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Research paper
Causes of heterozygosity excess: The case of Mexican populations of
Populus tremuloides
Javier Hern
andez-Velasco
a
,
b
, Jos
e Ciro Hern
andez-Díaz
c
,
Sergio Leonel Simental-Rodríguez
a
, Juan P. Jaramillo-Correa
d
, David S. Gernandt
e
,
Jos
e Jesús Vargas-Hern
andez
f
, Ilga Porth
g
, Roos Goessen
g
,
M. Socorro Gonz
alez-Elizondo
h
, Matthias Fladung
i
, Cuauht
emoc S
aenz-Romero
j
,
Jos
e Guadalupe Martínez-
Avalos
k
, Artemio Carrillo-Parra
c
, Eduardo Mendoza-Maya
a
,
Arnulfo Blanco-García
l
, Christian Wehenkel
c
,
*
a
Programa Institucional de Doctorado en Ciencias Agropecuarias y Forestales, Universidad Ju
arez del Estado de Durango, Constituci
on 404 sur. Zona Centro,
C.P. 34000, Durango, Mexico
b
Universidad Intercultural de Baja California (UIBC), San Quintín, Baja California, C.P. 22930, Mexico
c
Instituto de Silvicultura e Industria de la Madera, Universidad Ju
arez del Estado de Durango, Constituci
on 404 sur. Zona Centro, C.P. 34000, Durango,
Mexico
d
Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Aut
onoma de M
exico, 04510, CDMX, Mexico
e
Departamento de Bot
anica, Instituto de Biología, Universidad Nacional Aut
onoma de M
exico. 3er. Circuito Exterior, Ciudad Universitaria, C.P. 04510,
Coyoac
an, CDMX, Mexico
f
Postgrado en Ciencias Forestales, Colegio de Postgraduados, Montecillo, Texcoco, 56264, Estado de M
exico, Mexico
g
Institute for System and Integrated Biology (IBIS), Universit
e Laval, Charles-Eug
ene-Marchand Pavilion, 1030 Avenue de la M
edecine, Quebec City, G1V
0A6, Qu
ebec, Canada
h
Instituto Polit
ecnico Nacional, CIIDIR Unidad Durango, Sigma 119 Fracc. 20 de Noviembre II, 34234, Durango, Durango, Mexico
i
Thünen Institute of Forest Genetics, Sieker Landstr. 2, D-22927, Grosshansdorf, Germany
j
Instituto de Investigaciones sobre los Recursos Naturales (INIRENA), Universidad Michoacana de San Nicol
as de Hidalgo (UMSNH). Av. San Juanito
Itzícuaro s/n, Col. Nueva Esperanza, Morelia Michoac
an, 58337, Mexico
k
Instituto de Ecología Aplicada, Universidad Aut
onoma de Tamaulipas, Divisi
on del Golfo 356, Col. Libertad, Ciudad Victoria, 87019, Mexico
l
Facultad de Biología, Universidad Michoacana de San Nicol
as de Hidalgo (UMSNH). Av. J. Mújica s/n, Ciudad Universitaria, Morelia, Michoac
an, C.P. 58030,
Mexico
article info
Article history:
Received 16 April 2024
Received in revised form
25 December 2024
Accepted 27 December 2024
Available online xxx
Keywords:
Quaking aspen
Diploid
Triploid
Asexual reproduction
Adaptation
Deleterious SNPs
abstract
The presence of heterozygous individuals in a population is crucial for maintaining genetic diversity,
which can positively affect fitness and adaptability to environmental changes. While inbreeding gener-
ally reduces the proportion of heterozygous individuals in a population, polyploidy tends to increase the
proportion. North American Populus tremuloides is one of the most widely distributed and ecologically
important tree species in the Northern Hemisphere. However, genetic variation in Mexican populations
of P. tremuloides, including the genetic signatures of their adaptation to a variety of environments, re-
mains largely uncharacterized. The aim of this study was to analyze how inbreeding coefficient (F
IS
) and
ploidy are associated with clonal richness, population cover, climate and soil traits in 91 marginal to
small, isolated populations of this tree species throughout its entire distribution in Mexico. Genetic
variables were determined using 36,810 filtered SNPs derived from genome re-sequencing. We found
that F
IS
was approximately between 0 and e1, indicating an extreme heterozygosity excess. One key
contributor to the observed extreme heterozygosity excess was asexual reproduction, although ploidy
levels cannot explain this excess. Analysis of all neutral SNPs showed that asexual reproduction was
positively correlated with observed heterozygosity (H
o
) but negatively correlated with expected het-
erozygosity (H
e
). Analysis of outlier SNPs also showed that asexual reproduction was positively correlated
*Corresponding author.
E-mail addresses: javier.hernandez@ujed.mx,hernandez.javier@uibc.edu.mx (J. Hern
andez-Velasco), jciroh@ujed.mx (J.C. Hern
andez-Díaz), sergio.simental@ujed.mx
(S.L. Simental-Rodríguez), jaramillo@ecologia.unam.mx (J.P. Jaramillo-Correa), dgernandt@ib.unam.mx (D.S. Gernandt), jjesus.vargashernandez@gmail.com (J.J. Vargas-
Hern
andez), ilga.porth@sbf.ulaval.ca (I. Porth), roosje.goessen.1@ulaval.ca (R. Goessen), herbario_ciidir@yahoo.com.mx (M.S. Gonz
alez-Elizondo), matthias.fladung@
thuenen.de (M. Fladung), csaenzromero@gmail.com (C. S
aenz-Romero), jmartin@uat.edu.mx (J.G. Martínez-
Avalos), tecnologia.madera.fcf@gmail.com (A. Carrillo-Parra),
eduardo.mendoza@ujed.mx (E. Mendoza-Maya), arnulfo.blanco@umich.mx (A. Blanco-García), wehenkel@ujed.mx (C. Wehenkel).
Peer review under the responsibility of Editorial Office of Plant Diversity.
Contents lists available at ScienceDirect
Plant Diversity
journal homepage: http://www.keaipublishing.com/en/journals/plant-diversity/
http://journal.kib.ac.cn
https://doi.org/10.1016/j.pld.2024.12.006
2468-2659/Copyright ©2024 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This
is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Plant Diversity xxx (xxxx) xxx
Please cite this article as: J. Hern
andez-Velasco, J.C. Hern
andez-Díaz, S.L. Simental-Rodríguez et al., Causes of heterozygosity excess: The case of
Mexican populations of Populus tremuloides, Plant Diversity, https://doi.org/10.1016/j.pld.2024.12.006
with H
o
and negatively correlated with H
e
, although this latter correlation was not significant. These
findings support the presence of a Meselson effect.
Copyright ©2024 Kunming Institute of Botany, Chinese Academy of Sciences. Publishing services by
Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
The presence of heterozygous individuals in a population is
crucial for maintaining genetic diversity, which can positively affect
fitness and adaptability to environmental changes (Reed and
Frankham, 2003;Kremer et al., 2012). Heterozygosity excess in a
population leads to higher genetic diversity, which may improve
the population's ability to adapt to changing environmental con-
ditions due to the presence of a reservoir of different alleles that
may prove favourable under different selection pressures. However,
in essentially all naturally outbreeding species heterozygosity
excess can also have negative effects if accompanied by inbreeding
depression, in which deleterious recessive alleles are more likely to
accumulate and be expressed in closely related individuals. This can
lead to reduced fitness (Wright, 1977;Charlesworth and
Charlesworth, 1987;Thornhill, 1993;Frankham, 1995a;Falconer
and Mackay, 1996), lower reproductive success, a general popula-
tion decline (Balloux et al., 2004) and an increased risk of extinction
(Frankel and Soul
e, 1981;Frankham, 1995b;Newman and Pilson,
1997).
