Genome Skimming Reveals Widespread Hybridization in a Neotropical Flowering Plant Radiation

Abstract

The tropics hold at least an order of magnitude greater plant diversity than the temperate zone, yet the reasons for this difference are still subject to debate. Much of tropical plant diversity is in highly speciose genera and understanding the drivers of such high species richness will help solve the tropical diversity enigma. Hybridization has recently been shown to underlie many adaptive radiations, but its role in the evolution of speciose tropical plant genera has received little attention. Here, we address this topic in the hyperdiverse Bromeliaceae genus Vriesea using genome skimming data covering the three genomic compartments. We find evidence for hybridization in ca. 11% of the species in our dataset, both within the genus and between Vriesea and other genera, which is commensurate with hybridization underlying the hyperdiversity of Vriesea, and potentially other genera in Tillandsioideae. While additional genomic research will be needed to further clarify the contribution of hybridization to the rapid diversification of Vriesea, our study provides an important first data point suggesting its importance to the evolution of tropical plant diversity.

Full text

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ORIGINAL RESEARCH
published: 28 May 2021
doi: 10.3389/fevo.2021.668281
Edited by:
Danilo M. Neves,
Federal University of Minas Gerais,
Brazil
Reviewed by:
Clarisse Palma-Silva,
State University of Campinas, Brazil
Tim Barraclough,
University of Oxford, United Kingdom
*Correspondence:
Oriane Loiseau
oriane.loiseau@unil.ch
†Deceased
Specialty section:
This article was submitted to
Biogeography and Macroecology,
a section of the journal
Frontiers in Ecology and Evolution
Received: 15 February 2021
Accepted: 30 April 2021
Published: 28 May 2021
Citation:
Loiseau O, Mota Machado T,
Paris M, Koubínová D, Dexter KG,
Versieux LM, Lexer C and Salamin N
(2021) Genome Skimming Reveals
Widespread Hybridization in a
Neotropical Flowering Plant Radiation.
Front. Ecol. Evol. 9:668281.
doi: 10.3389/fevo.2021.668281
Genome Skimming Reveals
Widespread Hybridization in a
Neotropical Flowering Plant
Radiation
Oriane Loiseau1,2*, Talita Mota Machado3, Margot Paris4, Darina Koubínová1,
Kyle G. Dexter2, Leonardo M. Versieux5, Christian Lexer6†and Nicolas Salamin1
1Department of Computational Biology, University of Lausanne, Lausanne, Switzerland, 2School of Geosciences, University
of Edinburgh, Edinburgh, United Kingdom, 3Programa de Pós Graduação em Biologia Vegetal, Universidade Federal
de Minas Gerais, Belo Horizonte, Brazil, 4Department of Biology, Unit Ecology and Evolution, University of Fribourg, Fribourg,
Switzerland, 5Departamento de Botânica e Zoologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte,
Natal, Brazil, 6Department of Botany and Biodiversity Research, University of Vienna, Vienna, Austria
The tropics hold at least an order of magnitude greater plant diversity than the temperate
zone, yet the reasons for this difference are still subject to debate. Much of tropical plant
diversity is in highly speciose genera and understanding the drivers of such high species
richness will help solve the tropical diversity enigma. Hybridization has recently been
shown to underlie many adaptive radiations, but its role in the evolution of speciose
tropical plant genera has received little attention. Here, we address this topic in the
hyperdiverse Bromeliaceae genus Vriesea using genome skimming data covering the
three genomic compartments. We find evidence for hybridization in ca. 11% of the
species in our dataset, both within the genus and between Vriesea and other genera,
which is commensurate with hybridization underlying the hyperdiversity of Vriesea, and
potentially other genera in Tillandsioideae. While additional genomic research will be
needed to further clarify the contribution of hybridization to the rapid diversification of
Vriesea, our study provides an important first data point suggesting its importance to
the evolution of tropical plant diversity.
Keywords: Bromeliaceae, genome skimming, phylogenomics, hybridization, Vriesea
INTRODUCTION
Hybridization, the exchange of genetic material between lineages, is a widespread evolutionary
phenomenon of crucial importance in the evolution of diversity (Marques et al., 2019). Gene flow
between species may have deleterious effects on species cohesion by breaking species boundaries
and causing genetic homogenization. There is however increasing evidence that hybridization can
promote genetic diversity, adaptation and speciation, although the exchange of genetic material
per se might not be sufficient to create new diversity without a key role of natural selection
acting on the relevant parts of the genome to maintain them (Schumer et al., 2018). In particular,
hybridization has been shown to be involved in many adaptive radiations, such as Darwin’s
finches (Grant and Grant, 2019), African rift lakes cichlid fishes (Meier et al., 2017;Irisarri et al.,
2018;Svardal et al., 2020), Heliconius butterflies (Kozak et al., 2021), and Hawaiian silver swords
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Loiseau et al. Widespread Hybridization in Vriesea
(Barrier et al., 1999). In comparison with the low rate of
mutation, hybridization can immediately elevate the standing
genetic variation upon which selection can act (Seehausen, 2013).
This has the potential to accelerate the adaptive divergence of
populations in face of environmental variability, novel habitats
or previously underutilized niches. Elevated rates of speciation
following hybridization can create a positive feedback as the
evolution of many closely related species in one area may in turn
provide new opportunities for hybridization (Seehausen, 2004).
Interspecific gene flow can promote diversification through
the direct generation of a new species via hybrid speciation or
through adaptive introgression, the latter being the transfer of
only parts of the genome which may confer a selective advantage
(Harrison and Larson, 2014). The identification of these
mechanisms has been helped by two important advances in the
field. First, the emergence of the genome scale datasets that are
becoming standard in evolutionary studies provided the raw data
to tackle these questions. Second, the development of statistical
methods that can formally test the presence of introgression
against a null model that involves only incomplete lineage sorting
(ILS; e.g., Joly et al., 2009;Green et al., 2010;Blanco-Pastor
et al., 2012;Blischak et al., 2018). A remaining open question is
the prevalence of hybridization or adaptive introgression in the
context of diversifying lineages (Pfennig, 2021).
