Foliar endophytic fungi are a complex and ubiquitous
microbiome that associates with the leaf tissues in the
phyllosphere of all known plant species (in contrast to
leaf surfaces), without causing symptoms of a disease
(Carroll 1986). These fungi form highly diverse infec-
tions in the leaves of all woody plants (Rodriguez et al.
2009), being structured by the environment over large
spatial scales (Arnold & Lutzoni 2007; Zimmerman &
Vitousek 2012) and by host genetic properties (Cordier
et al. 2012; B
alint et al. 2013; Lamit et al. 2014). Foliar
endophytes are assumed to be horizontally transmitted
among leaves. This means that they are not transmitted
among plant generations via seeds, but rather via the
yearly infection of leaves from an environmental spore
pool (Arnold & Herre 2003; Saikkonen 2007; Rodriguez
et al. 2009). It was shown that foliar endophytes influ-
ence the susceptibility of host trees to pathogens, either
contributing to the defences (Arnold et al. 2003; Ragha-
vendra & Newcombe 2013), or increase pathogen symp-
tom severity (Busby et al. 2013). We investigated how
the foliar endophytic fungal microbiome of a North
American foundation tree species (balsam poplar –
P. balsamifera) reacts to two plausible impacts of climate
change – the human-assisted relocation of trees to high
latitudes and increased air temperatures at high lati-
tudes. The populations of balsam poplar belong to dis-
tinct geographic demes (Keller et al. 2010) and show
adaptations to the local climate along a latitudinal gra-
dient (Soolanayakanahally et al. 2009; Keller et al. 2011,
2012; Olson et al. 2013). This makes the species prone to
the effects of climate change and an ideal target to
understand how environmental conditions interact with
the genetic identity of host plants in microbiome assem-
bly under climate change conditions. We searched
answers for the following questions: (i) How does the
relocation of the host to high latitudes influence the
diversity, community structure and potential ecological
functioning of the foliar endophyte microbiome? (ii)
What is the effect of high-latitude warming? and (iii) To
what extent does the host genetic identity alter microbi-
ome responses to relocation and warming?
Materials and methods
Experimental set-up
The balsam poplar genotypes used in this study belong
to the AgCanBaP genotype collection of the Agriculture
and Agri-Food Canada (AAFC), Agroforestry Develop-
ment Centre, Indian Head, Canada. The collection was
established between 2005 and 2006 in Indian Head, in a
common garden close to the southernmost distribution
limit of the species (Fig. 1) (Soolanayakanahally et al.
2009). Cuttings from the southern collection were used
in 2010 to establish a new common garden on the cam-
pus of University of Alaska, Fairbanks (Olson et al.
2013), close to the northernmost edge of the species’ dis-
tribution (Fig. 1, Keller et al. 2010). For this experiment,
we considered one clone of each of 51 poplar genotypes
that was grown in the southern garden and two clones
that were grown in the northern garden (N = 153, Table
S1, Supporting information). The clonal pairs in the
north were randomly divided into two groups, with one
group being grown under natural conditions and the
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 C L I M A T E A N D H O S T I D E N T I T Y S H A P E M I C R O B I O M E S 237

Fig. 1 Experimental set-up. Cuttings from 51 balsam poplar genotypes from the southern–north-western distribution of the species
were rooted and grown in a common garden in the south. Two cuttings of these genotypes were rooted and grown in a common
garden in the north. Stars mark common gardens. Half of the rooted specimens (one from each genotype) were grown under natural
conditions in the north, the other half was exposed to atmospheric warming. Warming increased atmospheric temperatures by
~2.45 °C around the heated specimens during the experiment (~2 months). Circles mark the source populations of the 51 genotypes.
The populations belong to two distinct geographic demes of balsam poplar (Keller et al. 2010). Populations of the southern deme are
marked with light green, and populations of the northern deme are marked with dark green.

other group being exposed to artificial atmospheric
warming (Fig. 1, Robertson 2012). The clones were
planted in an 8 9 20 m grid, with 2.5 m spacing. Clonal
pairs were planted adjacent to one another randomly
across the garden. For each pair, one individual was
randomly selected to be grown under ambient condi-
tions, and the other individual was passively warmed.
The warmed individuals were surrounded by a 1.0-m-
diameter plastic chamber on 3 May 2010 and 3 May
2011, about 1.5 weeks before the average date of bud
flush for local poplar populations. The open-top cham-
bers were made from 0.7-mm clear plastic sheets. Day-
time solar heat was amplified/stored using black
containers filled with water (3.8 L), placed inside the
chambers. The trees remained in the chambers until
September 20 each year (about 2–3 weeks after the aver-
age date of the first frost). We used iButton Thermo-
chron dataloggers (Maxim Integrated Products, San Jose,
CA, USA) for an hourly recording of the air and soil
temperatures of 50 randomly selected trees. The temper-
ature difference between the naturally grown and
warmed trees was ~2.45 °C (variance r2 = 1.61) between
the time of the bud flush and the sampling (7 August
2011). We sampled one healthy, similarly sized leaf
(N = 153) from each tree in both gardens (south: 7 July
2011; north: 5 August 2011). The timing of the sampling
reflects bud flush that starts about 1 month earlier in
Indian Head, compared to Fairbanks (Olson et al. 2013).
The one leaf per tree sampling reduced the damage car-
ried out to poplar individuals. Our sampling focused on
replication among the experimental treatments to cap-
ture microbiome variability caused by the relocation
shift and warming (number of replicates per treatment
NT = 51), rather than on investigating the variability of
communities within single trees. The saplings were ~30–
250 cm high during the experiment. Given the small
canopy size of the sampled trees, we assume that can-
opy effects (Cordier et al. 2012) or microclimate (Schol-
tysik et al. 2013) has no influence on fungal assemblages.
The leaves were immediately dried on silica gel and
kept frozen until DNA extraction.
Description of the phyllosphere fungal community
We assayed the fungal communities of healthy poplar
leaves with the high throughput sequencing the ITS
region of the rRNA operon. Leaf surfaces were steril-
ized prior to DNA extraction by immersing them in 4%
sodium hydrochloride solution for 1 min. After immer-
sion, the leaves were washed twice in sterile water. The
sterilizing solution contained 0.1% of Tween 20 to break
surface tension. DNA was extracted with a KingFisher
Flex (Thermo Scientific, Germany) using KingFisher
DNA Pure Plant Kits (Thermo Scientific, Germany),
according to the manufacturer’s protocol. We amplified
the first part of the internal transcribed spacer (ITS1)
with primers ITS1FI2 (Schmidt et al. 2013) and ITS2
(White et al. 1990). For the PCR amplification, we used
the KAPA 3G Plant PCR kit (Peqlab, Germany). The 25-
lL reactions contained 0.5 U KAPA 3G Plant DNA
Polymerase, 12.5 lL buffer (29), 8.3 lL water, 0.5 lL
BSA (10 mg/mL), ~5 ng poplar leaf DNA (0.2 lL),
0.3 lM (0.75 lL) of each primer and 0.86 lM blocking
primer (2 lL, Poplar-bl-F: GGAAGGATCATTGTCGAA

