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\begin{document}

\title{Documenting homoploid hybrid speciation}



\author[1]{Zhiqin Long}%
\author[1]{Loren Rieseberg}%
\affil[1]{The University of British Columbia}%


\vspace{-1em}



  
  \date{June 13, 2024}


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\selectlanguage{english}
\begin{abstract}
Homoploid hybrid speciation is challenging to document because
hybridization can lead to outcomes other than speciation. Thus, some
authors have argued that establishment of homoploid hybrid speciation
should include evidence that reproductive barriers isolating the hybrid
neo-species from its parental species were derived from hybridization.
While this criterion is difficult to satisfy, several recent papers have
successfully employed a common pipeline to identify candidate genes
underlying such barriers and (in one case) to validate their function.
We describe this pipeline, its application to several plant and animal
species, and what we have learned about homoploid hybrid speciation as a
consequence. We argue that----given the ubiquity of admixture and the
polygenic basis of reproductive isolation----homoploid hybrid speciation
could be much more common and more protracted than suggested by earlier
conceptual arguments and theoretical studies.%
\end{abstract}%



\sloppy


\textbf{Title:} Documenting Homoploid Hybrid Speciation

\textbf{Authors:} Zhiqin Long, Loren H. Rieseberg

\textbf{Affiliations} :

Department of Botany and Biodiversity Research Centre, University of
British Columbia, Vancouver, BC, Canada

\textbf{Running Title} : Documenting homoploid hybrid speciation

\textbf{Correspondence} : Loren H. Rieseberg, Department of Botany and
Biodiversity Research Centre, University of British Columbia, Vancouver,
BC, Canada

Email: lriesebe@mail.ubc.ca

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_

\textbf{Abstract}

Homoploid hybrid speciation is challenging to document because
hybridization can lead to outcomes other than speciation. Thus, some
authors have argued that establishment of homoploid hybrid speciation
should include evidence that reproductive barriers isolating the hybrid
neo-species from its parental species were derived from hybridization.
While this criterion is difficult to satisfy, several recent papers have
successfully employed a common pipeline to identify candidate genes
underlying such barriers and (in one case) to validate their function.
We describe this pipeline, its application to several plant and animal
species, and what we have learned about homoploid hybrid speciation as a
consequence. We argue that----given the ubiquity of admixture and the
polygenic basis of reproductive isolation----homoploid hybrid speciation
could be much more common and more protracted than suggested by earlier
conceptual arguments and theoretical studies.

\textbf{Keywords:}

Hybrid speciation, genomics, introgression, phylogenomics, positively
selected genes

\textbf{Introduction}

Speciation is often defined as a process in which one species splits
into two. However, new species can also form as a result of
hybridization between different species (Abbott, 1992; Rieseberg, 1997;
Mallet,~2007; Soltis \& Soltis, 2010). This occurs most commonly when
hybridization is accompanied by whole genome duplication (i.e.,
allopolyploid speciation). The genome duplication provides partial
reproductive isolation between the new allopolyploid lineage and its
parental species, which eases establishment (Baack et al., 2015). Less
commonly, a new hybrid lineage establishes and becomes reproductively
independent without a change in ploidy level (i.e., homoploid hybrid
speciation). The ecological and evolutionary conditions necessary for
homoploid hybrid speciation are more restrictive than for allopolyploidy
because of the lack of a universal mechanism for creating reproductive
isolation between the hybrid lineages and its parental species (Buerkle
et al., 2000, 2003; Schumer et al., 2015; Comeault, 2018; Blanckaert et
al., 2018, 2023).

Allopolyploidy is also easier to document than homoploid hybrid
speciation because hybridization and whole genome duplication are
readily detected from cytological and genomic data. Thus, the prevalence
of allopolyploidy is fairly well established (Wood et al., 2009; Mayrose
et al., 2011; Barker et al., 2016). In contrast, the frequency of
homoploid hybrid speciation remains unclear, mainly because
hybridization can lead to outcomes other than speciation, including
maladaptive, neutral, or adaptive introgression, genetic swamping,
outbreeding depression, stable hybrid zones, reinforcement, etc., and it
can be difficult to distinguish between these different outcomes (Abbott
et al., 2013; Taylor \& Larson, 2019; Bock et al., 2023). Also, these
outcomes are not mutually exclusive. For example, while some
introgressions may be maladaptive and contribute to outbreeding
depression, others from the same donor species could enhance local
adaption and/or reproductive isolation (Schumer et al., 2018).