Inbreeding usually leads to a reduction in the proportion of
heterozygous individuals in a population, as it increases the prob-
ability that two alleles in an individual come from the same
ancestor and are thus identical by descent, which is referred to as
the inbreeding coefficient (F)(Wright, 1969;Crow and Kimura,
1970). Therefore, the inbreeding coefficient of a population (F
IS
)is
a useful parameter for analyzing various aspects of the evolution of
plant mating systems, e.g., the relationship between inbreeding
history and certain traits, such as self-fertilization capacity or
probability (Schultz and Willis, 1995). F
IS
is highly dependent on the
rate of asexual reproduction within a population (Stoeckel et al.,
2006). For instance, asexuality limits the segregation of alleles,
conserves ancestral heterozygosity through generations, and in-
creases heterozygosity, since alleles of the same gene locus may
independently accumulate mutations over generations, in a phe-
nomenon also known as the Meselson effect (Judson and Normark,
1996;Stoeckel and Masson, 2014). The mutations only accumulate
if they are (almost) neutral and there is no overdominance (i.e.,
there is an additive effect of the mutations on the phenotype). In
addition, progressive or balanced selection can promote the over-
lapping of heterogeneous strains/clones (Haigh and Smith, 1974;
Pavlidis and Alachiotis, 2017).
In general, polyploid individuals and populations retain a
higher degree of heterozygosity and exhibit less inbreeding
depression than their diploid ancestors and can therefore tolerate
a higher degree of self-fertilization (Soltis and Soltis, 2000).
Polyploidy is frequent in angiosperms, occurring in more than 50%
of species (Weiss-Schneeweiss et al., 2013). Indeed, it is believed
that environmental fluctuations can lead to adaptive responses via
the formation of polyploids (Van de Peer et al., 2017), especially in
connection with stress (Van de Peer et al., 2021). Polyploidy can
improve tolerance to abiotic and biotic stress factors and boost
disease resistance, which can have positive effects on plant
growth and net production (Greer et al., 2018;Blonder et al., 2020;
Tossi et al., 2022). The high levels of genetic diversity in polyploids
can help individuals and populations to colonize regions and
persist under strong climate variation (Dynesius and Jansson,
2000).
Populus tremuloides Michx. (quaking aspen) is one of the most
widely distributed and ecologically important tree species in the
Northern Hemisphere (Wang et al., 2016). The species distribution
ranges from Alaska through Canada and the United States to central
Mexico, covering a wide range of ecosystems and elevations (Little,
1971;Perala, 1990). Quaking aspen is an early successional tree that
is adapted to natural disturbances (e.g., fire and disease); it is highly
tolerant of environmental stress and is capable of colonizing new
areas and rejuvenating existing stands (Swift and Ran, 2012;
Goessen et al., 2022). These capacities are due to the reproductive
flexibility of the species, which can reproduce sexually (through
seeds and pollen suitable for wide-ranging wind dispersal) and
asexually (through root suckers) (Barnes, 1966;Mitton and Grant,
1996), and to ploidy-level variation (Mock et al., 2012;Goessen
et al., 2022). The quaking aspen, together with the European
aspen, has the highest level of intraspecific genetic diversity ever
recorded in a plant species (Cole, 2005;Callahan et al., 2013;Wang
et al., 2016), which also contributes to its great adaptability.
However, the genetic diversity and structure of Mexican pop-
ulations of Populus tremuloides, including the genetic signatures of
their adaptation to a variety of environments, remain largely
uncharacterized (Ouborg et al., 2010;Callahan et al., 2013). Previous
studies used AFLP markers to identify significant large-scale spatial
genetic structure in P. tremuloides (Qui~
nones-P
erez et al., 2014) and
found that P. tremuloides had lower AFLP diversity than other
Mexican tree species, as well as significantly positive associations
between neighbor tree species diversity and genetic diversity
(Simental-Rodríguez et al., 2014). In addition, one study reported
that almost all P. tremuloides individuals in each of seven forest
sample plots in the Mexican Sierra Madre Occidental were genet-
ically different concluding that vegetative reproduction probably
only plays a secondary role in quaking aspen in this region
(Wehenkel et al., 2014). These findings are partially in accordance
with those reported by Goessen et al. (2022), who investigated the
entire Mexican genetic cluster by using single nucleotide poly-
morphism (SNP) markers. These researchers found that: (i) a large
number of clonemates are located in Mexican aspen stands and (ii)
several SNPs are under selection and impacted by temperature and
precipitation variation across the four main genetic clusters in
northeastern North America, northwestern North America, the
western United States and Mexico.
The aim of this study was to analyze how inbreeding coefficient
and ploidy level are associated with clonal richness, climate and
soil traits in 91 marginal to small, isolated populations of Populus
tremuloides throughout its entire distribution in Mexico (i.e., Baja
California, Sierra Madre Oriental, Sierra Madre Occidental, and the
Trans-Mexican Volcanic Belt), by using SNP markers derived from
genotype by sequencing (GBS). We hypothesized that: (i) asexual
reproduction will increase the expected allelic diversity within
populations and will lead to more negative F
IS
, i.e., extreme het-
erozygote excess (Stoeckel and Masson, 2014), and (ii) progressive
selection against deleterious recessive alleles will result in less
negative F
IS
values in adaptive loci (Mitton (1989) in Stoeckel et al.
(2006)). We also hypothesized that the rate of asexual propagation
J. Hern
andez-Velasco, J.C. Hern
andez-Díaz, S.L. Simental-Rodríguez et al. Plant Diversity xxx (xxxx) xxx
2
affects ploidy and that higher ploidy levels tend to occur in more
extreme environmental conditions (Dynesius and Jansson, 2000;
Goessen et al., 2022).
2. Materials and methods
2.1. Study area and sampling
Leaf samples were collected from 809 spatially georeferenced
Populus tremuloides trees in 91 natural populations in Mexico.
These populations were distributed in the Sierra de San Pedro
M
artir (SSPM; Baja California), Sierra Madre Occidental (SMO;
Durango, Chihuahua and Sonora), Sierra Madre Oriental (SMOr;
Coahuila, Nuevo Le
on and Tamaulipas) and the Trans-Mexican
Volcanic Belt (Hidalgo, Michoac
an and Queretaro, in central
Mexico) (Fig. 1). Detailed information about the location of the
sampled stands is provided in Table S1. The population cover (in
hectares) was determined using Google Earth Pro v.6 (http://www.
google.com/earth/index.html [Accessed 10 November 2023]) (see
Table S2 for further details).
2.2. DNA extraction
DNA was extracted from dried leaves obtained in the field. The
Nucleospin 96 Plant II kit (MachereyeNagel, Bethlehem, PA) was
used following the manufacturer's protocol for the centrifugation
process, with modifications regarding the cell lysis step (PL2 buffer
was heated at 65
C for 2 h instead of 30 min). DNA concentrations
were adjusted to 10 ng/
m
l before library preparation and random-
ization of samples.
