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Research
Cite this article: Tea Y-K, Hobbs J-PA, Vitelli
F, DiBattista JD, Ho SYW, Lo N. 2020 Angels in
disguise: sympatric hybridization in the marine
angelfishes is widespread and occurs between
deeply divergent lineages. Proc. R. Soc. B 287:
20201459.
http://dx.doi.org/10.1098/rspb.2020.1459
Received: 19 June 2020
Accepted: 15 July 2020
Subject Category:
Evolution
Subject Areas:
ecology, evolution, genetics
Keywords:
Pomacanthidae, coral reef fish, biogeography,
hybridization, sympatry, parapatry
Author for correspondence:
Yi-Kai Tea
e-mail: yi-kai.tea@sydney.edu.au
Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.
c.5075138.
Angels in disguise: sympatric
hybridization in the marine angelfishes
is widespread and occurs between
deeply divergent lineages
Yi-Kai Tea1,2, Jean-Paul A. Hobbs3, Federico Vitelli4, Joseph D. DiBattista2,5,
Simon Y. W. Ho1and Nathan Lo1
1
School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales 2006, Australia
2
Australian Museum Research Institute, Australian Museum, 1 William Street, Sydney, New South Wales 2010,
Australia
3
School of Biological Sciences, University of Queensland, Brisbane, Queensland 4069, Australia
4
Edith Cowan University, 270 Joondalup Drive, Joondalup, Western Australia 6027, Australia
5
School of Molecular and Life Sciences, Curtin University, Perth, Western Australia 6102, Australia
Y-KT, 0000-0002-2146-2592; J-PAH, 0000-0003-0331-354X; FV, 0000-0003-3289-9594;
JDD, 0000-0002-5696-7574; SYWH, 0000-0002-0361-2307; NL, 0000-0002-5407-2005
Hybridization events are not uncommon in marine environments where
physical barriers are attenuated. Studies of coral reef taxa have suggested
that hybridization predominantly occurs between parapatric species distribu-
ted along biogeographic suture zones. By contrast, little is known about the
extent of sympatric hybridization on coral reefs, despite the large amount of
biogeographic overlap shared by many coral reefspecies. Here, we investigate
if the propensity for hybridization along suture zones represents a general
phenomenon among coral reef fishes, by focusing on the marine angelfishes
(family Pomacanthidae). Although hybridization has been reported for this
family, it has not been thoroughly surveyed, with more recent hybridization
studies focusing instead on closely related species from a population genetics
perspective. We provide a comprehensive survey of hybridization among the
Pomacanthidae, characterize the upper limits of genetic divergences between
hybridizing species and investigate the occurrence of sympatric hybridization
within this group. We report the occurrence of hybridization involving
42 species (48% of the family) from all but one genus of the Pomacanthidae.
Our results indicate that the marine angelfishes are among the groups of
coral reef fishes with the highest incidences of hybridization, not only between
sympatric species, but also between deeply divergent lineages.
1. Introduction
The biological species concept, which positsthat species are reproductively isolated
units with limited genetic exchange with other species [1], is among the most
widely considered and debated concepts in biology. In marine systems, where
physical barriers are attenuated and mechanical prezygotic barriers are mostly
lacking, secondary contact due to range expansion of recently diverged taxa
occurs more frequently, particularly for organisms capable of long-distance disper-
sal [2,3]. As a result, many closely related coral reef taxa have adjacent or even
overlapping distributions [4], blurring the role of biogeographic isolation as drivers
of speciation [5]. Yet, in the presence of few physical isolating mechanisms, coral
reefs persist as some of the most diverse ecosystems in the world [4].
Once regarded as a rare phenomenon, hybridization (the production
of viable offspring between two different species) is now known to involve
more than 173 species of coral reef fishes [6]. Although the lack of spatial sep-
aration in marine environments undoubtedly plays a key role, hybridization is
most frequently observed between closely related species that (i) have not had
© 2020 The Author(s) Published by the Royal Society. All rights reserved.
sufficient time to diverge, (ii) exhibit weak behavioural iso-
lation, (iii) have large ecological overlap or (iv) are rare at
range edges. Most studies of hybridization in coral reefs
have been conducted in regions of biogeographic importance
known as ‘hybrid zones’[7] or ‘biogeographic suture zones’
[5,8–10]. These areas connect regional biotas, promoting
opportunities for hybridization by allowing allopatric or
parapatric species to come into secondary contact with each
other [5,7]. Often, one or both parents are rare as a result of
distributional limits, and so heterospecific mating increases
in frequency in the absence of suitable mates [5,7,11–13].