In an attempt to reduce this uncertainty, Schumer et al. (2014) put
forward three criteria for establishing homoploid hybrid speciation: (1)
reproductive isolation from the parental species; (2) evidence of
previous admixture; and (3) demonstration that reproductive barriers are
derived via hybridization. While criteria one and two are relatively
straightforward to demonstrate, showing that hybridization led to the
formation of reproductive isolation is more challenging. Indeed, Schumer
et al. (2014) argued that only one butterfly species (Maverez et al.
2006) and three sunflower species (Rieseberg et al., 2003, but see Owens
et al., 2023) satisfied all three criteria. Others have worried that the
criteria may be too stringent and that a focus on reproductive isolation
might shift attention away from other important aspects of the process
such as ecological divergence and the generation of evolutionary novelty
(Nieto Feliner et al., 2017; Wang, Xu et al., 2022). Also, in some
instances biological mechanisms of reproductive isolation may not be
critical to homoploid hybrid speciation, such as in the Oxford ragwort
(\emph{Senecio Squalidus} ), where the homoploid hybrid is allopatric
with its parental species (James \& Abbott, 2005; Nieto Feliner et al.,
2017). These concerns have led to the suggestion that homoploid hybrid
species be classified as type 1 when reproductive isolation is a direct
result of hybridization and type 2 when reproductive isolation is a
by-product of other processes (Ottenburghs, 2018).

An important consequence of this debate has been an increased focus on
the potential role of hybridization in the evolution of reproductive
isolation between hybrid lineages and their parental species (Schumer et
al., 2018; Ottenburghs, 2018; Brennan et al., 2019). Partly as a result,
there now are more than a dozen cases of homoploid hybrid speciation in
which the evolution of reproductive barriers has been directly linked to
hybridization (Salazar~et al., 2010; Hermansen et al., 2014; Lukhtanov
et al., 2015; Leducq et al., 2016; Lamichhaney et al., 2018; Brennan et
al., 2019; Masello et al., 2019; Sun et al., 2020; Wang et al., 2021;
Stevison et al., 2022; Wang, Kang et al., 2022; Zou et al., 2022; Wu et
al., 2023; Zhang et al., 2023). More importantly, genomics approaches
have been developed that offer a straightforward means for showing that
the reproductive barriers isolating new homoploid hybrid species from
their parents are indeed derived from hybridization (Sun et al., 2020;
Wang et al., 2021). In this comment, we discuss a particularly effective
strategy for making such connections that was developed by Jianquan Liu
and his colleagues (Figure 1), the application of this method to several
plant and animal species, and what we have learned about homoploid
hybrid speciation as a consequence (Wang et al., 2021; Wang, Kang et
al., 2022; Zou et al., 2022; Wu et al., 2023; Zhang et al., 2023).

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/777211/articles/899245-documenting-homoploid-hybrid-speciation}

\textbf{Figure 1. Framework for documenting homoploid hybridization
speciation employed Wang et al. (2021) for \emph{Ostryopsis
intermedia}(hazel-hornbeam) and subsequently applied to other species.}
Satisfying the three criteria for establishing homoploid hybrid
speciation (Schumer et al., 2013): \textbf{(} A) Demographic modelling
and phylogenomic analyses providing evidence for previous admixture in
hybrid species; (B) Quantifying and inferring reproductive isolation
between the hybrid species and parents, including example from
\emph{Heliconius}butterflies in which the combination of parental wing
patterns in the hybrid led to premating isolation with its parents
(Mav\selectlanguage{ngerman}árez et al. 2006); and (C) Identifying positively selected genes
(PSGs) associated with reproductive barriers, determining the ancestry
of the candidate genes ,and performing functional validation of these
genes; (D) Applying this framework to other four examples of homoploid
hybrids, including\emph{Carpinus cordata} (heartleaf hornbeam; a species
from sect.\emph{Distegocarpus} ), \emph{Ursus thibetanus} (Asiatic black
bear),\emph{Rhinopithecus brelichi} (gray snub-nosed monkey) and
\emph{Macaca fascicularis} (long-tailed macaque; a species from
the\emph{fascicularis} group). Grey arrows show geographic location of
homoploid hybrid species.