Although hybridization has been detected across a wide
range of organisms, it is particularly frequent in plants, and is
acknowledged as one of the main drivers of the evolution of
angiosperms (Soltis and Soltis, 2009). However, little is known
about the contribution of hybridization in the evolution of
diverse tropical clades which make up a large part of plant
diversity. In this study, we focus on the Bromeliaceae, which
is, with ca. 3,500 species, one of the most diverse and iconic
plant families of the Neotropical flora, particularly abundant
in Andean and Atlantic rainforests (Smith and Downs, 1974;
Benzing, 2000;Gouda et al., 2018). The family harbors several
exceptionally diverse young clades, such as the core tillandsioids
and the tank-forming epiphytic bromelioids (Givnish et al.,
2014). The increased rates of diversification which underlie
these rapid radiations may have been spurred by several key
innovations such as the tank habit, CAM photosynthesis or
hummingbird pollination (Givnish et al., 2014;Silvestro et al.,
2014). Hybridization is also thought to have promoted bromeliad
diversity (Palma-Silva et al., 2016) but although both artificial
(Vervaeke et al., 2004;Wagner et al., 2015;de Souza et al., 2017)
and natural hybrids have been reported (e.g., Palma-Silva et al.,
2011), the frequency and exact contribution of this phenomenon
to bromeliad diversity are still to be determined. While some
studies suggest that hybridization generates intraspecific genetic
diversity in isolated populations (Lexer et al., 2016;Neri et al.,
2018;Mota et al., 2019), it may also prevent genetic differentiation
and blur species boundaries (Goetze et al., 2017). To date,
most of the research on hybridization in bromeliads focuses
on the population level and studies with a macroevolutionary
perspective are lacking.
Here, we take a phylogenomic approach to the study of
hybridization in the Tillandsioideae, the largest of the three
subfamilies of bromeliads, with a focus on the diverse genus
Vriesea (231 species, Gouda et al., 2018). Using genome
skimming we provide an unprecedented molecular dataset
for 108 species of Vriesea and 39 species from related
genera. We use phylogenetic inference and coalescent-based
approaches to test for the presence of hybridization during
the evolutionary history of the group. Our results provide
evidence for clear cyto-nuclear discordance in the phylogenetic
history of Tillandisoideae. We further detect signatures of ancient
hybridization at both the inter- and intrageneric level. Our study
suggests that hybridization has occurred repeatedly throughout
the evolutionary history of bromeliads and may have played an
important role in triggering the rapid evolution of this group.
MATERIALS AND METHODS
Genomic Dataset
We used an extended version of the chloroplast genomic
dataset generated with genome skimming by Machado et al.
(2020) containing nuclear, chloroplast and mitochondrial data.
Genome skimming, also referred as low-coverage whole genome
sequencing, allows easy and cost-effective obtention of DNA
sequences for phylogenomic analyses across a wide range of
evolutionary divergences (Dodsworth, 2015). This technique is
particularly effective for the high-depth sequencing of the high-
copy fraction of the genome such as plastids and ribosomal
nuclear DNA (Coissac et al., 2016). It also offers the possibility
to obtain shallow coverage of single copy nuclear DNA, and
thousands of low-depth nuclear markers can be obtained with
a sufficient sequencing effort. Sampling included 147 species of
11 genera in the subfamily Tillandsioideae, plus Ananas comosus
as an outgroup. In total, there were 108 species of Vriesea
s.l., representing c. 46% of the total diversity of the genus.
For the other genera the number and proportion of species
were as follows (species numbers according to Gouda et al.,
2018): Alcantarea (11 species; 26% of the diversity), Goudaea
(2; 100%), Guzmania (4; 1%), Lutheria (2; 50%), Mezobromelia
(1; 20%), Racinaea (1; 78%), Stigmatodon (4; 22%), Tillandsia
(9; 1%), Werauhia (3; 1%), Zizkaea (1; 100%). Protocols of
DNA extraction, library preparation and sequencing, as well as
the bioinformatic pipeline for quality checking of reads, and
SNP calling are described in Machado et al. (2020). Briefly,
library preparation was performed using KAPA LTP library
preparation kits (Roche, Basel, Switzerland) and unique barcodes
for each library were chosen from a set of 60 dual-index
primers designed in Loiseau et al. (2019) to allow multiplexing.
The use of a high number of Illumina dual-indexed primers,
designed with a minimum edit distance of 4 between each
of the 7-bp indexes, reduces the probability of conversion by
sequencing, amplification errors and index hopping (Kircher
et al., 2012;Costello et al., 2018). After sequencing in an
Illumina HiSeq 3000 Genome Analyzer (Illumina, San Diego,
California, United States), quality controlling and read trimming,
pair-end reads (2 ×150 bp) were mapped onto the Tillandsia
adpressiflora Mez pseudo-reference genome built in de la Harpe
et al. (2020) using BOWTIE2 v.2.2.5 (Langmead and Salzberg,
2012). After identification of PCR duplicates, realignment of
reads around indels and base calibration, SNPs were called
using UnifiedGenotyper of GATK v.3.6 (McKenna et al.,
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Loiseau et al. Widespread Hybridization in Vriesea
2010) and the EMIT_ALL_SITES option to recover both
variant and invariant sites. We used Vcftools v.0.1.13 (Danecek
et al., 2011) to filter only high-quality sites called with Q
values higher than 20 and less than 50% missing data. For
phylogenomic inferences invariant sites were filtered out and
separate alignments of SNPs were produced for the nuclear,
chloroplast and mitochondrial genomes.
Phylogenetic Inferences
We performed phylogenetic reconstruction using both
maximum-likelihood and coalescent-based methods. Since
using only variable sites for phylogenetic inference can lead to
incorrect estimation of branch lengths and topology (Leaché
et al., 2015), we employed methods designed for SNP datasets.