Although there has been rapid growth in research on
hybridization in the marine context, several aspects remain
poorly understood. In particular, little is known about the
prevalence of hybridization between sympatric species and
the extent of genetic divergence between heterospecific parents
that are able to produce viable hybrid offspring. Given the
ephemeral nature of isolating mechanisms in marine systems,
and the high dispersal capabilities and mode of external ferti-
lization in marine fishes, we expect hybridization between
sympatric species to be a common occurrence on coral reefs.
This can occur as a result of disassortative mating, in which
one species deliberately chooses to interbreed with another
species (e.g. opportunistic sneak spawning), or through acci-
dental fertilization [6]. Although some studies have
demonstrated that sympatric hybridization does indeed
occur in coral reef fishes [4,6,13–15], much of the research on
this topic has been limited to species that are also largely para-
patric and that also occur along hybrid zones [5,6,10,16,17].
In most terrestrial and freshwater systems, a difference of
less than 2% in mitochondrial DNA is generally permissive
for hybridization [18,19]. Greater divergences have been
reported for hybridizing marine species, but they usually fall
between 2 and 6% [5,20–22]. Although studies of the
butterflyfishes (Chaetodontidae) have shown that the degree
of genetic differentiation between contributing parents can
influence the directionality of mitochondrial inheritance and
extent of introgression [23], the genetic threshold for viable
hybridization has remained poorly characterized [22] (but
see [24]).
Among coral reef fishes, considerable research effort has
been directed towards the Chaetodontidae (butterflyfishes).
Previous studies have indicated that up to 39% of butterfly-
fishes are capable of hybridizing, and that up to 90% of
these involve parapatric species at biogeographic suture
zones [10]. Similar scenarios of hybridization along suture
zones are seen in numerous other taxonomic groups [8–10].
One other family renowned for a high proportion of hybri-
dizing species is the Pomacanthidae (marine angelfishes),
the sister group to butterflyfishes (but see [25]). The marine
angelfishes are a diverse family of fishes with a long evol-
utionary history dating to the Eocene [26]. Earlier studies
suggested that hybridization among the Pomacanthidae
might be more prevalent than previously thought [14,27],
potentially even more so than in the butterflyfishes. Yet,
recent studies of hybridization in this group are few, focusing
instead on the population genetics of closely related species
[21,28]. Consequently, the upper limits of genetic divergence
between hybridizing species are not well known.
To investigate whether the high levels of hybridization
between parapatric species of marine fishes along hybrid
zones represent a general phenomenon in coral reefs, or if
hybridization events are also common occurrences between
sympatric species, we surveyed incidences of hybridization
among the marine angelfishes. We identified putative
hybrids on the basis of intermediate coloration patterns and
compared mitochondrial genetic distances between hybri-
dizing parents. Further, we tested the reliability of visual
identification for hybrid detection with a mito-nuclear
analysis of one mitochondrial gene and three nuclear genes
on a natural hybrid between two divergent species of angel-
fishes, Paracentropyge venusta and Pa. multifasciata. Our results
indicate that up to 48% of marine angelfishes show evidence
of hybridization, with the majority occurring between
sympatric pairs, and often between highly divergent species.
2. Material and methods
(a) Survey of hybridization in the Pomacanthidae
We identified naturally occurring, putative hybrids of marine
angelfishes on a case-by-case basis according to photographs
taken in the field and aquaria, the literature and unpublished
personal records by the authors. We considered a total of 87
species within the Pomacanthidae as valid. Owing to taxonomic
contention, the following species were excluded from the study
(see [28,29]): Centropyge cocosensis,C. woodheadi,Chaetodontoplus
cephalareticulatus and Ch. chrysocephalus. Hybrids were identified
on the basis of aberrant or intermediate coloration patterns
between parent species (figure 1). The soundness of this
approach has been validated by numerous studies, where ana-
lyses of genetic data have supported the identification of
hybrids on the basis of intermediate coloration and morphologi-
cal characters [14,16,30,31]. To test for reproducibility and the
reliability of this method, we performed a mito-nuclear analysis
of two hybrid Paracentropyge angelfish identified on the basis of
intermediate coloration (see §2c).