\textbf{A framework for documenting homoploid hybrid speciation}

This strategy was initially applied to the documentation of the
homoploid hybrid origin of \emph{Ostryopsis intermedia} (Wang et al.,
2021; also see commentary by Owens, 2021). \emph{Ostryopsis} is a member
of the birch family (Betulaceae) and includes three species of shrubs
--\emph{O. davidiana} , \emph{O. intermedia} , and \emph{O. nobilis} --
all of which are endemic to China. The three species are mostly
allopatric, although \emph{O. intermedia} and \emph{O. nobilis}
occasionally co-occur in Southwestern China. As implied by its name,
\emph{O. intermedia} is intermediate phenotypically relative to the
other two members of the genus, and an earlier analysis of admixture
based on 20 microsatellites and 11 nuclear loci supported a hybrid
origin approximately 0.5-1.2 million years ago (mya), associated with
Quaternary climate oscillations (Liu et al., 2014).

In their more recent and comprehensive investigation of the system, Wang
et al. (2021) demonstrated that \emph{O. intermedia} was reproductive
isolated from its parental species (criterion 1 of Schumer et al. 2014),
more robustly validated the hybrid origin of \emph{O.
intermedia}(criterion 2), and directly linked the origin of reproductive
barriers to hybridization (criterion 3). While we primarily focus on the
latter component of the study, the findings of the first two components
provide useful background information. Briefly, Wang et al. (2021) found
that the parental species were primarily isolated by two premating
barriers (differential habitat adaptation and flowering time), whereas
the hybrid was isolated from one parent (\emph{O. davidiana} ) by
habitat (mainly soil iron concentration) and the other parent (\emph{O.
nobilis} ) by flowering time. There also was a partial crossability
barrier between the hybrid and parents, with total cumulative isolation
exceeding 0.87 in all comparisons, even without considering
ecogeographic isolation.

Next, Wang et al. (2021) used whole genome sequencing data and standard
phylogenomic and admixture analyses (e.g., PhyloNet and ABBA-BABA tests)
to more robustly document the hybrid origin of \emph{O. intermedia} , as
well as the timing of admixture. Consistent with the previous study, the
two parental species were found to have diverged \textasciitilde{}9.8
mya, with their hybrid originating approximately 1.8 mya. While this
analysis was largely confirmatory of earlier work, such phylogenomic
studies are useful because they can detect previously unsuspected cases
of admixture (e.g., Green et al., 2010, and refine (Nevado et al., 2020;
Owens et al., 2023) or even reject (Zalmat et al., 2021; Goulet-Scott et
al., 2021) earlier hypotheses of homoploid hybrid speciation.

To show that hybridization contributed to the evolution of reproductive
barriers isolating \emph{O. intermedia} from its parental species, Wang
et al. (2021) employed the following strategy (Figure 1). First, they
searched for candidate genes underlying the key habitat (soil iron
concentrations) and flowering time barriers. For tolerance to high soil
iron concentrations, a combined population genomic data set of \emph{O.
intermedia} and \emph{O. nobilis} was compared against \emph{O.
davidiana} , whereas for flowering time, population genomic data
of\emph{O. intermedia} and \emph{O. davidiana} was compared
against\emph{O. nobilis} . Candidate isolation genes were expected to be
under positive selection as indicated by Hudson--Kreitman--Aguade´ tests
(Hudson et al., 1987), outliers with respect to divergence (in the top
2.5\% of number of fixed non-synonymous mutation sites), and have
functional annotations consistent with the putative control of variation
in soil tolerance or flowering time. Three candidate genes (\emph{ZIP5,
FRO4,} and \emph{018656} ) satisfied these criteria for the comparison
of\emph{O. intermedia} + \emph{O. nobilis} to \emph{O. davidiana} ; all
were annotated as being involved in iron reduction. Similarly, for the
comparison of \emph{O. intermedia} + \emph{O. davidiana} to \emph{O.
nobilis,} three candidate genes (\emph{LHY} , \emph{PIE1} ,
and\emph{FPA} ) were under positive selection and had annotations
consistent with their involvement in the control of flowering.