2.3. Library preparation and sequencing
Libraries were prepared at the «Plateforme d'analyses
g
enomiques, Institut de Biologie Int
egrative et des Syst
emes»(IBIS,
Universit
e Laval, Qu
ebec, Canada). Libraries were sequenced on an
Illumina Novaseq 6000 S4 (1 lane) sequencing system (Centre
d'expertise et de services G
enome Qu
ebec in Montr
eal, Canada),
and 150-bp paired-end were obtained in the FastQ format (as
described in Poland et al. (2012), with some modifications
following Goessen et al. (2022).
2.4. Assembly, quality filtering and SNP calling
DNA was digested using the restriction enzymes PstI, NsiI and
MspI. Stacks v.2.4 was used to identify loci in the data and call ge-
notypes (https://github.com/enormandeau;Catchen et al., 2013;
Rochette et al., 2019). Cutadapt v.1.8.1 was used to remove adapters
from raw sequences (Martin, 2011). The process_radtags module in
Rv.4.2.1 software (R Core Team, 2022) was used to demultiplex the
libraries and perform quality trimming on reads of length 125 bp.
Sequenced reads were aligned to the P. tremuloides reference
genome (v.1.1; “Potrs01b,”~480 Mbp of sequence) available from
the Populus Genome Integrative Explorer website (http://www.
popgenie.org/;Sj€
odin et al., 2009;Sundell et al., 2015).
Chloroplast, mitochondrial and contaminant scaffolds were
removed from the original reference genome. The unwanted scaf-
folds consisted of 214 sequences and 211,552 nucleotides, which
accounted for 0.06% of the genome. Reads were aligned to the
reference genome on the basis of sequence similarity, determined
using the Burrows-Wheeler alignment tool, BWA v.0.7.17 (Li and
Durbin, 2009). SNPs were then called. The gstacks module was
first used to assemble and merge paired-end contigs and call
variant sites in the populations and genotypes in each sample (with
the argument –max-clipped 0.1). The populations module was then
run to filter the data (-p 200, -r 0.01, –vcf, –hwe, –smooth, –fasta-
loci) and export it to variant call format (VCF).
For all subsequent analyses, VCFtools v.0.1.14 (Danecek et al.,
2011) was used to generate a “final”SNP dataset, from which
indels and problematic individuals with >20% missing data were
removed. In addition, SNPs that did not meet the following criteria
were filtered out: (i) minimum allele count (MAC) of 2, (ii) minimal
depth (minDP) of 10, and (iii) missing rate of higher than 15%.
2.5. Ploidy levels in samples
The FastPloidy script was used to detect ploidy variation in in-
dividuals resulting from filtering. FastPloidy, developed by Goessen
et al. (2022), is available at https://github.com/RGoess/Ploidy_
detection/blob/main/FastPloidy.R. The correct identification of
ploidy levels by the FastPloidy script has already been verified (see
also fig. 3 in Goessen et al., 2022) using flow cytometry and
microsatellites on 105 individuals provided by Mock et al., (2012).
These individuals, along with our material, were part of the dataset
used by Goessen et al. (2022).
FastPloidy reads of VCF in Rv.4.2.1 (R Core Team, 2022) allows:
(i) extracting the allele depth for reference and alternative alleles
(minimum depth of 18); (ii) dividing the depth of the reference
allele by the total depth to obtain the allelic ratio of the reference
allele and only retaining heterozygous SNPs (allelic ratio within
0.1e0.9); and (iii) assigning each SNP to a class depending on the
value of the allelic ratio (A: 0.273e0.393, B: 0.44e0.56, C:
0.607e0.727, ‘Other’would refer to a value anywhere outside the
range). Triploids should have a higher allelic ratio, as most SNPs
should be found within class A and C, while diploids should include
most SNPs in class B, and thus, have a lower ratio. A comparison of
FastPloidy and gbs2ploidy was performed to evaluate the efficiency
of each (Gompert and Mock, 2017), and a slight advantage in ac-
curacy for assigning ploidy by the FastPloidy package was observed
(for more details see Goessen et al., 2022). The population cover
(ha) and clonal richness, as well as the relative frequency of diploid
and triploid individuals per population were then calculated in
order to test the univariate association between polyploidy and
bioclimatic and edaphic variables. Since there was probably only
one tetraploid individual in the study (see also in Goessen et al.,
2022), we merged it with the triploids for all analyses.
2.6. Assessment of clonal richness
The pairwise Jaccard similarity index (across all SNPs) (Jaccard,
1908) was used to assign an individual to a clone (genotype), and
a similarity matrix was constructed using the vcf2Jaccard.py script
developed by Rowe (2019) (available at: https://github.com/carol-
rowe666/vcf2Jaccard.git). Consequently, samples from the same
population with similarities 0.99 were assigned as belonging to
the same clone (Blonder et al., 2021). In subsequent analyses to
determine clonal richness within each study site (genotype), the
clonal values were multiplied by a correction term (N∕(N1))
(Gregorius, 1978), where Nrefers to sample size per population.
2.7. Linkage disequilibrium
Natural selection, which supports adaptation to local environ-
mental conditions, may increase the overall linkage disequilibrium
(LD) caused by differences in allele frequencies between pop-
ulations when alleles at different loci are favoured (Wright, 1940;
Ohta, 1982a,1982b;Agapow and Burt, 2001;Slatkin, 2008). LD
analyses were computed with Tomahawk version 0.7.0 (Klarqvist,
2019; available from https://github.com/mklarqvist/tomahawk).
The LD metrics (r
2
and D
0
) were calculated across all pairwise
J. Hern
andez-Velasco, J.C. Hern
andez-Díaz, S.L. Simental-Rodríguez et al. Plant Diversity xxx (xxxx) xxx
3
Fig. 1. Spatial distribution maps of (A) relative frequency of ploidy levels, and (B) clones in the 91 Mexican Populus tremuloides populations under study.
J. Hern
andez-Velasco, J.C. Hern
andez-Díaz, S.L. Simental-Rodríguez et al. Plant Diversity xxx (xxxx) xxx
4
combinations of variant sites by using the following criteria: (i) P
(Fisher's exact test/Chi-squared cutoff p-value)¼0.0001 and, (ii) r
2
(Pearson's R-squared minimum cut-off value ¼0.0001).
2.8. Influence of putative adaptation on H
o,
H
e
and F
IS
To examine the influence of selection on F
IS
in the natural
populations of Populus tremuloides, putative outlier loci in the
populations under study were identified using the multivariate
method implemented in the pcadapt v.4.1.0 package (Luu et al.,
2017) and Latent Factor Mixed Models 2.0 (LFMM2)(Caye et al.,
2019) from the LEA package (Gain and François, 2021)ofRv.4.2.1
(R Core Team, 2022).
Simulations showed that the pcadapt package compares
favourably to other genome scanning software (BayeScan,hapflk,
OutFLANK,sNMF), particularly in the presence of admixed in-
dividuals (data not shown). This method involves: (i) identification
of outlier loci in admixed or continuous populations, (ii) determi-
nation of the population structure by principal component analysis
(PCA) (rather than classifying individuals in admixed or continuous
populations), and (iii) identification of SNPs under putative selec-
tion as those that are strongly correlated with population structure.
