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Angels in disguise: sympatric hybridization in the marine angelfishes is widespread and occurs between deeply divergent lineages



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 reef species. 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.
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:
Received: 19 June 2020
Accepted: 15 July 2020
Subject Category:
Subject Areas:
ecology, evolution, genetics
Pomacanthidae, coral reef fish, biogeography,
hybridization, sympatry, parapatry
Author for correspondence:
Yi-Kai Tea
Electronic supplementary material is available
online at
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
School of Life and Environmental Sciences, University of Sydney, Sydney, New South Wales 2006, Australia
Australian Museum Research Institute, Australian Museum, 1 William Street, Sydney, New South Wales 2010,
School of Biological Sciences, University of Queensland, Brisbane, Queensland 4069, Australia
Edith Cowan University, 270 Joondalup Drive, Joondalup, Western Australia 6027, Australia
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
[5,810]. 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 [5,7,1113].
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 [4,6,1315], 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% [5,2022]. 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 [810].
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 zoneis 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 Proc. R. Soc. B 287: 20201459
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 organisms 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 S2S5.
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.
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.) Proc. R. Soc. B 287: 20201459
We extracted DNA using the DNeasy Blood and Tissue kit
(Qiagen, Hilden, Germany) following the manufacturers 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
steps over a total of 5 × 10
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
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
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
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.) Proc. R. Soc. B 287: 20201459
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 (11.111.3%)
and Po. imperator ×Po. semicirculatus (11.211.7%). 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 7.78.4% 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. Proc. R. Soc. B 287: 20201459
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 (7.78.4% 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 (53120 m
versus 770 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 Proc. R. Soc. B 287: 20201459
hybridization occurs in coral reef fishesthe 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 (MN51886692) of Paracentropyge used in this study
are availablein the sequence data files, availablefrom the Dryad Digital
Repository: [55].
Authorscontributions. 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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Supplementary resource (1)

... After the initial instance of hybridisation, F 1 hybrids may subsequently backcross with parental species, potentially leading to widespread introgression with evolutionary implications for parental species (Bernal et al., 2017). In coral reef fishes, hybrids are commonly detected through their intermediate physical attributes of their parental species, a method that has been supported in many studies by subsequent genetic analyses (e.g., Tea et al., 2020;Yaakub et al., 2006. However, with the progression of backcrossing, hybrids become morphologically indistinguishable from their parent species, making them difficult to identify Yaakub et al., 2006). ...
... While various mechanisms have been identified that may promote the chances of hybridisation, relatively few studies have examined the formation or maintenance of hybrid or suture zones. However, several major potential drivers have been identified: (a) Hybridisation in coral reef fishes has been reported between deeply divergent lineages Tea et al., 2020), but it occurs more readily between closely related species (DiBattista et al., 2015;Montanari et al., , 2016. ...
... Islands by , nine crosses represented hybridisation between Indian and Pacific Ocean sister species. While hybridisation has been described between distantly related species Tea et al., 2020), there is still a broad agreement that limited genetic distance and potential for heterospecific mating are strongly intertwined. Closely related taxa are more likely to share biological, ecological and behavioural traits, which can increase the probability of interbreeding and production of fertile offspring . ...
Full-text available
Our main aim was to identify the distribution of, and potential mechanisms underpinning, hybrid‐rich zones – regions with a disproportionate number of unique interspecific hybrids. We investigated whether coral reef fish hybrids coincided with factors such as phylogenetic relatedness, biogeographic barriers, species richness, geographic isolation, endemism, and oceanic currents. Global. Contemporary. Coral reef fishes. We conducted a literature review and mapping to assess the taxonomic and global prevalence of hybridisation in coral reef fishes. We then fit Generalised additive models using a full‐subsets and Bayesian framework to assess which variables are associated with hybrid‐rich zones. We found 143 unique interspecific coral reef fish hybrids involving 204 species – which accounts for approximately 7% of coral reef fish species, indicating that hybridisation is as common in the sea as it is on land. Characteristic coral reef fish families were not homogeneously represented in our dataset, with particularly colourful groups standing out. Mapping our dataset revealed that coral reef fish hybrids are found worldwide, though some ecoregions (e.g., the Christmas and Cocos (Keeling) Islands, South Kuroshio, Hawaii, and Eastern Philippines) are more hybrid‐rich than others. Our analysis revealed that mean surface current velocity, phylogenetic relatedness, and geographic isolation were the best predictors of hybrid richness in a given location. Phylogenetic distance between coral reef fish species may serve as a pre‐condition for hybridisation to occur, lying between introgression and reproductive incompatibility. We also propose a novel mechanism, with oceanic currents driving long‐distance larval dispersal events, transporting stray species to geographically remote sinks to maintain hybrid‐rich zones.