First, we used RAxML v.8.2.10 (Stamatakis, 2014) to infer
separate phylogenetic trees for the alignments of chloroplast,
mitochondrial and nuclear SNPs. For each analysis we applied
a GTR +G model of substitution and an ascertainment bias
correction to account for the absence of constant sites in the
alignments. Bootstrap replicates were performed using the
-autoMRE option which executes a maximum of 1,000 bootstrap
replicate searches but stops if support values reach convergence
earlier. Secondly, to account for any potential effects of ILS
in phylogeny estimation, we used SVDquartet (Chifman and
Kubatko, 2014, 2015), a coalescent-based method designed for
SNP data implemented in PAUP∗(Swofford, 2001) to infer the
nuclear phylogenetic tree. SVDquartet is a coalescent method
for inferring phylogenetic relationships based on phylogenetic
invariants which are mathematical functions representing the
expected frequencies of site patterns in an alignment. It first
infers the quartet topology of all subsets of four taxon in the
dataset and then agglomerate them in order to obtain the
species tree with all taxa. Although SVDquartet can also estimate
phylogenetic relationships for non-recombining loci, we did not
run it on our chloroplast or mitochondrial datasets because they
contained <10,000 SNPs and the performance of this method is
highly dependent on the number of SNPs.
Divergence Time Estimates
We temporally calibrated the nuclear phylogenetic tree using
penalized-likelihood implemented in the program treePL v1.0
(Smith and O’meara, 2012). We chose this approach because
joint estimation of the topology and divergence times from SNP
data (e.g., SNAPP, Bryant et al., 2012) was computationally too
intensive to be applied to our large dataset. Given the absence
of fossil data for bromeliads, we used a secondary calibration to
perform the divergence time estimation. We implemented the
age estimate of the crown node of core Tillandsioideae from
Bouchenak-Khelladi et al. (2014) as a calibration for the root node
of our tree, which encompasses the core Tillandsioideae, using
the boundaries of the 95% HPD as minimum and maximum
constraints (7.2417–12.984 My). Although their study is a
monocotyledon-wide analysis, their sampling of Tillandsioideae
is as good as other studies focusing on Bromeliaceae, and their
dating analysis is based on nine fossil calibration points. Their
sampling did not include any member of the tribe Vrieseeae,
and we were unable to calibrate any shallower node in our
tree. We performed cross-validation to estimate the value of
the best-fitting smoothing parameter whose value can vary
between 0 (each branch has its own substitution rate) and 1
(strict clock model).
Hybridization Detection
In order to estimate levels of hybridization across our dataset,
while accounting for ILS, we used the program HyDe v0.4.3
(Blischak et al., 2018), a coalescent method which tests for
hybridization in the presence of ILS using phylogenetic invariants
(Kubatko and Chifman, 2019), similarly to the D statistics
(Patterson et al., 2012). HyDe can detect both recent and
ancient hybridization without the need for an a priori hypothesis,
considers ambiguous sites, and can be applied to large genomic
datasets. For all possible quartets in a dataset comprising one
outgroup population, two parental populations (P2 and P3) and
one hybrid population (P1), HyDe estimates the amount of
admixture using a gamma statistic. Gamma values are comprised
between 0 and 1, where γ=0.5 means 50:50 hybrid and values
close to 0 or 1 indicate a low level of asymmetrical admixture by
introgression. We tested all four-taxon combinations comprising
one outgroup and a triplet of ingroup taxa. For all tests Ananas
comosus was set as the outgroup taxon using the python script
run_hyde.py1. The program applies a conservative Bonferronni
correction and outputs the combinations with a significant signal
for hybridization at a level of <0.05. Unless it applied to
population data, this approach does not determine whether
the significantly introgressed individuals represent the results
of a hybrid speciation event, but suggests at a minimum
that these individuals have genetic material from at least two
different lineages in the phylogeny. We summarized the result
of HyDe by recording the genera of the two parental species
for each significant triplet, and counted for each hybrid the
frequency of the different generic combinations. This allowed us
to evaluate whether the detected hybrids most likely reflected
intra- or intergeneric hybridization. We then constructed a
network diagram using the fonction qgraph from the R package
Qgraph v.1.5 (Epskamp et al., 2012) in order to display the
frequency at which each genus was involved as a parental
species (P2 or P3) for hybrids in other genera across all
significant combinations.
RESULTS
Sequencing
After base calling and filtering, 496,594 high-quality sites were
obtained for the nuclear genome with a coverage depth of 22.2
reads per position and 19.3% missing data. For the chloroplast
and mitochondrial genomes, 68,854 high-quality bases at 53.9X
(5.8% missing data) and 19,403 high quality bases at 11.2X
(18.2% of missing data) were recovered, respectively. From the
SNP calling 116,478 SNPs were obtained for the nuclear, 5,171
SNPs for the chloroplast and 1,069 SNPs for the mitochondrial
genomes. Sites presenting a pattern of a single nucleotide and
an ambiguity code are considered by RaxML as invariant sites
and we excluded them from the alignments, which led to a total
1https://github.com/pblischak/HyDe
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Loiseau et al. Widespread Hybridization in Vriesea
dataset of 103,244 nuclear SNPs, 3,913 chloroplast SNPs, and 970
mitochondrial SNPs.