Second, Wang et al. (2021) analyzed the ancestry of \emph{O.
intermedia}alleles for each of these six genes. As expected, alleles
for\emph{ZIP5, FRO4,} and \emph{018656} were mainly derived from
\emph{O. nobilis} , whereas those for \emph{LHY} , \emph{PIE1} , and
\emph{FPA} were mainly derived from \emph{O. davidiana} . However, there
were several instances in which a few genotypes of \emph{O. intermedia}
were heterozygous for alleles from both parental species, possibly due
to post-speciation introgression.

Lastly, Wang et al. (2021) validated the function of alleles for three
of the candidate premating barrier genes (\emph{FRO4} , \emph{ZIP5} ,
and\emph{LHY} ) using a combination of gene expression analyses,
enzymatic activity assays (for \emph{FRO4} and \emph{ZIP5} ), and
functional complementation of mutations in \emph{Arabidopsis thaliana.}
In transgenic \emph{Arabidopsis} plants, alleles of \emph{FRO4}
and\emph{ZIP5} from \emph{O. intermedia} or \emph{O. nobilis}
outperformed those from \emph{O. davidiana} under high iron
concentrations, with opposite effects under low concentrations, strongly
supporting their involvement in adaptation to different soil iron
concentrations. Likewise, transgenic plants with LHY alleles from
\emph{O. intermedia}or \emph{O. davidiana} flowered earlier than those
with \emph{O. nobilis} alleles, further confirming their contribution to
the flowering time barrier isolating \emph{O. intermedia} from \emph{O.
nobilis} .

\textbf{Application to other species}

Can this strategy be applied to document homoploid hybrid speciation in
other systems (Figure 1)? The short answer is ``yes'', with an
additional plant example (Wang, Kang et al., 2022), as well as examples
from charismatic megafauna including bears (Zou et al. 2022) and
primates (Wu et al., 2023; Zhang et al., 2023). The second plant example
also comes from the Betulaceae, but involves hybridization between two
different genera of trees and shrubs, \emph{Ostrya} and
\emph{Carpinus}(Wang, Kang et al., 2022)\emph{,} which diverged 23-33
mya. Hybridization between them gave rise to a new lineage
(\emph{Carpinus}section \emph{Distegocarpus} ), which has since
diversified into three species. Diversification following hybridization
does not seem to be uncommon, especially in adaptive radiations (e.g.,
Meier et al., 2017; Owens et al., 2023; Stull et al., 2023; Wogan et
al., 2023). Phylogenomic analyses indicate that section
\emph{Distegocarpus} is ancient as well, with an origin (17--26 mya)
fairly close in time to the divergence of the parental lineages.
Searches for positively selected and highly differentiated genes using
the pipeline of Wang et al. (2021) identified genes likely involved in
habitat and flowering time barriers from the alternate parental
lineages, thereby tentatively linking hybridization to the development
of reproductive barriers. In addition, because homoploid hybrid
speciation was so ancient in this case, the authors validated their
results with a parallel pipeline based on indels, which are expected to
be less vulnerable to homoplasy than nucleotide substitutions (Rokas \&
Holland, 2000).