The following criteria were applied: (i) exclusion of loci with global
MAF <0.05, (ii) an optimal K¼2 (according to a scree plot (Jackson,
1993;Fig. S1) and (iii) identification of outliers for different in-
dividuals by the significance of Mahalanobis distances for each SNP,
using a Benjamini-Hochberg (HB) correction for a false discovery
rate (FDR) of p<0.05 (Thissen et al., 2002).
Fig. 2. Histogram of observed heterozygosity in 809 individuals from the 91 Populus
tremuloides populations under study using the “DartR”package in Rv.4.2.1, with all
filtered SNPs.
Fig. 3. Comparison of expected heterozygosity (H
e
) vs. inbreeding coefficient (F
IS
) with neutral vs. 146 outlier SNPs (excluding deleterious SNPs within outlier SNPs).
J. Hern
andez-Velasco, J.C. Hern
andez-Díaz, S.L. Simental-Rodríguez et al. Plant Diversity xxx (xxxx) xxx
5
Since LFMM2 does not allow missing data, imputation was car-
ried out with the impute (.) command of the Rpackage “LEA”
(Frichot and François, 2015). This imputation method replaces
missing genotypes with predicted genotypes based on the popu-
lation structure inference made in the snmf function of the package
(Gain and François, 2021). We decided to run LFMM2 with two
latent factors (according to a scree plot Fig. S1). The HB correction
was used to adjust false-positive rates. SNPs with a corrected p
value <0.05 were retained.
The results of pcadapt and LFMM2 were processed using VCFtools
to generate two VCF files, one for the putatively neutral SNPs and
the other one for the putatively outlier SNPs. Only SNPs that were
found to be putatively outlier SNPs by both methods and were also
not identified as deleterious SNPs (more details in section 2.9.)
were used further. Each dataset was analyzed separately to calcu-
late values of observed heterozygosity (H
o
), expected heterozy-
gosity (H
e
) and F
IS
in the Rpackage hierfstat (Goudet and Jombart,
2022), and to test univariate associations with bioclimatic and
edaphic variables, population cover and clonal richness. Given that
F
IS
¼1eH
o
/H
e
(Wright, 1969), F
IS
and H
e
are positively correlated
when H
o
is constant.
2.9. Gene annotation and association of deleterious mutations with
heterozygous excess and clonal reproduction
Gene annotation was performed to categorize non-synonymous,
synonymous and deleterious SNPs based on their location and
impact using SnpEff v.5.2c and Sequences Ontology (SO) (http://
www.sequenceontology.org/) for accurately annotating genetic
sequences. The deleterious SNPs included the SO categories, in
particular: (i) missense mutations with moderate impact, (ii) SNPs
that cause the loss or disruption of the start codon of a gene
(start_lost), (iii) SNPs that lead to the formation of a premature stop
codon (stop_gained), (iv) SNPs that have both stop-gained and
splice region effects (stop_gained&splice_region_variants), (v)
SNPs that cause the loss of a stop codon (stop_lost), and finally (vi)
SNPs that have both stop-loss and splice region effects (sto-
p_lost&splice_region_variants). The SNP categories (ii) to (vi) are
classified as high impact (Cingolani et al., 2012). To construct the
input database for this gene annotation, we used the reference
genome of Populus tremuloides, available at https://plantgenie.org/
FTP?dir¼Data%2FPlantGenIE%2FPopulus_tremuloides%2Fv1.1.
By comparing the accumulation of heterozygous deleterious
SNPs due to heterozygous excess and clonal reproduction, these
findings helped to decipher not only the causes but also the con-
sequences of excessive heterozygosity.
2.10. Estimation of edaphic variables
For determination of 25 soil variables, four subsamples of 500 g
of soil at a depth of 0e15 cm were randomly collected in each
population. The subsamples were then mixed to produce a sample
of 2 kg for each population. The techniques used to estimate the
variables are described below. The concentrations of K
þ
(ppm),
Ca
2þ
(ppm), Mg
2þ
(ppm), Na
þ
(ppm), Cu
2þ
(ppm), Fe
3þ
(ppm),
Mn
þ2
(ppm), Zn
2þ
(ppm), texture (relative proportion of sand, silt,
and clay) and pH (CaCl
2
0.01 M) in each soil sample were deter-
mined using the methods described by Castellanos et al. (1999). The
concentration of P (kg/ha) was determined using the method
detailed by Olsen et al. (1954). The relative organic matter content
(%OM) was obtained by the Le
on and Aguilar (1987) method, and
that of nitrates (NO
3
-) (kg/ha) by the Baker (1967) method. Elec-
trical conductivity (CE) (dS/m) was determined according to
V
azquez-Alarc
on and Aguilar-Noh (2020). The hydraulic conduc-
tivity (HC) (cm/h) and saturation percentage (Sat%) were
determined as described by Mualem (1976) and Herbert (1992),
respectively. Finally, the relative proportions (%) of H
þ
,Ca
2þ
,Mg
2þ
,
K
þ
,Na
þ
and other bases (%OB) were obtained by calculating the
cation exchange capacity (meq 100 g soil) (CEC) using the ammo-
nium acetate method (pH 8.5).
2.11. Estimation of bioclimatic variables
The Rehfeldt's (2006) climate model was used to estimate 22
bioclimatic variables in each sampling site. The model is based on
the Thin Plate Spline (TPS) method (Hutchinson, 1991) and has
previously been used for climate-change research in Mexico (S
aenz-
Romero et al., 2010). The estimations of this model are based on the
reports from more than 1700 Mexican weather stations for the
period 1961e1990, estimating the standardized monthly mean
values of minimum and maximum temperatures and precipitation
(Table S1). The geographic coordinates (latitude, longitude and
elevation) of each site were uploaded to a public database (available
at http://forest.moscowfsl.wsu.edu/climate/[accessed 22 October
2023]) to obtain point estimates of the bioclimatic variables.
2.12. Statistical analysis
The non-parametric post-hoc Kruskal Wallis test (Tukey and
Kramer method) (Kruskal and Wallis, 1952;Sachs, 2013) was used
to test for any statistically significant differences in the medians of
H
o
,H
e
and F
IS
between SNP datasets (Table S3). The tests were
performed in Rv.4.2.1 (R Core Team, 2022), using the PMCMR
package (Pohlert, 2014).
Once the population cover, relative frequency of diploids and
triploids, clonal richness, number of heterozygous deleterious
SNPs, bioclimatic and edaphic variables and H
o
,H
e
and F
IS
were
calculated for each sampling site, the degrees of association
(measured by the Spearman's r
s
) and the corresponding p-values
were determined with the fuzzySim package (Barbosa, 2015).
To test the potential multivariate associations between both F
IS
and ploidy level and the bioclimatic and edaphic variables, popu-
lation cover and clonal richness, only the non-collinear variables
and with an absolute value of the nonparametric Spearman cor-
relation coefficient lower than 0.7 were selected for modelling
eight machine learning algorithms (MLM), all including cross-
validation (CV). The modelling methods used were: (i) Random
Forest (rf), (ii) Regularized Random Forest (RRF), (iii) Tree Models
from Genetic Algorithms (evtree), (iv) Multi-Layer Perceptron
(mlpWeightDecay), (v) Bayesian Regularized Neural Networks
(brnn), (vi) Model Averaged Neural Network (avNNet), (vii) Linear
Regression (lm), and (viii) Neural Network (nnet). These methods
were applied using the caret package together with the train
function (Venables and Ripley, 1999;Williams et al., 2015;http://
topepo.github.io/caret/index.html)inRsoftware. The models
were tested by 10-fold cross-validation, with 30 repetitions. The
goodness-of-fit of the regression models was determined by the
mean square error (MSE), root mean square error (RMSE) and the
(pseudo) coefficient of determination (R
2
). Analysis was performed
using the F
IS
values separately for neutral, outlier and all SNPs.