© 2014 John Wiley & Sons Ltd


LINT ET AL.
238 M . B A
ACC, primer developed for the current study). The
blocking primer was required to repress the amplifica-
tion of the poplar ITS1 products. The samples were first
denatured at 95 °C for 4 min, followed by 35 cycles of
95 °C at 20 s, 52 °C for 20 s, 72 °C for 15 s and a final
elongation of 72 °C for 3 min. The amplicons were
SPRI-purified with the Agencourt AMPure XP kit (Beck-
man Coulter, Germany). The fragments in each sample
were combinatorially labelled on both ends in a second
PCR. These 25-lL reactions contained 0.65 U TaKaRa
ExTaq (Clontech Laboratories, Inc. USA), 2.5 lL buffer
(109), 16 lL water, 2.0 lL dNTP mixture (2.5 mM each),
10 ng amplicon (2.5 lL), 0.22 lM (0.5 lL)-labelled for-
ward and reverse primer and 0.43 lM (1 lL) blocking
primer. We used multiplexing labels published by
Gloor et al. (2010). The reactions were performed with
the following cycle conditions: initial denaturation
95 °C for 4 min followed by 28 cycles of 95 °C for 30 s,
52 °C for 20 s, 72 °C for 20 s and a final elongation at
72 °C for 5 min. The end-labelled amplicons were SPRI-
purified, concentration-normalized (using Qubit Fluoro-
metric Quantitation, Life Technologies, Germany),
SpeedVac-dried and paired-end-sequenced (2 9 150 bp)
on an Illumina MiSeq sequencer at the Biomedical
Genomics Center of the University of Minnesota, USA.
The quality pruning of the sequence data was per-
formed according to B
alint et al. (2014). All reads with
an average quality score below 20 phred were removed.
We trimmed both ends of the remaining sequences with
PRINSEQ (Schmieder & Edwards 2011), whenever a base
with a quality score below 20 phred was encountered.
The read pairs were assembled with default settings in
PANDASEQ (Masella et al. 2012). We discarded all reads
that contained unknown nucleotides. Reads were then
reoriented into 50 -30 directions. Demultiplexing was per-
formed with FQGREP (https://github.com/indraniel/
fqgrep, accessed on 1 August 2012). We did not allow
mismatches in the label and primer sequences. Initial
denoising was performed with a 99% similarity cluster-
ing with the heuristic clustering algorithm UCLUST 2.1,
implemented in USEARCH v.6.0.203 (Edgar 2010). De novo
chimera detection was performed with the UCHIME algo-
rithm (Edgar et al. 2011). OTU picking was performed
at 97% sequence similarity with UCLUST 2.1. The most
abundant sequence types served as clustering seeds.
We excluded OTUs with less than 10 reads. Fungal
ITS1 reads were extracted from the centroid sequences
of each cluster with FUNGALITSEXTRACTOR (Nilsson et al.
2010). The ITS1 fragments were compared with a BLAST
search (Altschul et al. 1997) against the entire GenBank
nucleotide database (ftp://ftp.ncbi.nlm.nih.gov/blast/
db/nt*, downloaded on 18 October 2012). We parsed
the BLAST outputs in MEGAN 4 (Huson et al. 2011) for
initial taxonomic screening (min. support: 1, min. score:
170, top per cent: 5) and retained OTUs with supported
fungal origin for downstream analysis.
Statistical analyses of microbiome diversity
We compared the diversity of the fungal communities
in the south and north (under both natural and warmed
conditions) with Hill’s series of diversity. Diversity
indicators are differentially sensitive to rare and abun-
dant taxa; thus, the result of such comparisons may
depend on the choice of the index. Hill’s series of diver-
sity (Hill 1973) provide a measure accounting for the
abundance of taxa along to three scale parameters. The
series consists of three numbers: N0 is the number of
species in a sample; N1 is the antilogarithm of the
Shannon diversity (representing the abundant species in
a sample); and N2 is the inverse Simpson diversity
(representing the very abundant species in a sample).
Consequently, N1 and N2 emphasize evenness by putt-
ing more weight on abundant taxa. Communities can
be considered more diverse if their diversity ranks
higher at all three scale parameters. Hill’s numbers
were calculated in R v3.0.2 (R Core Team 2013) with the
VEGAN v2.0-7 package (Oksanen et al. 2013).
We used linear models to explain Hill’s N0, N1 and
N2 with the square root of sequencing read numbers
obtained for a sample, experimental parameters and
two indicators of the genetic identity of balsam poplar
genotypes. The square root of read numbers stands for
an observational bias stemming from differential
sequencing success in different samples with equal
sequencing effort, inherent of high-throughput sequenc-
ing (further referred as ‘sequencing bias’). By incorpo-
rating this predictor into the models as the first
explaining variable, we can explicitly account for this
bias. The explicit incorporation of the sequencing bias is
strongly advocated over the current practice of rarefy-
ing sequence read numbers (McMurdie & Holmes
2014). The three treatments (trees grown in the south,
relocated to the north, warmed in the north) served as
experimental predictors in the models. Both indicators
of host genetic identity are related to the phylogeo-
graphic history of the balsam poplar. The first indicator
consists of two single nucleotide polymorphism-defined
geographic demes of balsam poplar to which all experi-
mental genotypes belonged (Keller et al. 2010). The sec-
ond indicator is the latitude of the original poplar
source populations as poplars show adaptation to local
conditions along this latitudinal gradient (Soolanayaka-
nahally et al. 2009; Keller et al. 2011, 2012; Olson et al.
2013). We compared several nested models with
increasing complexity in a stepwise manner with
ANOVA-based F-tests, until the more complex model did
not statistically significantly explain more variation in