In bears, phylogenomic analyses showed that Asiatic black bears arose
via hybridization between the ancestor of polar/brown/American black
bears and the ancestor of sun/sloth bears (Zou et al. 2022). Simulations
of demographic further suggested that the parental lineages diverged
\selectlanguage{english}~5.91 mya, with the origin of Asiatic black bears occurring just 0.25
mya later. Using the Wang et al. (2021) pipeline for detecting
positively selected genes, Zou et al. (2022) identified candidate genes
underlying the intermediate body size of Asiatic black bears. As in the
previous two plant examples, candidate genes were inherited from the
alternate parental lineages and may contribute to assortative mating in
bears. Such alternate inheritance patterns were also observed for
positively selected genes underlying reproductive processes, including
male fertility, fertilization, and sperm development. Zou et al. (2022)
argue that these may represent Bateson-Dobzhansky-Muller (BDM)
incompatibilities, but this hypothesis remains to be confirmed by
functional analyses.

The situation in primates is surprisingly similar to that found for
bears. In macaques, which are the most widespread primates after humans,
it was found that ancient hybridization between the \emph{sinica}
and\emph{silenus} groups in Southeast Asia gave rise to
the\emph{fascicularis} group, which includes four closely related
species. Similar to bears, the \emph{fascicularis} lineage originated
shortly after (\selectlanguage{english}~0.35 mya) the divergence of the parental lineages
(\textasciitilde{}3.86 mya). Positively selected genes underlying a
mosaic of parental traits, including sexually selected traits, were
found to be inherited from the different parental lineages. The authors
argue that such a distinct mix of sexual phenotypes could result in
assortative mating, thereby enabling the establishment of the new hybrid
lineage. Likewise, phylogenomic analyses of snub-nosed monkeys
(\emph{Rhinopithecus} ) from China revealed that the gray snub-nosed
monkey (\emph{R. brelichi} ) originated by hybridization between the
golden snub-nosed monkey and the ancestor of black and black-white
snub-nosed monkeys (Wu et al. 2023). As with the macaques and bears,
hybrid origin (\selectlanguage{english}~1.87 mya) was close in time to parental lineage
divergence (1.98 mya). The distinct coat coloration of the hybrid
species is thought to contribute premating isolation with its parental
species. Using the Wang et al. (2021) pipeline, candidate genes
underlying coat coloration were found to be derived from alternate
parental lineages, providing a link between hybridization and premating
isolation in this system as well.

Together these five studies highlight the power of the Wang et al.
(2021) approach for documenting homoploid hybrid speciation (Figure 1).
Their strategy can be applied to any system, such as putative cases of
homoploid hybrid speciation recently published in this journal (Cuevas
et al., 2021; Li et al., 2021; Popoli Yazdi et al., 2022). However, keep
in mind that the strength of the resulting inferences will depend on the
extent of background information about the kinds and importance of
different reproductive barriers in a given system and the genes that
underlie them. For example, the case for homoploid hybrid speciation
of\emph{Ostryopsis intermedia} is especially strong because state of the
art methods were used to identify and order the reproductive barriers
isolating it from its parental species \emph{and} to functionally
validate key genes underlying the reproductive barriers. In the other
four papers, while reasonable inferences were made about likely
reproductive barriers and the underlying genes, the level of proof is
lower.

\textbf{What can we learn about homoploid hybrid speciation from these
five studies?}

First, all five examples involved hybridization that took place deep in
time (from \selectlanguage{english}~1.9 to \selectlanguage{english}~22.5 mya; Figure 2). This not only demonstrates the
power of modern genomic approaches for identifying ancient reticulations
(Stull et al., 2022), but it also helps assuage earlier worries that
hybridization and hybrid speciation were mainly a consequence of
anthropogenic disturbance and therefore unlikely to have a major
evolutionary role (Schemske, 2000). More broadly, these studies add to
growing evidence that hybridization is the rule rather than the
exception during the divergence of plant and animal lineages (Taylor \&
Larson, 2019; Daglis et al., 2022), and that it can contribute to
post-hybridization diversification (Meier et al., 2017; Stull et al.,
2023).