Photographs of all identified hybrids taken personally by the
authors and contributors are provided in electronic supplemen-
tary material, figure S1. A complete list of putative hybrids is
presented in electronic supplementary material, table S1. Mito-
chondrial COI sequences of parent species of the putative
hybrids were obtained from GenBank or Barcode of Life Data
System (BOLD). Sequence data from parent species were only
considered from geographical regions away from known
hybrid zones. We did this because hybrid introgression can
sometimes be masked by parental phenotypes (in which case
intermediate coloration is not obvious; see [21]), particularly in
backcrossed individuals where introgressed haplotypes are
retained [21,28]. Pairwise distances (uncorrected p-distances)
were calculated for each species pair in Geneious Prime
2019.1.1 (Biomatters, Auckland).
(b) Distributional and biogeographic data
We identified regions of biogeographic overlap for each hybrid
based on distribution records of the parent species. Distributional
and depth data were obtained from the International Union for
Conservation of Nature [32] and personal observations of the
authors (Y.-K.T., J.-P.A.H., F.V. and J.D.D.). In the context of
hybridization, the term ‘hybrid zone’is sometimes used to
refer to an area in which sympatric species within overlapping
geographic distributions undergo hybridization (see [10]). This
is problematic for several reasons, particularly because it does
not discriminate between (i) species that are geographically sym-
patric but ecologically separated (termed microallopatry); and
(ii) species that overlap completely in biogeography and habitat
niche. Hybridization in both instances is possible, but the under-
lying mechanisms are different. For example, truly sympatric
species (i.e. overlapping geography and ecology) might form
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 287: 20201459
2
rare hybrids as a result of accidental fertilization [14,33], and so
are not constrained by an arbitrary zonation. This is in contrast
with sympatric species that are partitioned by ecological niche
(i.e. microallopatry), which aligns more closely to the traditional
concept of hybrid zones.
The latter (microallopatry) is problematic because it conflates
a number of geographical and ecological concepts. The defining
characteristic of allopatry is the separation of species by discrete,
extrinsic barriers to dispersal, and not by an organism’s intrinsic
biology or ecological preference [2]. In this context, gene flow is
restricted by a biological rather than a mechanical barrier.
Although the argument is largely semantic depending on
whether a population genetic or biogeographic concept of sym-
patry is considered, its implication is particularly sensitive in
the context of hybridization. For the purpose of this study, we
restrict the definition of hybrid zones to a strictly biogeographic
context. Species were considered sympatric if their distributions
overlap by more than 50%, or parapatric if the distributions of
the parent species overlap by between 10 and 50% (see electronic
supplementary material, figure S2). Parapatric pairs that align to
previously defined biogeographic suture zones were identified
following definitions provided by Hobbs et al. [10].
(c) Mito-nuclear analysis of Paracentropyge hybrids
The genus Paracentropyge (see [34]) comprises three species dis-
tributed across the Pacific Ocean and eastern Indian Ocean: the
multibarred angelfish (Pa. multifasciata), distributed throughout
the Pacific Ocean and eastern Indian Ocean; the purple mask
angelfish (Pa. venusta), restricted to southern Japan, south to
Taiwan and the northern Philippines; and the peppermint angel-
fish (Pa. boylei), restricted to very deep reefs in the French
Polynesian Islands [29]. The identification of hybrids among
coral reef fishes has traditionally relied heavily on the inference
of intermediate coloration. To ensure that our visual methods
of identification in this study were reliable, we tested their
robustness on naturally occurring hybrids between Pa. venusta
and Pa. multifasciata using a combination of mitochondrial and
nuclear markers. These hybrids exhibit a striking pattern of inter-
mediate markings between the parent species (figure 2; electronic
supplementary material, figure S3), making them a suitable can-
didate to investigate the validity of this methodology. Previous
studies on marine angelfishes have suggested that the two
species are genetically well separated [34]. Therefore, we chose
to focus on these hybrids to investigate the occurrence of hybrid-
ization between divergent species.
(i) Taxon sampling, PCR and molecular sequencing
Putative hybrids between Pa. multifasciata and Pa. venusta were
collected from Cagayan, northern Philippines (n= 2) and depos-
ited in the Australian Museum, Sydney (AMS I.48937-001 and
AMS I.48938-001). Tissue samples were obtained from muscle
or fin, preserved in 100% ethanol, and stored at −20°C prior to
DNA extraction. In addition to the putative hybrids, we
sequenced six samples of Pa. venusta from Kagoshima, southern
Japan (KAUM-I 72089; KAUM-I 90127; KAUM-I 101525; KAUM-
I 103864; KAUM-I 122604 and KAUM-I 122034). Sequences for
the remaining samples were obtained from GenBank or BOLD.
Collection localities and accession numbers for all specimens
are provided in electronic supplementary material tables S2–S5.