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 C L I M A T E A N D H O S T I D E N T I T Y S H A P E M I C R O B I O M E S 239
Hill’s three numbers. The most complex model can be
written as Hill ~ sqrt(readNumbers) + experiment +
identity1 + identity2. We used ANOVA (F-test) to evalu-
ate the contribution and significance of the explanatory
variables in explaining diversity variation in the best-
performing model. We compared the partial residuals
of Hill’s numbers among the three treatments with Tu-
key’s HSD in R, after accounting for the variation
caused by differential sequencing success.
Statistical analyses of microbiome structure
The phyllosphere fungal microbiome is highly diverse,
but the occurrence of many OTUs may be restricted to
only few samples. It is unlikely that reliable conclusions
can be drawn about the effects of the predictors from
infrequently recorded OTUs. For example, an OTU that
is sporadically recorded in a few trees may not inform
us about the effect of poplar relocation and warming,
with each treatments consisting of 51 tree clones. These
infrequent OTUs may be informative about other fac-
tors that are not within the scope of the experiment, for
example by herbivore attacks, accidental damage to a
poplar specimen, etc. For this reason, we identified a
set of core OTUs that are present in a larger number of
poplar clones. We used OTU incidence–abundance rela-
tionships to define an incidence threshold. We fitted a
generalized additive model (GAM) on the logarithms of
average read abundance/tree of OTUs against the num-
ber of trees where the OTU was encountered with the R
package mgcv v1.7-24 (Wood 2011). Based on the result-
ing curve (Fig. S1, Supporting information), OTUs pres-
ent in more than 30 poplars out of all poplars were
defined as core OTUs and used in the evaluation of
community structure.
We evaluated the experimental factors shaping the
core fungal community composition with multispecies
generalized linear models (GLM). GLMs explicitly
model the special mean–variance relationship character-
istic of ecological counts, assuming a negative binomial
distribution of the read observations (Warton et al.
2012). The multispecies GLMs were fitted with the R
package MVABUND v3.7.0 (Wang et al. 2012). The same
explanatory variables were considered as above: the
square root of ITS sequence counts, three experimental
treatments and the two indicators of the host genetic
identity. We performed a forward model selection start-
ing with the ITS sequence numbers as sole predictors of
the community structure. The other terms were sequen-
tially added up, and finally, possible interactions were
considered. This resulted in nested models, which were
then compared with likelihood-ratio tests (ANOVA, Monte
Carlo resampling, 100 bootstraps). The most complex
tested model can be written as core microbiome ~ sqrt
© 2014 John Wiley & Sons Ltd
(readNumbers) + experiment*identity1*identity2. Once
the best-performing model was identified, we tested
and quantified the effect of all predictors and their
interactions (ANOVA, likelihood-ratio test, Monte Carlo
resampling, 300 bootstraps). Summary statistics (likeli-
hood-ratio test, Monte Carlo resampling, 300 bootstraps)
was used to disentangle the effect of warming trees in
the north in contrast to relocating trees to the north. We
estimated the robustness of the results by performing
community comparisons with OTUs delimited at 95%
sequence similarity. As distance-based methods are reg-
ularly used in community ecology, we also performed a
PERMANOVA of the model presented above (Anderson
2001). Sample distances were calculated with the Bray–
Curtis distance measure (Bray & Curtis 1957), and the
statistical significance was estimated with 999 permuta-
tions with the vegan package in R. We note that to date
all known distance metrics assume a mean–variance
relationship free of overdispersion, and this assumption
is violated by typical ecological abundance data (War-
ton et al. 2012). We also noted strong overdispersion in
our OTU data, violating the assumptions of all dis-
tance–distance matrices that can be used in PERMANOVA
to date (Fig. S2, Supporting information). GLMs are not
affected by this problem and they are recommended for
these type analyses, as they readily model the particular
mean–variance relationship of ecological count data
(Warton et al. 2012). The results of the multispecies
GLMs were visualized with nonmetric multidimen-
sional scaling (NMDS) in three dimensions, with 100
random starts, with the vegan package in R. For the
NMDS, Bray–Curtis distances among the samples were
computed after the square root transformation of the
core OTU counts. We emphasize that the NMDS only
serves for the visualization of the statistically tested
GLM results.
The statistical significance of univariate ANOVA tests
for each core OTU was returned after adjusting for the
familywise error rates with Holm’s step-down multiple
testing procedures (Holm 1979). We selected all OTUs
that were significantly affected by the relocation and
warming (at P < 0.01). To evaluate significant differ-
ences among the three treatments, we tested the GLM-
based abundance predictions of these OTUs with post
hoc Tukey’s HSD tests. To evaluate possible ecological
functions in the phyllosphere communities, we
attempted a functional assignment of the statistically
significantly affected OTUs to ecologically distinct
groups with the following procedure: (i) we performed
a BLAST search with the representative (centroid) OTU
sequence against the UNITE fungal ITS database (Aba-
renkov et al. 2010); (ii) we parsed the BLAST results in
MEGAN 4 (Huson et al. 2011); and (iii) in case of BLAST hits
that fell within the upper 5% threshold from the highest