Phylogenetic Relationships
Intergeneric phylogenetic relationships were well-supported
in all inferred trees. The chloroplast topology is similar to
the one recovered in Machado et al. (2020), with subtribe
Cipuropsidinae (composed of the genera Goudaea, Zizkaea,
Lutheria, and Werauhia) sister to subtribe Vrieseinae (composed
of Vriesea s.str., Stigmatodon, and Alcantarea,Figure 1). Our
study adds new results for the mitochondrial phylogeny,
which has a similar topology to the plastid tree. However,
the nuclear topology resolves the subtribe Cipuropsidinae
as sister to the tribe Tillandsieae (Figure 1). Furthermore,
within the Cipuropsidinae, Lutheria is recovered as sister to
Werauhia in the chloroplast and mitochondrial trees (with
bootstrap support of 100 and 98, respectively, Supplementary
Figures 1, 2), whereas in the nuclear tree, it forms a
clade together with Goudea,Zizkaea and the Cipuropsis-
Mezobromelia complex (with bootstrap support of 98; Figure 1
and Supplementary Figure 3). The genus Goudea, represented
by the two species G. ospinae and G. chrysostachys, is
recovered as monophyletic in the nuclear phylogeny but not
in the chloroplast and mitochondrial trees (Figure 1 and
Supplementary Figures 1–3). The coalescent-based phylogenetic
tree obtained with SVDquartet from the nuclear data has
a similar topology to the phylogenetic tree inferred with
RAxML but an overall lower bootstrap support (Supplementary
Figure 4). It differs, however, in the position of Werauhia,
which is found in a sister position to the tribe Tillandsieae,
and Guzmania, which is recovered nested within Tillandsia
(with low support, Supplementary Figure 4). The monophyletic
Vriesea s. str is characterized by small branch lengths and low
internal support in all phylogenetic trees, yet most of the 12
clades described in Machado et al. (2020), which correspond
to morphological or geographic groups, are recovered in
the chloroplast and nuclear phylogenies, but not in the
mitochondrial phylogeny.
Divergence Times
The best smoothing parameter found by treePL was 0.01,
which allows for substantial substitution rate variation among
lineages. The estimated crown age of the core Tillandsioideae
was 12.98 My. The two main clades, the tribe Tillandsieae +the
Cipuropsidinae and subtribe Vrieseinae have crown ages of 11.24
and 7.66 My, respectively (Figure 2). The genus Vriesea s. str
has a crown age of 6.18 My and following the initial divergence
aV. drepanocarpa, the rest of the group began to diversify
3.91 My ago and most of its diversification occurred during
the Pleistocene.
FIGURE 1 | Phylogenetic incongruence in core Tillandsioideae. Summary cladograms of the chloroplast (left) and nuclear (right) phylogenetic trees. The red dot
indicates the incongruent node associated with the placement of subtribe Cipuropsidinae. Genera indicated as follow (from up to down): V. s. str.,Vriesea sensu
stricto; Sti,Stigmatodon; Alc,Alcantarea; Cip- Mez,Vriesea—Cipuropsis/Mezobromelia complex; Gou,Goudeae; Ziz,Zizkaea; Lut,Lutheria; Wer,Werauhia;
Till,Tillandsia; Guz,Guzmania. Colors indicate genera where hybrids were detected (see Figure 2). Photos by Eric Gouda (Goudaea, Mezobromelia and Zizkaea)
and Talita Mota Machado (Alcantarea, Stigmatodon, Tillandsia and Vriesea s. str.).
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Loiseau et al. Widespread Hybridization in Vriesea
FIGURE 2 | Left: Dated nuclear phylogeny of coreTillandsioids (intrageneric branches not shown). Colors indicate clades were hybrids have been detected. Right:
network diagram of the putative origin of the detected hybrids across all significant triplets identified by HyDe. The width and color of the arrows are proportional to
the frequency at which one genus is detected as a parental lineageaccross all hybrids.
Introgression and Hybridization
The HyDe analyses detected significant introgression in 287
out of the 1,687,425 possible triplets, after the conservative
Bonferroni correction (Supplementary Table 1). For simplicity,
we will call these cases “hybrids,” although we fully recognize that
it is difficult to distinguish between hybrids and introgressive
hybridization from the analyses performed. These 287
combinations contain 17 species identified as potentially of
hybrid origin, which included nine species of Vriesea s.str., the
species V. dubia that belongs to the Vriesea-Cipuropsis clade,
three species of Tillandsia as well as Goudaea chrysostachys,
Guzmania berteroniana, Werauhia gladioliflora, and Zizkaea
tuerckheimii (Table 1). Nine of them represented products
of intergeneric hybridization. The boxplots of gamma values
from all significant tests for each of the 17 hybrids are shown
in Figure 3. Nine of these (representing V. medusae plus the
intergeneric hybrids, except Vriesea roberto-seidelli) are centered
around 0.5, indicating that these taxa are potentially 50:50
hybrids (Figure 3). The remaining hybrids were characterized by
a smaller introgression proportion as indicated by their gamma
values. Six hybrids had median gamma values between 0.5 and
0.7 and two (Vriesea friburgensis and Zizkaea tuerckheimii) had
median values above 0.7 indicating a low level of admixture
(Figure 3). Vriesea billbergioides and Tillandsia juncea were
identified as the parental species of the hybrid Vriesea roberto-
seidelli. All other hybrids had multiple significant triplets
involving different P2 and P3, indicating that the parental species
of most hybrid taxa could not be unambiguously identified
(Table 1 and Supplementary Table 1). When HyDe identifies
multiple significant triplets involving several putative parental
taxa for a given hybrid, it points to ancestral hybridization
which remains detectable in several descendant lineages, even
after considerable evolutionary divergence. Although the
exact origin of the detected hybrids could not be uncovered,
most of them had a signal of admixture biased toward one
combination of parental lineages (Table 1). For example, the
majority of the significant combinations involved a species
from Vriesea s.str and a species from Guzmania in eight
out of the nine intergeneric hybrids (Figure 4), while four
hybrids of Vriesea had both parental species from Vriesea. s.
str in most of their combinations. This prevalence of some
lineages in the significant triplets is reflected in Figure 2
(right), where the thickness and darkness of the arrows is
proportional to the number of time a genera was identified
as a parental lineage for the hybrids in other lineages. This
network diagram clearly depicts Guzmania and Vriesea s.str as
the main putative parents for Vriesea s.str, Tillandsia, Goudaea,
and Werauhia hybrids.