Although hybrids have not been reported for Pa. boylei,we
include it as an ingroup species because its distribution overlaps
with that of Pa. multifasciata. Further, the species is the most
genetically divergent member of the genus, and we include
it in the study to further investigate the genetic thresholds
associated with hybridization.
(a)
(b)
(c)
(d)
Figure 1. Putative hybrids are identified on the basis of intermediate coloration between contributing parents. This approach has been adopted by several authors in
the context of the Pomacanthidae and has been demonstrated to be a sound approach in several other coral reef taxa. (a)Apolemichthys griffisi ×A. xanthopunc-
tatus;(b)Centropyge eibli ×C. flavissima;(c)Centropyge loricula ×C. ferrugata;(d)Genicanthus melanospilos ×G. bellus. All photos by Y.-K.T. (Online version in
colour.)
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 287: 20201459
3
We extracted DNA using the DNeasy Blood and Tissue kit
(Qiagen, Hilden, Germany) following the manufacturer’s protocol.
We amplified the mitochondrial cytochrome coxidase subunit I
(COI) marker using primer sets and PCR protocols described by
Chang et al. [36]. To test for biparental inheritance, we sequenced
three nuclear markers from the hybrids and the available speci-
mens of Pa. venusta: nuclear recombination-activating gene 2
(RAG2); the first intron of the S7 ribosomal protein (S7); and the
protein-coding locus, TMO-4C4. Primer sets and PCR protocols
for the nuclear markers follow those of DiBattista et al.[21].
Sanger sequencing for all four markers was outsourced to Macro-
gen (Seoul, South Korea). Forward and reverse contigs were
aligned and trimmed using Geneious Prime. In the case of the
three nuclear markers, we checked chromatograms for double
peaks, which indicate the presence of two different alleles at a locus.
(ii) Phylogenetic analysis of mitochondrial COI sequences
We analysed a total of 23 mitochondrial COI sequences from
Paracentropyge venusta (n= 11), Pa. multifasciata (n= 6), the putative
hybrid Pa. venusta ×Pa. multifasciata (n= 2) and Pa. boylei (n=4) as
ingroup samples, and Pygoplites diacanthus (n= 1) as an outgroup.
Sequences were aligned using the MUSCLE v3.8.31 algorithm [37].
The final sequence alignment had a length of 612 bp.
Phylogenetic analyses were performed using both Bayesian
inference and maximum likelihood. Bayesian analyses were
conducted in MrBayes v. 3.2.5 [38] with the GTR + G substitution
model. Markov chain Monte Carlo sampling was performed using
one cold and three heated Markov chains. Samples were recorded
every 5 × 10
3
steps over a total of 5 × 10
7
steps, with the initial 25%
of samples discarded as burn-in. The analysis was run in duplicate
and convergence was checked in the program Tracer v. 1.7.1 [39].
Maximum-likelihood analyses were conducted in RAxML v. 8.2.12
[40]. The analysis was performed in duplicate, each using 10
random starts. Node support was evaluated by bootstrapping
with 1000 pseudoreplicates of the data.
(iii) Haplotype networks
We used median-joining in PopART [41] to construct haplotype
networks for the study species based on the single mitochondrial
and three nuclear markers. Owing to the scarcity of samples for
these species, and the narrow geographical distribution of
Pa. venusta and Pa. boylei, we partitioned the sequences according
to species designation instead of sampling location. For the mito-
chondrial COI haplotype network, we used the 612 bp alignment
described above. For the nuclear markers, sequences generated in
this study were combined with other publicly available Paracen-
tropyge sequences on GenBank and BOLD. Sites in the nuclear
marker alignments that displayed ambiguous nucleotides (e.g.
R, Y or W) in one or more taxa were removed prior to haplotype
network construction, with the exception of the sequence from
the putative hybrid individual. In the case of the hybrid individ-
ual, ambiguous nucleotides (identified as double peaks in
sequence chromatograms) might provide direct evidence of
inheritance of different alleles from parent species, in the case
where the two parent species have distinct alleles. The final
alignment lengths were 407 bp for RAG2 (Pa. multifasciata,
n=3; Pa. venusta,n= 8; putative hybrid, n= 1 and Pa. boylei,
n= 3), 345 bp for TMO-4C4 (Pa. multifasciata,n=3; Pa. venusta,
n= 7; putative hybrid, n= 1 and Pa. boylei,n= 4) and 301 bp for
S7 (Pa. multifasciata,n=3; Pa. venusta,n= 5; putative hybrid,
n= 1 and Pa. boylei,n= 3).