Bioclimatic and edaphic variables, population cover, and clonal
richness were tested regarding their conditional importance (i.e.,
considering all other variables studied) in predicting F
IS
(all SNPs,
outlier SNPs and neutral SNPs) and ploidy levels, considering the
following criteria: (i) the multivariate association between F
IS
and
ploidy levels and the bioclimatic and edaphic variables, population
cover and clonal richness; and (ii) the multivariate association
between F
IS
and ploidy levels and the bioclimatic and edaphic
variables and the population cover. This procedure was performed
using the cforest algorithm in the random forest implementation in
J. Hern
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the party package (Strobl et al., 2009)inRv.4.2.1 (R Core Team,
2022). The packages “ggpubr”(Kassambara, 2023) and “ggplot 2”
(Wickham, 2016) were used in Rv.4.2.1 to display associations
graphically.
3. Results
3.1. SNP filtering, ploidy levels, clonal richness and pattern of
linkage disequilibrium
After sequencing, SNP calling and filtering for quality using
Stacks v.2.4, and script available in Stacks workflow, the raw VCF file
contained 1,556,064 singleton SNPs with an average depth of 16.7
per SNP and 932 individuals. After SNP filtering, 36,810 SNPs and
809 individuals from 91 populations were retained for subsequent
analyses. The histogram of H
o
is shown in Fig. 2.
FastPloidy script identified 718 diploids and 91 triploids. A total
of 293 unique genotypes were identified from among the 809 in-
dividuals (Table S2).
The distribution pattern of linkage disequilibrium (LD) (r
2
)
values between pairs of SNPs (in a total of 36,810 SNPs) ranged from
0.0 to 1.0. The pairwise mean values of r
2
decreased rapidly with
increasing physical distance 10 kbp. The overall mean LD value
across the genome, 0.46, supports the occurrence of selection
(Fig. S2).
3.2. Influence of putative adaptation on H
o,
H
e
and F
IS
Pcadapt and LFMM2 analyses identified 183 potential adaptive
outlier SNPs (optimal K¼2 according to a skew plot; Fig. S1),
excluding 37 of those outliers that were found to be deleterious
(Fig. S4). The potential adaptive outlier SNPs without the detected
deleterious SNPs (146 SNPs) were treated separately from the
remaining (neutral) 36,627 SNPs for estimating genetic diversity
and performing correlations with the environment and with indi-
vidual clonality (Tables S5, S6 and S7). Significance of the associa-
tions between SNPs and environmental factors across the genome
are shown in a Manhattan plot (Fig. S3).
The non-parametric post-hoc Kruskal Wallis test (Tukey and
Kramer method) revealed significant differences between the me-
dians of the 36,627 neutral SNPs and the 146 outlier SNPs for H
o
(p<0.0000001), H
e
(p<0.0000001), except for F
IS
(p¼0.54). All H
e
values using the outlier SNPs were much smaller than those using
the neutral SNPs (Fig. 3).
Spearman's correlation showed that although H
o
and H
e
were
not significantly correlated for neutral SNPs (r
s
¼0.02, p¼0.83), a
strong positive correlation was observed between H
o
and H
e
for the
146 outlier SNPs (r
s
¼0.89, p<0.0000001). There was also a pos-
itive, significant association between F
IS
(both for all neutral SNPs
and the 146 outlier SNPs) and clonal richness in the 91 populations
of P. tremuloides (r
s
¼0.88 and 0.79, p<0.0000001). By contrast,
ploidy level was negatively correlated (not significantly) with
clonal richness (r
s
¼0.10, p¼0.36). When all neutral SNPs were
considered, clonal richness was negatively correlated with H
o
(r
s
¼0.23, p¼0.03) but positively correlated with H
e
(r
s
¼0.87,
p<0.000001). When we analysed only the outlier SNPs, clonal
richness again was negatively correlated with H
o
(r
s
¼0.29,
p¼0.01) and positively correlated with H
e
, however, this positive
correlation was not significant (r
s
¼0.05, p¼0.65).
3.3. Associations of deleterious mutations with heterozygous excess
and clonal reproduction
Out of the total 36,810 filtered SNPs, 28,998 were detected in
gene regions, 3926 in intron regions, and 3886 in intergenic regions
according to the gene annotation. In total, 5697 SNPs were classi-
fied as deleterious mutations (Fig. S4).
As the total heterozygous SNPs per individual increased, the
total heterozygous deleterious SNPs per individual also increased
(r
s
¼0.97, p<0.00000001), but the relative proportion of total
heterozygous SNPs per individual significantly decreased
(r
s
¼0.38, p<0.00000001). At the population level (with H
o
per
population remaining almost constant), there were no significant
correlations between the relative proportion of total heterozygous
deleterious SNPs and either clonal richness or F
IS
. At the population
level, the relative proportion of heterozygous deleterious SNPs in
the total number of heterozygotic SNPs in clones varied from 0.11 to
0.14. This variation was slightly greater than in populations with
higher clonal richness (Fig. 4).
3.4. Associations between F
IS
, clonal richness, polyploidy and
selected environmental variables
By selecting variables classified as non-collinear, according to
the absolute value of the nonparametric Spearman correlation co-
efficient (ǀr
s
ǀ)<0.7, population cover (Table S1), clonal richness
(Table S2), five bioclimatic variables (Table S3) and eighteen
edaphic variables (Table S4) were retained.
Using all filtered SNPs, Spearman's correlation showed statisti-
cally significant associations of H
o
with relative proportion of other
bases in cation exchange capacity (%) in the soil (r
s
¼0.45, p¼
0.000008), mean annual precipitation (mm) (r
s
¼0.45, p¼
0.000008), mean temperature in the warmest month (
C)
(r
s
¼þ0.41, p¼0.0001), degree-days below 0
C (DD0) (r
s
¼þ0.29,
p¼0.0046) and relative frequency of triploids (r
s
¼þ0.60, p<
0.000000001) at the population level (91 populations). Using the
outlier SNPs, significant associations were also found between H
o
and the above-mentioned variables (p<0.05), except for H
o
vs. DD0
(p¼0.44). There were also significant correlations of clonal rich-
ness with phosphorus concentration in the soil (ppm) (r
s
¼0.35,
p¼0.0006), clay proportion in the soil (%) (r
s
¼þ0.31, p¼0.0032)
and winter precipitation (mm) (r
s
¼0.27, p¼0.0087) (Fig. 5). The
relative frequency of triploids was significantly associated with
mean temperature in the warmest month (r
s
¼þ0.29, p¼0.0046),
DD0 (r
s
¼þ0.27, p¼0.0098) (Fig. 5) and winter precipitation
(r
s
¼þ0.26, p¼0.01). However, statistical associations of H
o
, clonal
richness or the relative proportion of triploids with geographic
longitude or latitude were not observed (Fig. 1).