LINT ET AL.
240 M . B A
BLAST bit score of an OTU, we checked the references of
the databased sequence for plausible ecological func-
tions. This assignment relied on all BLAST hits that
were supported by at least one match of a query
sequence against a database sequence, and a bit score
over 170. We rendered all OTUs as ‘Not assigned’ if the
references did allow an equivocal functional assign-
ment, or if the assignment indicated mycorrhizal taxa,
which are not expected in the phyllosphere. We used
an NMDS ordination in vegan in two dimensions to
visualize the GLM-predicted abundances of the func-
tionally assigned OTUs among the three experimental
conditions.
Results
We generated 3 870 141 paired-end Illumina MiSeq
ITS1 reads. Of these, 2 708 348 reads passed the
demultiplexing step, and 1 759 757 quality-filtered read
pairs could be assigned to fungi. This represents a
mean of 11 731 (median 9621) fungal read pairs per
sample. We defined 2022 unique fungal operational
taxonomic units (OTUs). The abundance distribution of
the OTUs was strongly skewed, with a few very abun-
dant and many rare OTUs (Fig. S3, Supporting infor-
mation). On the class level, most OTUs were assigned
to Dothideomycetes (826), Sordariomycetes (210), Aga-
ricomycetes (194), Tremellomycetes (79), Eurotiomyce-
tes (76), Leotiomycetes (22), Microbotryomycetes (11)
and Sacharomycetes (8).
The effects of relocation and high-latitude warming on
microbiome diversity, community structure and
plausible ecological functions
The most complex model for any of the numbers in
Hill’s series included sequencing success, experimental
treatment and the first indicator of poplar genetic iden-
tity (geographic deme) (Hill ~ sqrt(readNumbers) +
experiment + identity1). The models revealed that varia-
tion in all three numbers in Hill’s series was strongly
and statistically significantly explained by the experi-
mental treatments (growing location and warming) after
accounting for differences in sequencing success
(Table 1). The series indicated more diverse microbio-
mes in poplars under both northern conditions when
compared to poplars in south (Tukey’s HSD, P < 0.05).
The series also indicated that microbiome diversity
decreases in poplars warmed in the north when com-
pared to poplars grown naturally in north (Fig. 2). This
decrease was statistically significant for N1 and N2
which emphasize abundant taxa (Tukey’s HSD,
P < 0.05), and statistically non-significant for N0 (aver-
age OTU number under natural conditions: 235.9,
average OTU number under warmed conditions: 234.5,
Tukey’s HSD, P = 0.37). Host geographic deme was a
statistically significant weak predictor of Hill’s N0, but
did not predict N1 and N2.
Of 2022 OTUs, 269 were present in more than 30
poplar clones and were defined as core OTUs. All core
OTUs were recorded in trees grown in the south. In
trees grown in the north, 267 core OTUs were recorded
under natural conditions, and 268 OTUs under
warmed conditions. The best-performing generalized
linear model of the core OTUs incorporated sequencing
depth, experimental treatments, indicators of the host’s
genetic identity and interactions among treatments and
genetic identity (Table 2). This model is written as mi-
crobiome ~ sqrt(readNumbers) + experiment*iden-
tity1 + experiment*identity2, the strongest predictors
of community structure being the experimental treat-
ments (see Fig. 1 for poplar geographic demes and the
latitude of source populations). The results of the PER-
MANOVA are mostly similar, the strongest predictor of
community structure being the sequencing bias, fol-
lowed by the experimental treatments (Table S2, Sup-
porting information). Within treatments, the summary
statistics show that the microbiome of poplars grown
in the south statistically significantly differs both from
the microbiome of poplars grown in the north under
natural conditions (likelihood-ratio score z = 377.36,
P < 0.003) and from the microbiome of poplars
warmed in the north (z = 338.99, P < 0.003). The mi-
crobiome of poplars warmed in the north also statisti-
cally significantly differed from the microbiome of
trees grown under natural northern conditions
(z = 436.11, P < 0.003). These results are visualized on
a NMDS plots in three dimensions, with a stress of
0.19 (Fig. 3). Community comparisons with OTUs
delimited at 95% sequence similarity (1586 fungal
OTUs) yielded similar results (Figs S5–S7, Table S5,
Supporting information).
Range shift and warming alone significantly affected
the abundance of 94 of the 269 core OTUs after account-
ing for multiple comparisons. Tukey’s HSD tests of the
GLM predictions showed that the abundance of most of
these OTUs statistically significantly differed among the
three treatments (80 OTUs between the southern and
northern natural conditions, 75 OTUs between the
northern natural and warmed conditions, 82 between
the southern and the northern warmed conditions
(Table S3, Supporting information).
We could assign 43 of the 94 OTUs to known gen-
era of plant endophytes, pathogens, saprotrophs and
mycoparasites (NMDS with stress 0.025, Fig. 4, Table
S3, Supporting information). Some OTUs were
assigned to unexpected taxa such as mycorrhiza. This
may be a result of an erroneous assignment (see simi-
© 2014 John Wiley & Sons Ltd
 C L I M A T E A N D H O S T I D E N T I T Y S H A P E M I C R O B I O M E S 241