Another interesting observation is the vast difference in the extent of
divergence of the parental lineages at the time of hybrid speciation,
with the animal hybrid lineages arising fairly recently after the
divergence of the parental species (Figure 2): snub-nosed monkey (\selectlanguage{english}~0.11
mya), bear (0.25 mya), and macaque (\selectlanguage{english}~0.35 mya). In contrast, the two
plant hybrid species arose \selectlanguage{english}~1.8 mya and \selectlanguage{english}~6.5 mya after parental lineage
divergence, respectively, which is 5-fold to 59-fold greater than the
seen for the animal hybrids. Does this mean that successful hybrid
speciation in animals is likely to involve more recently diverged
lineages than for plants? Probably not. Keep in mind that evolutionary
rates are slower in woody species compared to annual or perennial herbs
(Smith \& Donoghue, 2008), and homoploid hybrid species involving
annuals appear to have arisen closer in time to parental divergence than
for \emph{Ostryopsis} and section \emph{Distegocarpus} (Osborne et al.,
2013; Owens et al., 2023). Likewise, there is evidence that some animal
hybrid species have originated from more divergent parental lines than
the primates and bears discussed here (e.g., Ottenburghs et al., 2016;
Ravinet et al., 2018). Furthermore, on average, reproductive barriers
appear to be more rapidly established in plants than animals (Monnet et
al., 2023). A meta-analysis would be useful to assess the timing of
hybrid speciation more broadly, with the goal of asking whether
homoploid hybrid speciation is most common at intermediate levels of
divergence as predicted by models and experimental studies (Buerkle et
al., 2001; Chapman \& Burke, 2007; Comeault \& Matute, 2018; although
see Blanckaert et al., 2023).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. The timing of parental divergence and hybridization in
five examples of homoploid hybridization speciation.} The five homoploid
hybrids are displayed according to the timing of origin (from most
ancient to most recent): \emph{Carpinus cordata} (heartleaf hornbeam; a
species from sect. \emph{Distegocarpus} ), \emph{Ursus
thibetanus}(Asiatic black bear), \emph{Ostryopsis
intermedia}(hazel-hornbeam)\emph{,} \emph{Macaca fascicularis}
(long-tailed macaque; sapecies from the \emph{fascicularis} group),
and\emph{Rhinopithecus brelichi} (gray snub-nosed monkey). Branch
lengths are not to scale.

While the Wang et al. (2021) pipeline appears to be broadly applicable
to the documentation of homoploid hybrid speciation, some ambiguity will
remain. Reproductive isolation is typically polygenic, so natural
selection acting on an admixed population will likely take advantage of
alleles from both parents (Thompson et al., 2023). Thus, as admixed
populations diverge, even if the admixture were initially maladaptive or
neutral, it seems likely that they will (at some point) become homoploid
hybrid species according to the Schumer et al. (2014) criteria and
diagnosable following Wang et al. (2021). Given the ubiquity of
admixture, this line of reasoning implies that homoploid hybrid
speciation could be more common and protracted than suggested by
conceptual arguments and theoretical studies (Grant, 1981; Rieseberg,
1997; Buerkle et al., 2001, 2003; Schumer et al., 2015; Comeault, 2018;
Blanckaert et al., 2018, 2023). However, before such a conclusion can be
made, discussion is required about how much reproductive isolation must
derive from the minor parent of homoploid hybrid species and whether
that isolation must be critical to the initial establishment of the
homoploid hybrid species, as implied by the Ottenburghs' (2018)
classification scheme. Also, how divergent must the parental lineages be
at the time of hybrid origin? When would our classification of
evolutionary process shift from the `Grey Zone of Speciation' (Roux et
al. 2016) to homoploid hybrid speciation? Methods for more accurately
estimating the ages of alleles, the timing of admixture, and the timing
of selective sweeps will contribute importantly to such a debate (Y C
Brandt et al., 2022).

\textbf{Acknowledgements}

We thank Zefu Wang, Lei Zhang, Jiansheng Peng, Guiyun Li, Dongdong Wu,
and Jianquan Liu for photographs of the five homoploid hybrid species.

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\textbf{Author Contributions}

ZL developed the figures and wrote the figure legends; LHR wrote the
text.

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