DISCUSSION
Hybridization as a Widespread
Phenomenon in Bromeliaceae
Our study points to widespread hybridization in subfamily
Tillandsioideae, both within and among genera. By recovering
mitochondrial, chloroplast and nuclear DNA in a single
sequencing experiment, we were able to clearly demonstrate
incongruent evolutionary histories between the three genomic
compartments. Discrepancy between organellar and nuclear
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TABLE 1 | Frequency of the possible combinations for the generic origin of the
parental species for each hybrid across all significant triplets identified by HyDe.
Hybrid (P3) Genera of parental species
(P1,P2)
Number of
significant
triplets with
this
combination
Goudaea chrysostachys Vriesea s.str.—Guzmania 21
Guzmania—Tillandsia 3
Guzmania—Vriesea Cipuropsis 2
Guzmania—Zizkaea 2
Guzmania berteroniana Vriesea s.str.—Guzmania 18
Guzmania—Tillandsia 3
Guzmania—Zizkaea 2
Guzmania—Vriesea Cipuropsis 1
Tillandsia geminiflora Vriesea s.str.—Guzmania 23
Guzmania—Tillandsia 3
Guzmania—Zizkaea 2
Guzmania—Vriesea Cipuropsis 1
Tillandsia juncea Vriesea s.str.—Guzmania 27
Guzmania—Tillandsia 3
Guzmania—Vriesea Cipuropsis 3
Guzmania—Zizkaea 3
Tillandsia tenuifolia Vriesea s.str.—Vriesea s.str. 9
Vriesea s.str.—Tillandsia 2
Vriesea s.str.—Werauhia 2
Vriesea s.str.—Goudaea 1
Vriesea s.str.—Guzmania 1
Vriesea botafoguensis Vriesea s.str.—Vriesea s.str. 4
Vriesea s.str.—Goudaea 1
Vriesea s.str.—Tillandsia 1
Vriesea s.str.—Werauhia 1
Vriesea dubia Vriesea s.str.—Vriesea s.str. 3
Vriesea s.str.—Tillandsia 2
Vriesea s.str.—Guzmania 1
Vriesea friburgensis Vriesea s.str.—Vriesea s.str. 2
Vriesea s.str.—Tillandsia 1
Vriesea medusa Tillandsia—Tillandsia 1
Tillandsia—Vriesea Cipuropsis 1
Vriesea s.str.—Tillandsia 1
Vriesea oxapampae Vriesea s.str.—Guzmania 21
Guzmania—Tillandsia 3
Guzmania—Zizkaea 2
Guzmania—Vriesea Cipuropsis 1
Vriesea roberto-seindelli Vriesea s.str.—Tillandsia 1
Vriesea rodigasiana Vriesea s.str.—Guzmania 25
Guzmania—Tillandsia 3
Guzmania—Zizkaea 3
Vriesea saundersi Vriesea s.str.—Vriesea s.str. 2
Vriesea s.str.—Tillandsia 1
Vriesea saxicola Vriesea s.str.—Vriesea s.str. 6
Vriesea s.str.—Tillandsia 4
Vriesea s.str.—Goudaea 2
(Continued
TABLE 1 | Continued
Hybrid (P3) Genera of parental
species (P1,P2)
Number of significant
triplets with this
combination
Vriesea s.str.—Guzmania 2
Vriesea s.str.—Werauhia 2
Vriesea simulans Vriesea s.str.—Guzmania 21
Guzmania—Tillandsia 3
Guzmania—Zizkaea 2
Werauhia gladioliflora Vriesea s.str.—Guzmania 20
Guzmania—Tillandsia 3
Guzmania—Zizkaea 1
Zizkaea tuerckheimii Vriesea s.str.—Vriesea s.str. 2
Vriesea s.str.—Tillandsia 1
phylogenies is common in plants and can be caused either by ILS
or hybridization (Naciri and Linder, 2015). By applying a method
which uses phylogenetic invariants to detect hybridization in the
presence of ILS, we were able to detect a significant level of
admixture in the nuclear genome of 17 of the 148 species included
in our phylogeny. Four of these (Goudaea chrysostachys, Vriesea
dubia, Werauhia gladioliflora, and Zizkaea tuerckheimii) belong
to subtribe Cipuropsidinae and two of these originated from
ancestral hybridization between the Vriesea s.str. and Tillandsieae
lineages (Figure 4). This finding supports the hypothesis that
hybridization is responsible for the incongruent placement of
subtribe Cipuropsidinae which is recovered as either closely
related to the Vrieseinae (in the plastid and mitochondrial
phylogenies) or alternatively to the Tillandsieae (in the nuclear
phylogeny). In bromeliads, a similar example of cyto-nuclear
discordance was found in the genus Puya and was explained
by a mixture of ancient and recent hybridization events (Jabaily
and Sytsma, 2010;Schulte et al., 2010). Our results add to the
growing body of evidence that hybridization may have been a
widespread phenomenon and an important driving force in the
evolution of bromeliads. Indeed, interspecific and intergeneric
artificial crosses of bromeliads are a common horticultural
practice (Vervaeke et al., 2004;de Souza et al., 2017) and several
studies report the permeable nature of species boundaries among
bromeliads (Palma-Silva et al., 2011;Mota et al., 2019). For
example, a study on pre-pollination isolating mechanisms in
an assemblage of 42 sympatric species of bromeliads in Brazil
found that neither microhabitat preferences, flowering phenology
nor pollinators contributed to prezygotic reproductive isolation
(Wendt et al., 2008). Another study looked at post-pollination
barriers among 13 species of bromeliads and found that despite
pollen tube growth inhibition being an important mechanism,
nearly 25% of interspecific and intergeneric crosses lead to
normal pollen tube growth (Matallana et al., 2016). Additionally,
three sympatric species of Tillandsia in Mexico showed overlap
in flowering time, similarity in reproductive organs morphology
and manual crosses among them resulted in high fruit production
and viable seeds (Ramírez-Rosas et al., 2020). There is further
morphological and genetic evidence for natural introgression
or hybrid speciation in several bromeliad genera, including
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Loiseau et al. Widespread Hybridization in Vriesea
Aechmea (Goetze et al., 2017), Alcantarea (Versieux et al., 2012;
Lexer et al., 2016), Fosterella (Paule et al., 2017), Pitcairnia
(Palma-Silva et al., 2011;Mota et al., 2020), Puya (Jabaily and
Sytsma, 2010;Schulte et al., 2010), Tillandsia (Gardner, 1984;
Gonçalves and de Azevêdo-Gonçalves, 2009), and Vriesea (Matos
et al., 2016;Zanella et al., 2016;Neri et al., 2018).