Table 1 Variation of Hill’s series of diversity, explained by linear models. Hill’s N0 is species richness; N1 represents abundant spe-
cies (the antilogarithm of Shannon’s diversity); and N2 represents very abundant species (the inverse of Simpson’s diversity)

Hill’s N0 Hill’s N1 Hill’s N2

Degrees of
freedom Sum of squares F P Sum of squares F P Sum of squares F P

Read numbers 1 842 343 874.15 <0.001 62.23 5.35 0.02 9.86 6.91 0.009
Experiment 2 107 179 55.61 <0.001 1362.22 58.56 <0.001 87.78 30.76 <0.001
Geographic deme 1 7696 7.99 0.005 26.05 2.24 0.137 0.00 0.00 0.965
Residuals 145 139 723 1686.37 206.93

Fig. 2 Relocation and warming of poplars result in less diverse foliar fungal communities. Boxplots show the Hill diversity profile of
the foliar microbiome in the south, in the north under natural and warmed conditions. Diversity differences are consistent across the
Hill diversity profile. Poplars grown under northern natural conditions harbour the most diverse microbiomes. Diversity in the north
statistically significantly decreases if trees are heated at N1 and N2. Poplars in the south have statistically significantly lower diver-
sity in comparison with any northern conditions. Values indicate the partial residuals of Hill’s diversity series after accounting for
differential sequencing success in linear models. Letters mark statistically significant differences in Hill’s numbers evaluated with
Tukey’s HSD.

larities in Table S4, Supporting information), or they
may have accidentally entered the leaves. The phyllo-
sphere microbiomes in the south were dominated
mostly by OTUs that were assigned to six Mycosphae-
rella strains, four of them known as poplar-specific
pathogens (Fig. 4, Table S4, Supporting information).
These OTUs were present also in the north but at
much lower abundances (Fig. S4, Supporting informa-
tion). The northern conditions, both natural and
heated, were characterized by a functionally complex
mixture of other fungi (Fig. 4). Tukey’s HSD tests of
GLM abundance predictions showed that the abun-
dances of Mycosphaerella OTUs significantly differed
among all treatment pairs (Table S3, Supporting
information). According to the models, five of the six
Mycosphaerella OTUs are expected to be more highly
represented under the heated than under the natural
northern conditions (Fig. S4, Supporting information).
Phyllosphere microbiome responses to host genetic
identity under relocation and warming
Both indicators of host genetic identity (geographic
demes and the latitude of poplar source populations)
had statistically significant effects on the phyllosphere
microbiome (Table 2). These effects were about one
magnitude smaller in comparison to the effects of the
experimental treatments. We found a stronger and sta-

© 2014 John Wiley & Sons Ltd


LINT ET AL.
242 M . B A

tistically significant interaction effect among both host

Discussion
identity indicators and the experimental treatments
(Table 2). The results indicate that the relocation and
Relocation to high latitudes and high-latitude warming
warming more strongly influenced the phyllosphere
rearrange the foliar fungal endophytic microbiome
microbiome of poplar genotypes that belong to the
southern geographic deme in comparison with the mi- The class-level composition of the foliar fungal
crobiome of poplar genotypes of the northern deme endophytic microbiome was similar in poplars in
(Fig. 5). Although important for the entire microbiome, comparison with what was recorded by metabarcoding
there was little evidence for host genetic identity having in other tree species (Meiser et al. 2014). We recorded
statistically significant effects on the abundance of indi- lower OTU diversity in poplars grown in the south in
vidual OTUs. comparison with trees relocated to the north. This was
consistent and statistically significant for the entire Hill
diversity profile: in species richness, in abundant spe-
Table 2 Variation in the composition of the phyllosphere fun-
gal microbiome explained by multispecies GLMs. Models are cies and in very abundant species (Fig. 2). Warming
tested with ANOVA (likelihood-ratio test, 300 bootstraps). Only trees in the north decreased their phyllosphere diversity
the abundances of OTUs that were present in more than 30 in comparison with natural conditions along the entire
trees were modelled (N = 269). Read numbers (sequencing suc- profile (statistically significant for N1 and N2, but not
cess) were square root-normalized. The experiment consists of for N0, Fig. 2). This means that the phyllosphere
range shift from the south to the north and atmospheric warm-
microbiome is less even in the south than in the north,
ing in the north. The host genetic identity is represented by
and warming decreases the evenness in the north. Find-
two indicators of the balsam poplars’ phylogeographic history:
a southern and a northern demographic deme that formed ing higher phyllosphere microbiome diversity in the
during the late Pleistocene range expansion of the species and north was surprising, as existing studies record a nega-
the latitude of the locally adapted source populations tive latitudinal gradient in the diversity of foliar fungal
endophytes of several host tree species (Arnold &
Residual Lutzoni 2007; Meiser et al. 2014). However, we evalu-
degrees Degrees of Test
ated the endophyte diversity of a single host species.
of freedom freedom statistics P
We could also not include tropical microbiomes into
Read numbers 148 1 3954.2 <0.001 our comparisons. We speculate that the diversity differ-
Experiment 146 2 5061.7 <0.001 ences result from higher abiotic stress in natural high-
Geographic deme 145 1 469.0 0.007 latitude environments, which is predicted to increase
Source latitude 144 1 462.2 0.004 evenness in communities (Svensson et al. 2012).
Experiment 9 142 2 955.6 0.014 Most variation in the phyllosphere community com-
Geographic deme
position is explained by experimental factors in the
Experiment 9 140 2 814.5 0.02
multispecies GLM framework (~43% of the explained
Source latitude
variation, Table 2). This is similar to the results of the
1.0