3. Results
(a) Incidence of hybridization among the
Pomacanthidae
We expand the number of putative hybrids reported within
the Pomacanthidae from 11 [29] to 37, involving 42 species
(electronic supplementary material, table S1 and figure S1),
Pygoplites diacanthus JF494340
1/84
1/100
1/98
Paracentropyge multifasciata FJ582979
Hybrid Paracentropyge MN518873
Paracentropyge multifasciata KJ148953
Paracentropyge multifasciata KJ148952
Hybrid Paracentropyge MN518872
Paracentropyge venusta MN518869
Paracentropyge venusta MN518866
Paracentropyge venusta MN518867
Paracentropyge venusta MN518870
Paracentropyge venusta MN518868
Paracentropyge venusta MN518871
0.03 substitutions/site
Paracentropyge venusta KJ148975
Paracentropyge venusta KU944244
Paracentropyge venusta KJ148976
Paracentropyge venusta FJ582994
Paracentropyge venusta FJ582995
Paracentropyge multifasciata KJ148951
Paracentropyge multifasciata GBMTG027
Paracentropyge multifasciata GBMNA15030
(e) TMO-4C4
(d) S7
(a)(b)
N
2000 km
(c) RAG 2
59 mutations
42 mutations
Paracentropyge venusta
Paracentropyge multifasciata
Paracentropyge boylei
Paracentropyge venusta ×
Paracentropyge multifasciata
Paracentropyge boylei KJ148912
Paracentropyge boylei KJ148911
Paracentropyge boylei KJ148913
Paracentropyge boylei KJ148914
Figure 2. (a) Geographical distribution of species of Paracentropyge. Known localities of hybrids are indicated by a solid star. Overlapping regions between species
are denoted by coloured gradients. The Kuroshio Triangle sensu Chen & Shashank [35] is loosely reconstructed and represented by the dashed triangle. (b) Phy-
logenetic relationships and median-joining haplotype network among species of Paracentropyge based on mitochondrial COI. Phylogenetic relationships were inferred
using maximum likelihood and Bayesian inference. Numbers at nodes indicate posterior probabilities and likelihood bootstrap support. Haplotypes of hybrids are
indicated in light blue. Median-joining haplotype networks are also presented for (c)RAG2,(d)S7 and (e)TMO-4C4. Each circle represents a haplotype and its size is
proportional to its total frequency. Unless specifically stated, each black crossbar represents a single nucleotide change. In the haplotype networks for RAG2, two
haplotypes are presented for the single hybrid examined, corresponding to alleles inherited from each parent (see electronic supplementary material, figure S4).
Sites that displayed ambiguous bases in the sequence of one or more non-hybrid individuals were removed from alignments prior to network construction, and a
single haplotype consensus sequence per individual was used. Each network is, therefore, an underestimation of the true haplotype diversity at each nuclear locus for
the parent species. Photographs of angelfishes from top to bottom by: H. Senou, Y.-K.T., J. T. Williams and Y.-K.T. (Online version in colour.)
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 287: 20201459
4
representing 48% of the family. Except for the monotypic
genus Pygoplites, hybridization appears to occur in all
extant angelfish genera as currently defined. Of the 37 puta-
tive hybrids, 15 involved parapatric parent species. Of these,
nine aligned to well-defined biogeographic suture zones
outlined by Hobbs et al. [10], namely southern Japan,
Hawaii, Papua New Guinea-Micronesia and the eastern
Indian Ocean (including Cocos [Keeling] Islands, Christmas
Island, southern Indonesia and reefs off northwest Australia).
Of the surveyed species where abundance data were avail-
able, hybrids formed between parapatric pairs occurred in
regions where at least one of the parent species was uncom-
mon or rare (table S1). Sympatric hybrids were reported for
22 species pairs, of which only 4 pairs involved species that
are ecologically separated on the basis of depth. These
involved species that primarily occur in mesophotic coral eco-
systems, namely Centropyge multicolor,C. potteri and
Genicanthus bellus.
Genetic distance in mitochondrial COI for heterospecific
pairs ranged from 0% (in completely introgressed species;
Centropyge eibli × Indian Ocean C. flavissima) to 11.7%
(Pomacanthus imperator ×Po. semicirculatus). Not including
those with missing data, 37% and 23% of putative hybrids
were between species with less than 5% and greater than or
equal to 8% mitochondrial divergences, respectively. The high-
est genetic divergences were recorded from the genus
Pomacanthus:betweenPo. imperator×Po. annularis (11.1–11.3%)
and Po. imperator ×Po. semicirculatus (11.2–11.7%). Hybridiz-
ation within the Pomacanthidae appears to occur mostly
between sympatric species, with Pomacanthus exhibiting the
highest incidence of sympatric hybridization.