Despite this wealth of evidence for a strong hybridization
potential in bromeliads, population genetic studies generally have
found low levels of interspecific gene flow in extant populations
of bromeliads. For example, in Vriesea s.str., gene flow has been
reported between V. scalaris and V. simplex (Neri et al., 2018) and
between V. carinata and V. incurvata (Zanella et al., 2016) but
all species exhibited high genetic structure, a low proportion of
admixed individuals in the populations (between 8 and 12%) and
low level of introgression. In these cases, the interaction between
multiple reproductive barriers such as different reproductive
systems, variation in floral traits, temporal flowering differences
and low hybrid seed viability may have limited interspecific
crosses and helped to maintain species integrity (Zanella et al.,
2016;Neri et al., 2017). Similar results have been found in
other genera such as Pitcarnia (Palma-Silva et al., 2011;Mota
et al., 2020). However, these studies were done at the population
level and explicitly targeted recent or ongoing gene-flow within
small sympatric populations, whereas we focused on the long-
term evolutionary signature of introgression across the whole
subfamily. Our results indicate that 11% of the species in our
dataset exhibit a signature of ancient hybridization with high
levels of admixture. Therefore, while exchange of genetic material
seems to be restricted to low level of introgression in the extant
populations of bromeliads that have been studied so far, our study
provides evidence that gene flow occurred between the ancestors
of extant lineages of core Tillandsioideae that have considerably
diverged from each other. Whether these events represent cases
of hybrid speciation or introgressive hybridization remains to be
investigated. Nevertheless, our results suggest that hybridization
may have been more frequent in bromeliads than what has been
suggested so far. If this is the case, it could have played an
essential role in promoting speciation and generating diversity
in bromeliads over evolutionary time, similarly to what has
been demonstrated in other evolutionary radiations. In fact,
the emerging view of bromeliad evolution is that the existence
of generally strong, yet occasionally permeable reproductive
barriers maintains species cohesion, while allowing the spreading
of advantageous alleles (Neri et al., 2017). This is in line with the
“speciation with gene flow” concept or the idea of the “porous
genome,” in which some parts of the genome can be easily
introgressed while genes essential to species cohesion are resistant
to gene flow (Wu, 2001). Uniformity of chromosomal numbers in
Tillandsioideae may enhance chromosome rearrangements and
recombination, promoting speciation in the presence of gene flow
(Rieseberg, 2001).
Although our results suggest that hybridization is a
widespread phenomenon in Tillandisoideae, it does not
rule out the existence of ILS as an additional source of
phylogenetic incongruence. In fact, the lower branch support of
the phylogenetic tree inferred using a method which models ILS
(SVDquartet), compared to a method which does not (RAxML),
suggests that ILS is indeed present in the group. Yet, given that
HyDe is based on a theoretical frameworks that considers both
ILS and hybridization, the presence of ILS is unlikely to have
mislead our inference of hybridization. However, we cannot rule
out the possibility that other factors related to data acquisition
impacted our analyses. First, index hopping, an inherent source
of errors linked to sequencing technologies, can potentially lead
to mis-assignment of reads between multiplexed samples. In
this study, we used a set of 60 dual-indexed Illumina adapters
to limit the redundancy of the indexes and reduce this potential
error, as recommended by Costello et al. (2018).Ros-Freixedes
et al. (2018) also showed that index hopping, when present, has
little impact on SNP call accuracy from low-coverage sequence
data, and we are therefore confident that this potential bias has
a limited impact on our analyses. Bias toward reference allele
due to alignment also can have an effect on SNP calling, even
if it was shown to be negligible with genome skimming on
pigs (Ros-Freixedes et al., 2018). To limit this bias, we used the
Tillandsia adpressiflora pseudo-reference genome constructed in
de la Harpe et al. (2020) for mapping our samples. The method
consisted in incorporating specific variation of a T. adpressiflora
individual sequenced at high coverage into the high quality
Ananas comosus genome (Ming et al., 2015). This strategy has
the advantage of improving the global mapping efficiency of our
samples for both reference and alternative alleles, but we cannot
exclude that some of our SNP calls were impacted by biases
linked to the quality and distance of the reference genome. In
addition, because our genome skimming approach favors the
detection of the high copy fraction of the genome, our sequence
data may include duplicated genes or transposable elements. It
is possible that the recovery of alternative paralogs in different
lineages can affect the inference of phylogenetic trees and the
estimation of the proportion of introgression. A Vriesea de novo
genome assembly using long reads sequencing would be very
helpful to detect and filter such multicopy genomic regions.
The method that we used to detect introgression has been
shown through simulations to perform well in identifying
introgressed individuals but may select the wrong parental
species in the case of ancestral hybridization, by favoring
parental species belonging to the sister clade of the true parent
(Kubatko and Chifman, 2019). This is why here we focus
on the identification of the hybrids rather than on the exact
determination of their origin, which could also be affected the
unbalanced taxonomic sampling of our study. Although it is
unclear how the limited sampling outside of Vriesea may have
impacted the detection of hybridization, a deeper sampling
of all Tillandsioid genera would allow to further investigate
intergeneric hybridization and confirm that the repeated signal
for ancestral hybridization between Vriesea and tribe Tillandsieae
is not an artifact due to the lack of intrageneric sampling. Finally,
our results suggest that ancient hybrid speciation has occurred
in core Tillandsioids, but the analysis we performed does not
allow to discriminate between hybrid speciation and introgressive
hybridization. A deeper investigation of the exact origin of the
hybrids detected in this study would require additional analyses
based on whole genome data in order to carry out a proper test of
these two scenarios.