0.5

0.5
0.5

0.0

0.0
NMDS2

NMDS3

NMDS3
0.0

−0.5
−0.5
−0.5

−1.0
−1.0

−1.0 −0.5 0.0 0.5 1.0 −0.5 0.0 0.5 1.0 −1.0 −0.5 0.0 0.5 1.0
NMDS1 NMDS2 NMDS1

Fig. 3 NMDS plot of the phyllosphere microbiome of poplar hosts (stress 0.19). Circles represent the microbiome of polar genotype
cloned (N = 150) grown under southern (red), northern natural (blue) and northern warmed (orange) conditions. The size of the cir-
cles is proportional to the Shannon diversity of the microbiome. The 95% confidence intervals of the group centroids of each treat-
ment are marked with coloured ellipses. Lines link individual microbiomes to the treatment group centroids. The plot serves only
for the visualization of the GLM results about the relocation and warming.

© 2014 John Wiley & Sons Ltd

 C L I M A T E A N D H O S T I D E N T I T Y S H A P E M I C R O B I O M E S 243

PERMANOVA, where experimental factors are the second

Mycosphaerella
1.0 Other pathogens most important predictors of community composition
Mycoparasite after the sequencing bias (~15% of all variation, Table
Saprotroph
Not assigned
S2, Supporting information). Likelihood-ratio tests show
South that poplar clones moved to the northern environment
host-distinct microbial communities in comparison with
0.5
NMDS2

the clones of the same poplar genotypes grown in the

North, heated south (Fig. 3). Functionally, the phyllosphere microbi-
ome of the poplar hosts in the south was dominated by
six OTUs that were assigned to Mycosphaerella strains
0.0

(Fig. 4), although we note that it is difficult to distin-

guish pathogens from other closely related taxa using
North, natural sequence data alone. Mycosphaerella is a large genus of
−0.5

fungi that includes numerous plant pathogens, sapro-

−0.5 0.0 0.5 1.0
NMDS1
Fig. 4 Nonmetric multidimensional scaling (NMDS) ordination
of modelled and functionally assigned OTUs (stress 0.025). Cir-
cles represent OTUs in the southern garden and in the north-
ern garden under natural and warmed conditions. Only OTUs
with abundances directly explained by relocation and heating
are shown (N = 94 OTUs). The ordination is based on OTU
abundances predicted by the best-performing multispecies
GLM. Circles represent OTUs, with sizes scaled proportionally
by the corresponding read numbers.
Fig. 5 Host relocation and warming interact with host genetic
identity in shaping foliar fungal microbiome. Range shift and
warming effects are stronger on OTUs of poplars from the
southern geographic deme. The lines show the generalized lin-
ear model coefficients of the N = 269 core OTUs. Model coeffi-
cients represent the extent of change under a predictor in
comparison with the baseline conditions. The coefficients were
mean-centred according to the intercept (southern garden,
southern deme). *marks an extreme outlier at 823.36.
trophs and foliar endophytes (Crous 2009). Although it
is hard to decide whether the recorded Mycosphaerella
OTUs are truly pathogenic, several Mycosphaerella spe-
cies are known to lead facultative nonpathogenic life-
styles (Crous et al. 2008). The relocation of poplar
clones to the north results in a strong decrease in the
abundance of these OTUs, and the leaves become domi-
nated by a functionally more diverse mixture of fungi.
The results indicate that the northward relocation of
poplars may decrease the abundance of common south-
ern pathogens. This is in line with assumptions that
host relocation results in their release from enemies
(Van der Putten 2012). Studies of interactions among
plants, soil microbiota and herbivores show that hosts
may have increased fitness in a new habitat due to this
effect (Callaway et al. 2002; Klironomos 2002; Van
Grunsven et al. 2010). Although we did not measure
host fitness effects, we show that pathogen release may
also act in the phyllosphere. Slow range shifts may
influence the microbiomes differently compared to
assisted relocation, as the estimated climate-related nat-
ural range shift of macro-organisms is relatively slow
(~16.9 km per decade, Chen et al. 2011). This may pro-
vide more opportunities for endophytes to codisperse
with their hosts under climate change or to adapt to
new conditions.
Warming poplars hosts in the north results in a phyll-
osphere microbiome structure that is statistically signifi-
cantly different in comparison with the microbiome
structure of poplars grown under natural northern con-
ditions (Fig. 3). In line with this, the post hoc testing of
OTU abundance predictions shows that approximately
the same number of OTUs have statistically signifi-
cantly different abundances among the three experi-
mental conditions (Table S3, Supporting information).
Altogether, high-latitude warming results in a novel mi-
crobiome, which is different in comparison with both
low-latitude microbiomes and the microbiomes of the
natural high-latitude conditions. A metabarcoding study
of a tropical tree also found that environmental factors,