(b) Hybridization in Paracentropyge
Phylogenetic analysis of mitochondrial COI yielded a tree
with three strongly supported groups corresponding to the
three species of Paracentropyge (figure 2). Paracentropyge
multifasciata was placed with strong support as the sister
species to Pa. venusta in all analyses, with a pairwise genetic
distance of 7.7–8.4% in COI. Maternal inheritance was
unidirectional, with the putative hybrids sharing COI haplo-
types with Pa. multifasciata, thus resolving the identity of the
maternal contributor.
The status of the putative hybrid was further supported
in our analyses of the nuclear markers. A comparison of
RAG2 sequences of Pa. venusta,Pa. multifasciata and the
sequence chromatogram from the putative hybrid individual
revealed four sites (189, 212, 227, 358) at which double peaks
were found in the hybrid (electronic supplementary material,
figure S4). For example, at site 189, all representatives of
Pa. multifasciata possess a C nucleotide, while all representa-
tives of Pa. venusta possess a T nucleotide. The hybrid was
found to possess a double peak comprising both C and T
nucleotides at this site. The most parsimonious explanation
for this is the inheritance of C from Pa. multifasciata (maternal
contributor, based on the mitochondrial analyses) and a T
from Pa. venusta (paternal contributor). Haplotype networks
of all three nuclear markers indicated the sharing of
haplotypes by Pa. multifasciata,Pa. venusta and the putative
hybrid (figure 2), but potential inheritance from both
parent species was detected only in RAG2. At all three mar-
kers, Pa. boylei exhibited haplotypes that were distinct from
those of Pa. venusta and Pa. multifasciata.
4. Discussion
(a) Hybridization among the Pomacanthidae
Results from our survey suggest that Pomacanthidae is one of
the most prolifically hybridizing groups of fishes on coral
reefs, more so than the Chaetodontidae (48% in Pomacanthi-
dae versus 39% in Chaetodontidae). Some studies have
indicated a higher incidence of hybridization in the rabbit-
fishes (Siganidae, 55% [42]), but they are considerably less
speciose than the Pomacanthidae (about one-third in terms
of the number of valid species) and with fewer genera
(only one genus in Siganidae; Siganus). One caveat, however,
is that hybridization is more easily detected in conspicuous
groups of fishes, given that colour is the primary cue for
identification in the field. Therefore, we acknowledge that
the incidence of hybridization might be higher in small, cryp-
tic groups of fishes, or in groups with poorly known life
histories, taxonomy and distributional ranges.
Unlike the Chaetodontidae, only nine pomacanthid
hybrids were reported from biogeographic suture zones
defined by Hobbs et al. [10] (24% in Pomacanthidae versus
90% in Chaetodontidae). We identified four other regions
of biogeographic importance: the Ogasawara Islands, Baja
California, eastern Australia and the Philippines in the
Western Pacific. Although not explicitly recognized as hybrid
zones, these regions are suture points for several biogeographic
provinces, enabling hybridization between regional, parapatric
faunas. The Ogasawara Islands, Baja California and eastern
Australian coast are notable in harbouring several endemic
angelfish species (e.g. Genicanthus takeuchii in the Ogasawara
Islands and Holacanthus clarionensis in Baja). Eastern Australia
is markedly divided and represented, in part, by the Peronian
and Flindersian provinces [43], where the regionally endemic
Chaetodontoplus meredithi and Ch. conspicillatus occur. Given
the high levels of endemism and faunal distinctiveness, a
suture zone is likely to occur along the boundary of these
areas. In most parapatric examples, one of the contributing
parents was usually rare as a result of being at its distributional
limit, suggestingthat rarity between species plays an important
role in at least some hybridization events.
We found a markedly higher proportion of hybrids occur-
ring in regions of sympatry (59% versus 41% in parapatric
parents). Even in sympatric species separated by ecological
niche, hybridization accounted for only 11% of all reported
hybrids, contributed by only four species occurring in meso-
photic coral ecosystems. This finding is in contrast with
hybridization in the Chaetodontidae, where up to 90% of
hybrids occur between parapatric pairs in suture zones [10].
One possible explanation for this difference is in the contrasting
biology of both families. Except for a handful of gregarious
species [44], chaetodontids most frequently occur in monog-
amous, heterospecific pairs [45,46]. Among pomacanthids,
however, many species are protogynous hermaphrodites,
with females aggregating in species-specific harems that
are tended to by a single male (electronic supplementary
material, figure S5) [47]. This hierarchical social structure
is displayed most prominently in the genera Centropyge,
Paracentropyge,Apolemichthys and Genicanthus (and sometimes
in Pomacanthus [48]).