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FIGURE 3 | Histogram of the distribution of the gamma statistics across all significant triplets for each detected species. The dashed line indicate the value for 50/50
hybridization.
Biogeographic History of Tillandsioideae
and Opportunities for Hybridization
Kessous et al. (2020) inferred a broad ancestral area for
the core Tillandsioideae, spreading across the Atlantic Forest
(AF), the Andes and the Chacoan dominion (i.e., the South
American “Dry Diagonal,” Neves et al., 2015). The authors
hypothesized that tectonic and climatic events during the
Miocene (the formation of the Paranean Sea and the Dry
Diagonal) likely caused the vicariance between the Andean
lineages (Tillandsieae +Cipuropsidinae) and the Vrieseinae
in the AF (Kessous et al., 2020). Although the Andean and
Amazonian rainforests are at present separated from the AF
by the drier biomes of the Dry Diagonal, there is considerable
evidence in the literature that these wet forest biomes were
in contact during the Miocene and Pleistocene, allowing for
migration and biotic exchange (Batalha-Filho et al., 2013;
Trujillo-Arias et al., 2017). Our analysis revealed a strong signal
of hybridization between Vriesea s.str. and the tribe Tillandsieae
in nine species, a finding coherent with the biogeographic
history of the group. Indeed, hybridization between Vriesea
s.str. and Guzmania is unlikely to be the result of recent
hybridization given that these two lineages diverged from each
other more than 12 My ago and have their respective centers
of diversity in the Brazilian Atlantic Forest and the Andes.
It is therefore plausible that hybridization occurred between
ancestral populations of different core-tillandsioid genera with
overlapping broad distributions or between populations that had
geographically diverged between the Andes and the Amazon but
were in contact during the Miocene. More recent hybridization
events between Vriesea s.str and the Andean lineages are less
likely but cannot be ruled out given that Vriesea has dispersed
to the Amazonian and Andean regions during the Pleistocene
(a few species have a disjunct distribution spanning the Dry
Diagonal, with others being restricted to the Andes/Amazon
and absent from the AF). In contrast, we did not detect
hybridization between Vriesea and its two most closely genera,
Alcantarea and Stigmatodon, which are also distributed in the
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Loiseau et al. Widespread Hybridization in Vriesea
FIGURE 4 | Most likely ancestral intergeneric hybridization events detected by HyDe. Left: Cladogram of the intergeneric phylogenetic relationships within core
Tillandsioideae. Colored squares show genera where hybrids have been detected. Right: Most frequent combinations for the generic origin of the nine inter-generic
hybrids inferred by HyDe. Numbers below each quartet indicate the number of time the depicted combination was found over the total number of significant triplets
for that hybrid.
AF and have species occuring in sympatry, nor did we find
signal for intrageneric hybridization within these two genera.
This result could by explained either by our limited taxonomic
sampling, insufficient genomic data, limitation of the method
used to detect hybridization, or alternatively by the existence
of strong reproductive barriers preventing interspecific crosses
among Vriesea,Alcantarea and Stigmatodon. Little is known
about Stigmatodon, a recently segregated genus, but studies of
Alcantarea have demonstrated the existence of introgression
among several Alcantarea species, in spite of high population
genetic divergence (Barbará et al., 2009;Lexer et al., 2016).
However, because our phylogenetic approach does not include
intraspecific sampling and uses only a limited part of the genome,
it cannot detect such geographically localized and low level
of interspecific gene flow. Despite a difference in style length
which could act as a prezygotic reproduction barrier, natural
hybridization between Vriesea and Alcantarea cannot be ruled
out given that it is at least experimentally feasible under certain
conditions (e.g., with Alcantarea as the male donor; de Souza
et al., 2017). The main habitat of Alcantarea and Stigmatodon are
inselbergs, considered terrestrial islands due to their ecological
and spatial isolation which reduces the connectivity between
populations (Porembski, 2007). It has been proposed that low
level of introgressive hybridization could contribute to the
maintenance of the genetic diversity of these isolated populations,
thereby balancing the lack of intraspecific gene flow observed
in inselberg bromeliad species (Palma-Silva et al., 2011;Mota
et al., 2019). Thus, considering that Alcantarea, Stigmatodon
and Vriesea are often found in sympatry in the Atlantic Forest
inselbergs, hybridization between them seems at least plausible.
Further investigation is required to elucidate whether or not gene
flow between these lineages occurred at some point during their
evolutionary history.