Using the non-collinear bioclimatic and edaphic variables,
population cover and clonal richness, the Regularized Random
Forest (RRF) algorithm proved to be the best for estimating the
spatial pattern of F
IS
(from the dataset with neutral SNPs) in Populus
tremuloides populations, producing a pseudo R
2
of 0.95 and a RMSE
of 0.08 (Table 1). The results obtained with the cforest random
forest algorithm showed that clonal richness was strongly associ-
ated with F
IS
(with 70.6% of all included variables), population cover
(with 11.4%) and phosphorus concentration in the soil (with 7.7%).
Using the 146 outlier SNPs, RRF was the best for estimating F
IS
, with
a pseudo R
2
of 0.65 and RMSE of 0.23) (Table 2). In this case, the
cforest results obtained indicated that the variables most closely
associated with F
IS
were mean temperature of the warmest month
(with 34.6%), relative proportion of Mg in the cation exchange ca-
pacity (11.1%), and Julian date of the first freezing date of autumn
(8.4%). However, including the 37 deleterious SNPs together with
the outlier SNPs resulted in an R
2
of 0.76 and RMSE of 0.19. Here, the
cforest results were very similar to the findings using the neutral
SNPs.
When using the bioclimatic and soil variables and population
cover (i.e., excluding clonal richness), RRF proved to be the best
model for estimating F
IS
(from the dataset with neutral SNPs) in the
J. Hern
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populations, producing a pseudo R
2
of 0.35 and RMSE of 0.30
(Table S7). In this case, the cforest results obtained showed that the
variables most closely associated with F
IS
were population cover
(40.6%), soil phosphorus content (18.5%), and winter precipitation
(12.7%). Using the 146 outlier SNPs, RRF was found to be the best
model for estimating F
IS
, with a pseudo R
2
of 0.28 and RMSE of 0.34)
(Table S8). Here, the cforest results obtained indicated that the
variables most closely correlated with F
IS
were mean temperature
of the warmest month (with 27.6%), relative proportion of Mg in the
cation exchange capacity (14.6%), and Julian date of the first
freezing date of autumn (8.4%). Including the deleterious with the
neutral outlier SNPs together gave an R
2
of 0.38 and RMSE of 0.30.
The Tree Models algorithm in Genetic Algorithms (evtree)was
the best model for estimating the associations between ploidy level
and bioclimatic and edaphic variables, population cover and clonal
richness, yielding a pseudo R
2
of 0.26 and RMSE of 0.24 (Table 3).
Using the outlier SNPs, Regularized Random Forest (RRF) had the
best model for estimating F
IS
, with a pseudo R
2
of 0.30 and RMSE of
0.33. The results obtained with cforest showed that the variables
most closely (and positively) associated with ploidy level were
winter precipitation (39.6%), soil phosphorus content (18.4%) and
relative proportion of Mg
2þ
in CEC (16.8%).
4. Discussion
This study found that the values of the inbreeding coefficient
(F
IS
) in Mexican populations of Populus tremuloides were mostly
negative (Table S2). Comparing F
IS
levels between neutral and
outlier SNPs in 91 populations of P. tremuloides showed that the
median of putative outlier SNPs was not significantly larger (Fig. 3).
It can be assumed that: (i) the negative F
IS
can at first be attributed
to a bottleneck effect in the populations studied, with a single allele
Fig. 4. Relationships between: (A) total heterozygous SNPs and total heterozygous deleterious SNPs per Populus tremuloides individual, (B) total heterozygous SNPs and relative
proportion of total heterozygous deleterious SNPs per P. tremuloides individual, (C) relativeclonal richness (clonal richness per sample) and relative proportion of total heterozygous
deleterious SNPs per P. tremuloides population, and (D) inbreeding coefficient of a population (F
IS
) and relative proportion of total heterozygous deleterious SNPs per P. tremuloides
population; RF ¼relative frequency.
J. Hern
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retained for most loci, while the less common alleles represent
more recent mutations (Cole, 2005); (ii) later processes of genetic
drift led to accumulation of almost always recessive alleles derived
from mutation and absence of recombination in small populations
of trees with no gene flow between individuals of the same pop-
ulation or between populations; and (iii) natural selection forces
Fig. 5. Relationship between: (A) relative proportion of triploid individuals and observed heterozygosity (H
o
)(r
s
¼þ0.60, p<0.000000001), (B) clonal richness and H
o
(r
s
¼0.23,
p¼0.028), (C) relative proportion of other bases in cation exchange capacity (O.B., %) in the soil and H
o
(r
s
¼0.45, p¼0.0000 08), (D) mean annual precipitation (MAP, mm) and H
o
(r
s
¼0.45, p¼0.000008), (E) phosphorus concentration in the soil (ppm) and clonal richness (r
s
¼0.35, p¼0.0006), and (F) mean temperature in the warmest month (MT WM,
C) and relative proportion of triploid individuals (r
s
¼þ0.29, p¼0.005) in 91 Mexican Populus tremuloides populations under study; mean values (black lines) and the 95%
confidence level intervals for predictions (grey area) are based on linear models (LM), using all filtered SNPs.
Table 1
Best-fit models for predicting F
IS
in Populus tremuloides by the influence of various bioclimatic and edaphic variables, population cover and clonal richness, using 36,627neutral
SNPs from 91 populations.
F
IS
vs Bioclimatic, edaphic, Population size and clonal richness variables Algorithm RMSE R
2
MAE
~ map þmtwm þfday þdd0 þwinp þpH þCE þCaCO
3
þOM þDensity þSilt þClay þPþ
Fe þMn þZn þCu þmeq_Mg þmeq_Na þCIC þper_Mg þper_K þper_ob þPop_cover þ
R_clonal
RRF 0.084 0.945 0.055
evtree 0.089 0.938 0.062
rf 0.090 0.937 0.059
avNNet 0.170 0.804 0.121
mlpWeightDecay 0.183 0.771 0.132
brnn 0.260 0.548 0.213
lm 0.347 0.413 0.263
nnet 0.617 0.069 0.495
Note:map ¼Mean annual precipitation (mm); mtwm ¼Mean temperature in the warmest month (
C); fday ¼Julian date of the first freezing date of Autumn; dd0 ¼Degree-
days below 0
C (based on mean monthly temperature); winp ¼Winter precipitation (Nov þDec þJan þFeb) (mm); pH ¼pH; CE ¼Electric conductivity (dSmol); CaCO
3
¼
Calcium carbonate (%); OM ¼Organic material (%); Density ¼Density (gr/cm
3
); Silt ¼Silt (%); Clay ¼Clay (%); P¼Phosphorus (ppm); Fe ¼Iron (ppm); Mn ¼Manganese
(ppm); Zn ¼Zinc (ppm); Cu ¼Copper (ppm); meq_Mg ¼Magnesium milliequivalent; meq_Na ¼Sodium milliequivalent; CIC ¼Cation exchange capacity (meq/100 g soil);
per_Mg ¼Relative proportion of Mg in the cation exchange capacity (%); per_K ¼Relative proportion of K in the cation exchange capacity (%); per_ob ¼Rel. Proportion of
other bases in the cation exchange capacity (CEC; meq/100g soil) (%); Pop_cover ¼Population cover (ha); R_ clonal ¼Clonal richness.
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subsequently acted to eliminate these recessive alleles and pre-
serve rare beneficial alleles (Fig. 3), resulting in highly heterozygous
populations and maintaining negative F
IS
values (see the Meselson
effect; Judson and Normark, 1996;Stoeckel and Masson, 2014).