In hermaphroditic, haremic species, it is possible that
selection for monogamy is relaxed because multiple individ-
uals reproduce within the same group, thereby reducing the
constraint for mono- or heterospecific pair formation.
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 287: 20201459
5
Although each individual in a harem is capable of sex
change, the transition is usually supressed by a dominant
male, thus maintaining a sex ratio that is predominantly
female biased. This might present greater opportunities for
sympatric hybridization through accidental fertilization,
disassortative mating and sneak spawning, particularly
between mixed-species harems that spawn synchronously
on the same reef (see [49,50]). This is difficult to test, however,
because sympatric hybridization can also occur between
adjacent pairs of monogamous fishes.
Indeed, there is evidence to suggest that in some instances,
interspecies hybridization between harems is influenced by
mate choice rather than accidental fertilization [48,51]. How-
ever, these studies have been conducted on very closely
related species in species complexes along hybrid zones, and
so the influence of intentional versus unintentional hybri-
dization in distantly related sympatric species remains
uncertain. Nonetheless, many sympatric species of Genicanthus
and Centropyge often engage in mixed-species aggregations as
a result of sympatry between adjacent harems (electronic sup-
plementary material, figure S5). Our results indicate that the
majority of heterospecific parents exhibiting deep levels of gen-
etic separation (greater than or equal to 8% in mitochondrial
COI) occur between sympatric species, and that hybridization
can occur between parents from phylogenetically distant,
non-sister lineages (e.g. Pomacanthus semicirculatus ×Po.
maculosus;Po. imperator ×Po. semicirculatus; for phylogenetic
relationships, see [26]). This appears to be common in
Pomacanthus, where we report some of largest pairwise dis-
tances between parents. Although similar examples are not
well documented for marine taxa, studies conducted on
terrestrial Heliconius butterflies suggest that hybridization
does not occur between species with mitochondrial divergences
in excess of 10% [19]. While range overlap in hybrid zones
remains an important underlying factor, our results demon-
strate that broad sympatry plays an equal, if not more integral
role in hybrid formation, at least for the Pomacanthidae.
(b) Visual methods of hybrid identification and hybrid
ancestry of Paracentropyge
Our study brings the number of reported hybrids between
Pa. venusta and Pa. multifasciata in the literature to four.
Only two other examples have previously been reported
[27,52], but no detailed genetic studies have been performed
to confirm hybrid ancestry beyond visual and morphological
identification. As with the previous hybrid specimens, our
specimens were collected from the northernmost Philippines.
This region lies between southern Japan and the northern
Philippines, at the intersection of two important biogeo-
graphic regions: the Ryukyu Archipelago and the Coral
Triangle (figure 2; dubbed the Kuroshio Triangle [35]). This
represents a major hybrid zone for coral reef species, particu-
larly for the Chaetodontidae [10]. Here, several species are
close to their range limits, including Pa. multifasciata, which
becomes increasingly rare northwards into the Yaeyamas
and the rest of the Ryukyu Islands (figure 2) [53].
Molecular date estimates have shown that Pa. venusta and
Pa. multifasciata shared a most recent common ancestor 9 Ma
during the Miocene [34]. This deep temporal divergence is
reflected in the large mitochondrial divergence between
the two species (7.7–8.4% in COI). Our analysis of mito-
chondrial COI and nuclear RAG2 strongly suggests the
status of the hybrid, with maternal inheritance of COI from
Pa. multifasciata, and biparental inheritance of RAG2 from
Pa. multifasciata and Pa. venusta. The shared S7 and TMO-
4C4 haplotypes between the hybrid and both parent species
can be explained by the lower evolutionary rate or incomplete
lineage sorting of the nuclear genome. Although recurrent
introgression as a result of successive hybrid backcrosses
could also account for haplotype sharing, we note that this is
very unlikely, as our sampled individuals of Pa. venusta were
collected from Kagoshima, lying just outside the geographical
range of Pa. multifasciata [53]. Nonetheless, our combined ana-
lyses confirm hybrid ancestry for the first time between these
two species of Paracentropyge angelfishes. Importantly, the ear-
lier-diverging Pa. boylei (approx. 16 Ma [34]) remained distinct
from the other species of Paracentropyge at all of the analysed
markers. Although sample sizes were small, we emphasize
that natural hybrids between these species are exceedingly
rare, with these instances representing half of all known
specimens recorded in the literature.