© 2014 John Wiley & Sons Ltd


LINT ET AL.
244 M . B A
such as temperature and precipitation, strongly struc-
ture the foliar fungal endophytic microbiome (Zimmer-
man & Vitousek 2012). Regarding warming, most
studies to date focus on root-associated micro-organ-
isms (Compant et al. 2010). Results from root-associated
systems range from no compositional effects in Salix
arctica (e.g. Fujimura et al. 2007) to decreased fungal
diversity and changed composition in grassland soils
(e.g. Sheik et al. 2011). Understanding the ecological rel-
evance of foliar endophyte community shifts helps to
evaluate the effects of climate change on forest ecosys-
tems. This is important because (i) foliar endophytes
may play a role in tree defences against disease (Arnold
et al. 2003; Raghavendra & Newcombe 2013), they may
be latent pathogens themselves emerging with climate
change (Krabel et al. 2013), and (ii) numerous foliar en-
dophytes can be latent saprotrophs (Parfitt et al. 2010)
that may increase carbon cycling as warming increases
their action (M€ uller et al. 2014). The generalized linear
models predicted increased abundances for five of the
six Mycosphaerella OTUs under the warmed conditions,
indicating a possible increase in pathogen prevalence in
woody plants with high-latitude warming. This is simi-
lar to recent results about increased crop pathogen
prevalence at high latitudes with climate change (Beb-
ber et al. 2013).
Host identity defines microbiome response to relocation
and warming
The standalone effects of host genetic identity were rel-
atively small on the phyllosphere microbiome (although
statistically significant). However, we found relatively
strong interaction effects among the experimental treat-
ments and the host genetic identity (Table 2). The
effects of relocation and warming were the strongest
among the microbiomes of trees with genotypes that
belong to the southern geographic deme (Fig. 5). We
speculate that abiotic stress-related adaptation in pop-
lars at high latitudes may stand behind the observed
patterns. For example, stress may decrease the costs of
pathogen resistance in plants (Bergelson & Purrington
1996). Low temperatures induce defence gene expres-
sion against fungal pathogens in winter wheat (Gaudet
et al. 2011). Low temperatures induce plant hormone
accumulation relevant for systemic-acquired resistance
in conifers (Pedranzani et al. 2007). Northern deme pop-
lars are adapted to conditions typical of high latitudes
(Soolanayakanahally et al. 2009; Keller et al. 2011, 2012;
Olson et al. 2013), and this adaptation may additionally
influence how their microbiome reacts to the environ-
ment.
The results imply that if the genetic composition of
poplar populations is restructured as a result of climate
change or human-assisted relocations, this will strongly
influence the microbiome. The genetic composition of
populations may be rearranged by climate change-
related range shifts (Pauls et al. 2013). Even if the range
of a species is not altered by climate change, the genetic
composition of populations may change through the
spread of migrant genotypes and adaptation to new
climatic environments (Davis & Shaw 2001; Davis et al.
2005). This underlines the need to consider microbiome
ecology in a community genetics framework and to syn-
thesize aspects of both community ecology and evolu-
tionary biology (Rowntree et al. 2011). We can expect
that host-intraspecific genetic variation will affect the
reactions of the microbiome to climate change, just as
intraspecific variation in dispersal abilities and pheno-
logical responses affect macro-organism responses
(Pauls et al. 2013).
Differential sequencing success and distance-based
tools in the assessment of community structure
It is a common practice in metabarcoding to sequence
multiple samples in the same sequencing reaction. The
resulting read numbers may vary by orders of magni-
tude among the samples (e.g. Schmidt et al. 2013; this
study). The common practice to deal with this problem
is rarefying, which is a process composed of (i) select-
ing a read number threshold, (ii) discarding samples
with less reads than the threshold and (iii) subsampling
the reads in the remaining samples without replace-
ment to the selected threshold (Hughes & Hellmann
2005). However, rarefactions are statistically inadmissi-
ble because they (i) discard large fractions of valid data,
(ii) result in loss of power in statistical tests, (iii) fail to
deal with overdispersion (see also the discussion about
mean–variance relationships below), (iv) require an
arbitrary threshold selection for minimum sample size
and (v) add additional uncertainty to the data in the
form of random subsampling (McMurdie & Holmes
2014). The effects of differential sequencing depth
should be explicitly considered and quantified in all
analyses. In this study, the sequencing bias explains
most of the variation in Hill’s N0 (Table 1), and it is the
second most important variable explaining community
structure (Table 2). Tools are readily available to deal
with sequencing bias through R packages that are spe-
cifically tailored for gene expression analyses and suit-
able for microbiome data such as DESEQ (Anders &
Huber 2010), EDGER (Robinson et al. 2010) and METAGENO-
MESEQ (Paulson et al. 2013), or they ar PRINSEQ E devel-
oped for multispecies abundance matrices such as
MVABUND (Wang et al. 2012).
Typical ecological count data is overdispersed, mean-
ing that its variance increases with the mean. When
© 2014 John Wiley & Sons Ltd
 C L I M A T E A N D H O S T I D E N T I T Y S H A P E M I C R O B I O M E S 245
distance-based tools such as PERMANOVA are used for the
evaluation of ecological count data, implicit assump-
tions are made about the mean–variance relationship of
the data sets, and these are not met by the typical over-