Bromeliads Phylogenomics
Bromeliaceae are known for their low rate of molecular evolution
compared to other Poales (Gaut et al., 1992;Smith and
Donoghue, 2008), resulting in a lack of resolution in species-
level phylogenies reconstructed from a small number of plastid
and sometimes nuclear markers (e.g., Sass and Specht, 2010;
Versieux et al., 2012;Goetze et al., 2016). In this study, we aimed
at circumventing this limitation by using genome skimming,
a method which allows the obtention of a large amount of
DNA from different genomic compartments without the need
for upstream marker development. We obtained thousands of
SNPs from the chloroplast, mitochondrial and nuclear genomes
for nearly 150 species of Tillandsioideae, representing the largest
genomic dataset for bromeliads to date. Using these data to infer
a phylogeny, we were able to confidently resolve intergeneric
relationships, indicating that the method is well-adapted for
intermediate levels of divergence in bromeliads. However, within
genera, branch support values ranged from high (Alcantarea)
to intermediate (the Stigmatodon clade), to very low (Vriesea
s.str.). This lack of power is particularly critical in our chloroplast
dataset where only 8.3% of sites are variable despite a high
sequencing depth, 53.9x on average, obtained with our genome
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Loiseau et al. Widespread Hybridization in Vriesea
skimming method. Chloroplast genes have been the most
common markers used for phylogenetics analyses in bromeliads
so far, but our results suggest that even the full plastome would be
unlikely to contain enough information to resolve phylogenetic
relationships within Vriesea.Simpson et al. (2017) faced a similar
problem using genome skimming approach to obtain plastid
genomes for the phylogenomic analysis of a large clade of
Boraginaceae. Coissac et al. (2016) suggest that entire plastid
genomes obtained with genome skimming would not be enough
for “difficult” groups, and that the obtention of hundreds of
nuclear loci is likely to be required. They suggested that shallow-
pass nuclear loci obtained with genome skimming could provide
enough phylogenetic informative data in such cases. Despite
considerable genomic data in our study, we obtained low support
within the focal clade Vriesea s.str. This is probably due to (1)
the substantial amount of missing data (c. 22%) in the alignment
of nuclear SNPs, and (2) the fact that only 40% of the nuclear
SNPs are parsimony-informative. Indeed, even though 48% of
our detected high-quality nuclear sites are polymorphic, SNPs
were found mainly at low frequencies (median frequency of 5.9%)
and were rarely informative. Hence, while being advantageous
in term of cost and time, genome skimming is probably not the
most efficient approach for shallow-scale phylogenetic studies
in Bromeliaceae, particularly in young and speciose clades such
as Vriesea, which diversified into c. 300 species in less than 8
My. Obtaining more informative nuclear SNPs, would require
increasing the sequencing depth of each sample and therefore
dramatically increasing the cost for large scale phylogenomic
studies. For these reasons, we argue that more specific approaches
such as target-capture sequencing of low-copy nuclear genes
could be the way forward to obtain better-resolved phylogenies of
large bromeliad genera such as Guzmania,Tillandsia or Vriesea.
Even if the development of such target-capture kits is complex
and challenging (de La Harpe et al., 2019;Andermann et al.,
2020), it would benefit a wide research community working on
many fascinating groups of bromeliads.
CONCLUSION
By generating an unprecedented genomic dataset for the
largest sub-family of Bromeliaceae, our study sheds lights
on the evolutionary history of an important floristic
component of Neotropical rainforests. We pinpointed the
incongruence of the nuclear phylogeny with the chloroplast and
mitochondrial phylogenetic trees regarding the position of the
subtribe Cipuropsidinae.
Our finding of 17 substantially introgressed taxa, potentially
the products of hybrid speciation, in our dataset of 116,478
nuclear SNPs for 148 species of Tillandsioideae suggest that
hybridization is a plausible explanation for this incongruence.
Furthermore, the signal for hybridization between the
ancestral lineages of Vriesea and tribe Tillandsieae suggests
that hybridization may be widespread phenomenon in core
tillandsioids, and to a greater extent, in bromeliads. Thus, our
study adds to the growing body of evidence that hybridization
is ubiquitous across this diverse Neotropical plant family and
may have fueled the diversification of the most diverse clades
of Bromeliaceae such as the core Tillandsioideae and core
Bromelioideae. Future genomic studies with deeper sequencing
across a wide taxonomic range in bromeliads would likely
reveal more hybridization and help to tell apart the respective
contribution of hybrid speciation and adaptive introgression to
the evolution of bromeliad diversity. Further joint investigation
of hybridization at the micro- and macro- evolutionary level
will be necessary to clarify the exact mechanisms through
which it may have promoted genetic diversity, adaptation and
speciation, and ultimately contributed to the adaptive radiation
and ecological success of bromeliads.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories
and accession number(s) can be found below: NCBI
(accession: PRJNA554504).
AUTHOR CONTRIBUTIONS
NS and CL designed the study. TM performed fieldwork and
sample collection. MP led the sequencing experiments and post
sequencing bioinformatics. TM and MP did the labwork. DK
and OL did phylogenetic analyses. OL did hybridization test
and led the writing with significant contributions from all co-
authors. All authors commented and agreed on the last version
of the manuscript.
FUNDING
This work was funded by the Sinergia grant (CRSII3-147630)
from the Swiss National Science Foundation to NS and CL
as well as funding from the University of Lausanne. TM
was supported by Ph.D. fellowship grants from CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior) and CNPq [Conselho Nacional de Desenvolvimento
Científico e Tecnológico—CNPq-SWE (205660/2014-
2)/CNPq (142354/2016-3)]. LV received funding from the
CNPq (455510/2014-8 and 303794/2019-4). Computational
resources were supplied by the project “e-Infrastruktura
CZ” (e-INFRA LM2018140) provided within the program
Projects of Large Research, Development and Innovations
Infrastructures, and by the computing infrastructure (DCSR) of
the University of Lausanne.
ACKNOWLEDGMENTS
The authors thank the Botanical Gardens of the University of
Vienna (WU-HBV), Botanical Garden of Rio de Janeiro (RBvb),
Marie Selby Botanical Garden (SEL), and Jardin des Serres
d’Auteuil (P) for providing plant material, ICMBIO, and IEF-MG
for collection permits. TM thanks all the amazing people who
collaborated in the fieldwork and other stages of this work.
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Loiseau et al. Widespread Hybridization in Vriesea
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fevo.2021.
668281/full#supplementary-material
Supplementary Figure 1 | Phylogenetic tree of core Tillandsioideae inferred with
RaxML using the concatenated mitochondrial SNPs.
Supplementary Figure 2 | Phylogenetic tree of core Tillandsioideae inferred with
RaxML using the concatenated chloroplast SNPs.
Supplementary Figure 3 | Phylogenetic tree of core Tillandsioideae inferred with
RaxML using the concatenated nuclear SNPs.
Supplementary Figure 4 | Phylogenetic tree of core Tillandsioideae inferred with
SVDquartet using the concatenated nuclear SNPs.
Supplementary Table 1 | List of all significant triplets identified by HyDe.
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Conflict of Interest: The authors declare that the research was conducted in the
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