Genetic drift processes (claim (ii)) are supported by the strong
positive correlation found between the total number of heterozy-
gous SNPs per individual and heterozygous deleterious SNPs per
individual. The negative relationship between the relative propor-
tion of heterozygous deleterious SNPs per individual and the total
number of heterozygous SNPs per individual (Fig. 4) are consistent
with the action of natural selection forces (claim (iii)). Both con-
clusions correspond with the findings of low variation of the minor
relative proportion of heterozygous deleterious SNPs independent
of F
IS
and clonal richness at the population level. These two results
are in agreement if it is assumed that only a certain upper pro-
portion of deleterious SNPs ensures the fitness of the populations
and that only the fittest individuals could be found.
After constructing a population genetics model based on
genotypic states in a finite population (less than 400 individuals)
with mutations, Stoeckel and Masson (2014) concluded that
reproduction by asexuality increased the expected allelic diversity
within populations, as indicated by a more negative F
IS
index,
compared to similar, fully sexual populations. Asexuality fixes
heritability and genetic drift at the genotypic level and preserves
ancestral genetic states against drift, ultimately reducing allelic
identities within individuals.
There is a lack of a significant correlation between H
o
and H
e
for
neutral SNPs, in contrast to the strong positive and significant
correlation between H
o
and H
e
for outlier SNPs. Moreover, H
o
was
nearly always larger than H
e
for neutral and also for outlier SNPs,
and H
e
for outlier SNPs was much smaller than H
e
for neutral SNPs
(Fig. 3). The F
IS
association of the outlier SNPs with the environ-
mental variables studied was higher than those of the neutral SNPs
with these variables. These findings suggest that these outlier SNPs
found in Mexican populations of Populus tremuloides may be pri-
marily subject to balancing selection, in which heterozygous ge-
notypes have a fitness advantage over homozygous genotypes,
resulting in multiple alleles being maintained in the population due
to their adaptive advantages in different environments (Delph and
Kelly, 2014). This contrasts with a whole-genome resequencing
study from Canada and the USA by Wang et al. (2016), in which
purifying and positive selection strongly affected the patterns of
nucleotide polymorphism at linked neutral sites in P. tremuloides,
P. tremula and P. trichocarpa.
By analyzing polymorphic enzyme loci, Jelinski and Cheliak
(1992) also observed a substantial excess of heterozygotes in Pop-
ulus tremuloides (F
IS
of 0.10), attributing this result to the rather
dry climate in the western range of the species, which limits seed
germination, and results in predominantly asexual reproduction
through the formation of adventitious shoots on the roots. Cheliak
and Dancik (1982) also obtained a F
IS
of 0.24 for seven locations in
Alberta, Canada, indicating that asexual propagation could increase
genetic diversity by facilitating the accumulation of mutations in
offspring. By contrast, Latutrie et al. (2016) detected recent bottle-
necks with an excess of heterozygotes in six populations distrib-
uted in the northwestern range of aspen distribution between
Manitoba and Alaska.
Negative F
IS
values have also recently been reported in other
species of the genus Populus, e.g., Populus yunnanensis (F
IS
¼0.65;
Zhou et al., 2020) and Populus laurifolia (F
IS
¼0.11; Wiehle et al.,
2016), according to results based on the presence of high-frequency
gene flow. On the other hand, after performing a mutation drift
equilibrium test, Wu et al. (2020) reported significant heterozy-
gosity excess (F
IS
¼0.50) in Populus wulianensis, probably because
of recently experienced bottleneck effects in the study populations.
Similar results were observed in other forest tree species, e.g.,
Prunus avium L. (Stoeckel et al., 2006) and Tilia cordata Mill.
(Erichsen et al., 2019).
While polyploidy is frequently observed in Populus tremuloides
(Zhu et al., 1998;Mock et al., 2008), we reject the hypothesis that
the ploidy level affected F
IS
(r
s
¼0.07, p¼0.53), which contradicts
the claims made by Krieger and Keller (1998) and Ridout (2000).
This level of ploidy has no significant effect on the ratio between H
o
and H
e
, which is crucial for F
IS
(Wright,1969). Although triploids are
Table 2
Best-fit models for predicting F
IS
obtained from outlier SNPs in Populus tremuloides by the influence of various bioclimatic and edaphic variables, clonal richness and population
coverage, using 146 outlier SNPs (without deleterious SNPs) from 91 populations.
F
IS
vs Bioclimatic, edaphic, Population size and clonal richness variables Algorithm RMSE R
2
MAE
~ map þmtwm þfday þdd0 þwinp þpH þCE þ
CaCO
3
þOM þDensity þSilt þClay þPþFe þMn þZn þCu
þmeq_Mg þmeq_Na þCIC þper_Mg þper_K þper_ob þPop_cover þR_clonal
RRF 0.234 0.652 0.164
rf 0.236 0.653 0.166
evtree 0.263 0.569 0.201
mlpWeightDecay 0.361 0.259 0.300
brnn 0.368 0.235 0.299
avNNet 0.382 0.282 0.277
lm 0.476 0.153 0.354
nnet 0.572 0.130 0.426
Note: Abbreviations used for variables are given in Table 1.
Table 3
Best-fit models for predicting ploidy level in Populus tremuloides by the influence of diverse bioclimatic and edaphic variables, population cover and clonal richness, 91
populations (using all SNPs).
Ploidy level vs bioclimatic and edaphic variables, population cover and clonal richness Algorithm RMSE R
2
MAE
~ map þmtwm þfday þdd0 þwinp þpH þCE þCaCO
3
þOM þ
Density þSilt þClay þPþFe þMn þZn þCu þmeq_Mg þ
meq_Na þCIC þper_Mg þper_K þper_ob þPop_cover þR_clonal
evtree 0.224 0.258 0.150
RRF 0.233 0.161 0.164
rf 0.234 0.137 0.166
nnet 0.235 0.209 0.174
mlpWeightDecay 0.241 0.181 0.156
brnn 0.243 0.217 0.155
lm 0.255 0.153 0.182
avNNet 0.265 0.136 0.181
Note: Abbreviations used for variables are given in Table 1.
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thought to reproduce almost exclusively by asexual methods
(Mable, 2004;Baldwin and Husband, 2013;Chinone et al., 2014)
clonal richness was not significantly lower (r
s
¼0.10, p¼0.36).
A large number of clones was detected in this study (36% of the
individuals). Similar results were reported by Goessen et al. (2022)
for the entire Mexican aspen cluster (not at the population level)
using a large part of our dataset. Our results provide evidence that
asexual (clonal) reproduction is an indicator of extremely negative
F
IS
occurring within the study populations based on the strong
association between these variables (r
s
¼0.79e0.88,
p<0.0000001), as also discussed by Cheliak and Dancik (1982) and
Jelinski and Cheliak (1992). Therefore, it can be inferred that clonal
growth maintains or even increases the levels of heterozygosity and
adaptive capacity by mutation over generations (Judson and
Normark, 1996;Welch and Meselson, 2000;Delmotte et al., 2002).