dispersed macro-organismic (Warton et al. 2012) or
micro-organismic data sets (McMurdie & Holmes 2014;
Fig. S2, Supporting information in this study). It is not
the construction of the PERMANOVA test statistic that is
problematic in this respect, but the underlying distance
measure (Anderson & Walsh 2013). Violating the
assumptions about the mean–abundance relationship
may lead to confounded location and dispersion
effects, low detection power of a multivariate effect
expressed in low-variance taxa and difficulties in the
identification of taxa in which an effect is expressed
(Warton et al. 2012). GLMs properly model the mean–
variance relationships of ecological counts, and they are
clearly preferred over methods that rely on distance
measures.
Conclusions
Our results show that host relocation and warming
strongly influence the diversity, structure and possible
functions of the foliar fungal endophytic microbiome.
The relocation of trees to higher latitudes may result in
a release from pathogens. This effect may be compro-
mised by future warming at high latitudes, as this is
predicted to increase pathogen abundances. The climate
change reactions of the foliar fungal endophytic microb-
iome may largely depend on its ability to codisperse
with the hosts. To understand these reactions, more
data are needed about the biogeography and dispersal
abilities of foliar fungal endophytes.
The phyllosphere fungal communities are strongly
influenced by the interaction of environment and host
genetic properties. Similar interactions between host
identity and the environment are increasingly discov-
ered in model systems, for example in the gut microbio-
mes of mammals (Benson et al. 2010; Spor et al. 2011),
or in the root-associated prokaryotic microbiomes of
Arabidopsis (Bulgarelli et al. 2012; Lundberg et al. 2012).
Here, we show that environment–host genetic interac-
tions are also important in the assembly of the fungal
microbiome of trees. This suggests that host-associated
complex microbiomes are formed along the same eco-
logical principles in both bacterial and eukaryotic
domains of life and general assembly principles may
apply over different taxonomic domains.
Acknowledgements
We are grateful for the comments of Ingo Ebersberger,
Leho Tedersoo and three anonymous reviewers on a
© 2014 John Wiley & Sons Ltd
previous version of the manuscript. We are also grateful
for feedback from Axios Review during the review pro-
cess. Balsam poplar genotypes used in the current study
are part of a much larger collection of Agriculture and
Agri-Food Canada’s AgCanBaP collection. We are grate-
ful for the support of Agroforestry Development Centre,
Indian Head, Canada, for assistance in sampling of
leaves in the Indian Head common garden. The work
was funded by the research funding programme Lan-
des-Offensive zur Entwicklung Wissenschaftlich-Oko- €
nomischer Exzellenz (LOEWE) of Hesse’s Ministry of
Higher Education, Research, and the Arts, a US National
Science Foundation Plant Genome Research Program
grant (DBI-0701911 and 1137001) and a grant from
Alaska EPSCoR (NSF award #EPS-0701898 and the state
of Alaska). The findings and conclusions in this article
are those of the authors and do not necessarily represent
the views of the U.S. Fish and Wildlife Service.
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M.B., I.S., M.O. and A.L.R. designed research; M.B.,
L.B., J.O. and A.L.R. performed research; R.B.O., M.P.
and P.T. contributed new reagents or analytic tools;
M.B. analysed data; and M.B., I.S. and P.T. wrote the
manuscript.
Data accessibility
Illumina MiSeq sequence data were deposited in the
European Nucleotide Archive (Accession numbers
PRJEB7842, ERP008816). All sequences that passed qual-
ity filtering, the centroid sequences of all OTUs, the cen-
troid sequences of all fungal OTUs, the fungal OTU
abundance tables, the results of the functional assign-
ments and the R script for all ecological analyses are
provided as online Supporting Information.
Supporting information
Additional supporting information may be found in the online ver-
sion of this article.
Fig. S1 Generalized additive model of OTU incidences against
logarithmic abundances.
Fig. S2 The variance in OTU sequence counts strongly
increases with the mean. This type of mean-variance relation-
ship is typical of ecological abundance data.
Fig. S3 The abundance distribution of DNA reads assigned to
the 2,022 fungal OTUs.
Fig. S4 The abundance of modeled poplar-specific Mycosphae-
rella OTU counts in the south, and the north under natural and
heated conditions.
Fig. S5 Generalized additive model of 95% similarity OTU inci-
dences against logarithmic abundances.
Fig. S6 Non-metric multidimensional scaling of the poplar
hosts in two dimensions according to the 95% similarity OTUs.
Fig. S7 The effects of range shift and atmospheric warming on
95% similarity OTUs are modified by the phylogeographic his-
tory of the host.
Table S1 Poplar genotypes grown in the southern (Indian
Head, Saskatchewan, Canada) and northern (Fairbanks, Alaska,
USA) common gardens.
Table S2 Variation in the composition of the phyllosphere fun-
gal microbiome explained by PERMANOVA (Bray-Curtis dis-
tance, 999 permutations).
Table S3 Tukey HSD tests for the GLM predictions of 94 phyll-
osphere OTUs that were statistically significantly effected by
the south – north relocation, and the warming in the north.
Table S4 Taxonomic and functional assignment of the OTUs
defined by the southern and northern natural, and northern
warmed conditions.
Table S5 Variation in 95% similarity OTU communities
explained by multispecies generalized linear models (ANOVA,
likelihood-ratio test, 100 bootstraps).
Appendix S1. Sequences passing filtering, OTU representative
sequences, OTU abundance tables, functional assignments, R
script.
© 2014 John Wiley & Sons Ltd