Our results parallel those from a study of Chaetodon
trifasciatus ×Chaetodon lunulatus, where deep levels of genetic
separation between parent species are reflected in infrequent,
unidirectional hybridization events with infertile offspring
[23], leading to little or no adverse evolutionary outcomes, pro-
viding further support for the lack of recurring backcrosses
and introgression in either parent species. This stands in con-
trast with hybrids formed between genetically similar or
undifferentiated species, with ongoing backcrosses leading to
introgression. In the marine angelfishes, this scenario is well
documented for the Centropyge flavissima complex [21,29], but
the upper limits of genetic divergence between hybridizing
species have been poorly characterized.
Curiously, no hybrids have been reported between
Pa. mul tifasciat a and Pa. boylei despitetheir distributional overlap
in the French Polynesian Islands of Rarotonga and Moorea
(figure 2). However, unlike Pa. multifasciata and Pa. venusta,
Pa. boylei has a preference for much deeper waters (53–120 m
versus 7–70 m in Pa. multifasciata [30,54]), making any overlap
in the two species a rarity. A more parsimonious explanation
is that hybrids between the two species have not yet been
recorded, which is not unlikely given the geographical isolation
and preference of Pa. boylei for very deep reefs. Nonetheless, our
study provides novel insights into the outcomes of hybridiz-
ation between divergent species of marine angelfishes, with
results corroborating previous studies on hybridization
between other divergent lineages of coral reef fishes.
(c) Concluding remarks
We have presented evidence that hybridization among the
Pomacanthidae occurs mostly between sympatric species,
and between highly divergent lineages, but several questions
remain unanswered. In particular, the underlying mechanisms
of hybridization along hybrid zones remain unresolved, par-
ticularly for species that do not engage in monogamous pair
formation. Additionally, it is still unclear why some closely
related, sympatric species do not hybridize, but yet are able
to hybridize with more distantly related species. Whether
this has anything to do with hybrid fitness or prezygotic
incompatibility requires further investigation, though captive
breeding of marine angelfishes remains an area of great diffi-
culty. Nonetheless, our study provides a first step in tackling
key questions about the circumstances under which
royalsocietypublishing.org/journal/rspb Proc. R. Soc. B 287: 20201459
6
hybridization occurs in coral reef fishes—the largest group of
sympatric vertebrate species in the marine environment.
Data accessibility. Sampling locations and GenBank and BOLD accession
numbers for COI sequences of all other angelfishes used in calculating
pairwise distances are provided in electronic supplementary material,
table S2. GenBank accession numbers for all publicly available and de
novo sequences (MN518866–92) of Paracentropyge used in this study
are availablein the sequence data files, availablefrom the Dryad Digital
Repository: https://doi.org/10.5061/dryad.7pvmcvdpv [55].
Authors’contributions. Y.-K.T., J.-P.A.H., F.V., S.Y.W.H. and N.L. devised
the study. Y.-K.T. conducted the analyses and wrote the manuscript
with input from all authors. All authors approved the final version of
the manuscript and agree to be held accountable for the content.
Competing interests. The authors declare no competing interests.
Funding. Y.-K.T. was funded by a Research Training Program Scholar-
ship from the Australian Government and by an Australian Museum
Research Institute Postgraduate Award. J.-P.A.H. was funded by the
Australian Research Council (grant no. DE200101286). J.D.D. was
funded by a Curtin University Early Career Research Fellowship.
S.Y.W.H. and N.L. were funded by the Australian Research Council
(grant nos FT160100167 and FT160100463, respectively).
Acknowledgements. We thank J.K. Ong, Y.Z. Tay, S.K. Tea, K. Lim,
A. Hay, D. Pitassy and S. Reader for sample preparation and curator-
ial assistance. B.D. Greene provided helpful comments on angelfish
distribution records. H. Motomura and J.T. Williams provided
tissue samples for Paracentropyge venusta and P. multifasciata. S.K.
Tea, B. Shutman, L.A. Rocha, H. Senou, J.T. Williams, H. Debelius,
K. Kohen, J. Coppolino, Z. Lin, S. Kobayashi, C. Delbeek, W. P. Su,
J. Ma, R. Lanceley and P. Supanantananont provided colour photo-
graphs used in this manuscript, and L. Gentry provided map
vectors. The hybrid Paracentropyge specimens were collected and
donated by B. Shutman, under permits issued by Philippines
Bureau of Fisheries and Aquatic Resources, Department of Agricul-
ture, permit numbers OFNCR-MTP-10-09 and LTP40-QUES218-0226.
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