In addition, we detected a clear influence of bioclimatic and
edaphic variables on the prediction of F
IS
and ploidy level of pu-
tatively neutral and outlier SNPs (Tables 1, 2,S7 and S8). We can
confirm that F
IS
(from the dataset with all SNPs) was higher in
populations with lower soil phosphorus content and less winter
precipitation, but that triploids occurred in locations with higher
winter precipitation. Therefore, our study, which only included 23
environmental variables, did not support the general expectation
that polyploids should display a greater capacity than diploids to
adapt to a wide range of conditions (
Cern
a and Münzbergov
a,
2015). Our results contradict our hypothesis and the findings re-
ported by Goessen et al. (2022), who analyzed Mexican populations
as a single cluster. Thus, our results seem to be more consistent
with those reported by Benson and Einspahr (1967) and Greer et al.
(2018) as triploid individuals have more resource-oriented life
histories, and may have a higher risk of induced mortality in drier
environments (Dixon and De Wald, 2015;Blonder et al., 2021,
2022). Hence, the wide geographic range of the quaking aspen
(Wang et al., 2016) implies that natural populations grow in diverse
environmental conditions. This, in turn, leads to local adaptation
(Savolainen et al., 2007;Neale and Kremer, 2011;Aitken and
Whitlock, 2013). Thus, genotypes that originate from a particular
habitat exhibit greater fitness in that specific habitat than geno-
types from other habitats (Kawecki and Ebert, 2004), which in-
dicates strong local adaptation. This highlights that adaptation
processes rely on the emergence of advantageous mutations, which
are then either fixed or maintained at an intermediate frequency
through natural selection (Peischl and Kirkpatrick, 2012). This has
also been observed in P. tremula, in which Wang et al. (2018)
detected a locus (PtFT2) associated with an adaptive mutation
from the post-glacial recolonization of northern Scandinavia and
suggested that strong genetic drift at the front of the expansion
range caused surfing of the adaptive allele in the newly colonized
regions (Klopfstein et al., 2006;Excoffier and Ray, 2008).
In our study, the H
o
for putative neutral and outlier SNPs, was
positively and significantly correlated with clonal proliferation. This
finding supports the claim of the possible presence of a Meselson
effect, because accumulation of heterozygous loci was observed in
populations with fewer genotypes and in which asexual repro-
duction was more common and, therefore, counteracted the
inbreeding effect in small and isolated populations. However, the
simultaneous reduction in genetic diversity (H
e
) overwhelmed this
positive effect and could probably have an overall negative impact
on the survival of the species in Mexico from a genetic point of view
(Leimu et al., 2006;Aitken and Whitlock, 2013). The Meselson ef-
fect is generally considered a strong indicator of long-term evolu-
tion under strict asexuality (Hartfield, 2016;Brandt et al., 2021);
however, this phenomenon has also been shown to occur in rela-
tively recent lineages, of less than 100,000 years of age (Pellino
et al., 2013). Some researchers have suggested that quaking aspen
clones may be millions of years old and have survived several
glacial cycles (Barnes, 1966;Kemperman and Barnes, 1976;Mitton
and Grant, 1996).
Asexual reproduction in Mexican Populus tremuloides may play a
contributing role in the ability of this species to survive under
extreme ecological conditions (Goessen et al., 2022). For example,
quaking aspen has developed physiological and ecological adap-
tations to survive and proliferate in diverse ecological environ-
ments, sometimes even extreme environments, such as the
mountainous regions of the Sierra Madre Occidental in Mexico and
areas experiencing severe drought (Ding et al., 2017;Rogers et al.,
2020). Ding et al. (2017) considered the Sierra Madre mountain
range in northeastern Mexico one of the few locations where
quaking aspen populations show a moderate to high probability of
surviving multiple glaciations.
5. Conclusions
This study contributes to a better understanding of the survival
and adaptation to changing environmental conditions of natural
clonal plant populations, especially of Populus tremuloides, one of
the most widespread and ecologically important tree species in the
Northern Hemisphere.
However, further research on the Mexican populations of Pop-
ulus tremuloides is required, given the importance of obtaining in-
formation from natural populations located at the border of the
general climatic niche of the species, where projections are not at
all encouraging: (i) because global climate change is expected to
transform the distribution of large numbers of forest trees (Noss,
2001), and (ii) it is estimated that about 26% of the current
geographical distribution will no longer be suitable for quaking
aspen by 2060, and that the loss of habitat will be particularly
marked in the southern distribution range of the species, where the
Mexican populations are distributed (Worrall et al., 2013). It has
been suggested that these Mexican populations have developed
strategies for surviving adverse conditions that are unique in the
plant kingdom, in contrast to findings on populations of the same
species in the southwestern USA (Crouch et al., 2023). It is impor-
tant to continue exploring these adaptive mechanisms, as this
would enable prediction of the impacts of climate change, which
could be helpful for designing effective mitigation strategies.
CRediT authorship contribution statement
Javier Hern
andez-Velasco: Writing ereview &editing, Writing
eoriginal draft, Resources, Formal analysis, Data curation. Jos
e Ciro
Hern
andez-Díaz: Writing ereview &editing, Resources. Sergio
Leonel Simental-Rodríguez: Writing ereview &editing, Re-
sources. Juan P. Jaramillo-Correa: Writing ereview &editing,
Resources. David S. Gernandt: Writing ereview &editing, Re-
sources. Jos
e Jesús Vargas-Hern
andez: Writing ereview &editing,
Resources. Ilga Porth: Writing ereview &editing, Funding
acquisition, Formal analysis, Data curation. Roos Goessen: Writing
ereview &editing, Formal analysis, Data curation. M. Socorro
Gonz
alez-Elizondo: Writing ereview &editing, Resources. Mat-
thias Fladung: Writing ereview &editing. Cuauht
emoc S
aenz-
Romero: Writing ereview &editing, Resources. Jos
e Guadalupe
Martínez-
Avalos: Writing ereview &editing, Resources. Artemio
Carrillo-Parra: Writing ereview &editing, Resources. Eduardo
Mendoza-Maya: Writing ereview &editing, Resources. Arnulfo
Blanco-García: Writing ereview &editing, Resources. Christian
Wehenkel: Writing ereview &editing, Writing eoriginal draft,
Resources, Funding acquisition, Formal analysis, Data curation.
J. Hern
andez-Velasco, J.C. Hern
andez-Díaz, S.L. Simental-Rodríguez et al. Plant Diversity xxx (xxxx) xxx
11
Data availability
Afiltered variant of the SNP file used for the population genomic
analyses has been deposited in the Dryad repository under the
accession doi: XXXXX (available upon acceptance). Scripts used for
the analyses are available at https://github.com/XXXX (available
upon acceptance).
Declaration of competing interest
The authors have no competing interest to declare.
Acknowledgments
We thank the Mexican Consejo Nacional de Humanidades,
Ciencias y Tecnologías (CONAHCYT) for the financial support pro-
vided to the first author to carry out his training in the Institutional
Doctoral Program in Agricultural and Forestry Sciences (PIDCAF-
UJED) with Scholarship No. 334852 and financial support with
agreement number CONACYT-FRQ-2016: 279459 for the project
“Genome-wide scans for detecting adaptation to climate and soil in
Populus tremuloides as the most widely distributed tree species in
North Am
erica”. Dr. Jesús M. Olivas-García assisted in the sampling
in the state of Chihuahua, Mexico, and Katrin Groppe, Thünen
Institute of Forest Genetics, Germany, provided excellent lab work.
The Emerging Leaders of the Americas Program (ELAP) of the
Government of Canada awarded a scholarship and the Institute of
Integrative and Systems Biology (IBIS) of Laval University allowed
the use of its campus and contributed to the training of the first
author.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.pld.2024.12.006.
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