... In marine fishes, many studies have been conducted on natural hybridization, introgression, and speciation of small, colourful common reef fish families, such as the Acanthuridae Randall & Frische, 2000), Chaetodontidae (Montanari et al., 2012(Montanari et al., , 2014, Labridae (Yaakub et al., 2006(Yaakub et al., , 2007, Pomacanthidae (Pyle & Randall, 1994;Tea et al., 2020), and Pomacentridae (Gainsford et al., 2020;He et al., 2019). Eels (order Anguilliformes) are one of the abundant and important apex predators in different aquatic ecosystems; however, almost nothing is known about these processes in this large group of fishes. ...
... 2.2.3 | Mito-nuclear analyses to survey the incidence of hybridization Ambiguous sites of nuclear genes were recognized by the double peaks in the chromatograms, in which the secondary peaks were at least half of the level of the dominant peaks. These sites may indicate heterozygous alleles at a given locus, and a hybrid sequence could be further confirmed by checking the interspecific ambiguous sites of its possible parental species ( Figure S1; He et al., 2019;Tea et al., 2020). In the present study, only those individuals possessing an intermediate colouration pattern and sharing nuclear haplotypes with both parental species were referred to as "hybrids" (Tea et al., 2020). ...
... These sites may indicate heterozygous alleles at a given locus, and a hybrid sequence could be further confirmed by checking the interspecific ambiguous sites of its possible parental species ( Figure S1; He et al., 2019;Tea et al., 2020). In the present study, only those individuals possessing an intermediate colouration pattern and sharing nuclear haplotypes with both parental species were referred to as "hybrids" (Tea et al., 2020). Otherwise, those with the mito-nuclear discordance but purebred phenotypes which may be the result of introgressive hybridization were referred to as "introgressed" individuals (DiBattista et al., 2012;Montanari et al., 2014;Perea et al., 2016). ...
Phenovariant is a pair of populations or species with distinct phenotypes but little to no genetic divergence, which may have resulted from strong assortative mating, hybridization, or incomplete lineage sorting that changes in the phenotype have exceeded the genotype. Previous studies had mainly tackled this issue on small, colourful reef fishes, but little effort has been made to the barely seen and solitary taxa like the morays (family Muraenidae). In the present study, three species pairs of sympatric morays from Taiwanese waters revealed as phenovariant in the mitochondrial cytochrome oxidase I (COI) were examined from the molecular and morphological perspectives, with three dissimilar scenarios discovered: (1) no hybridization was found between the morphologically most distinct Gymnothorax intesi and Gymnothorax neglectus; (2) Gymnothorax kidako bidirectionally hybridized with Gymnothorax prionodon instead of its closely related species Gymnothorax pseudokidako; and (3) frequently bidirectional hybridization and introgression were detected between Gymnothorax pseudothyrsoideus and Gymnothorax reevesii, a species pair that overlapped in all the morphometric and meristic characters. Nevertheless, the principal component analysis indicated that each species pair has evolved significant morphological divergence, further supporting their taxonomic validities. Our results document the natural hybridization of marine eel taxa for the first time and reveal a diverse evolutionary process among the morays.
... In 1995, Veron challenged this view by proposing that hybridisation was the key mechanism in the reticulate evolution of corals. Subsequent research has provided evidence to support this idea, and more recently, there has been a surge in reports of hybridisation among other coral reef organisms (reviewed in Richards and Hobbs 2015;Tea et al. 2020). However, our knowledge of hybridisation and its possible impacts on species diversity is still largely limited to the use of traditional genetic markers and morphological data (Willis et al. 2006;Richards and Hobbs 2015). ...
... Similar searches in Google Scholar and Scopus produced a subset of these papers. We also searched the reference lists of hybrid review papers (Pyle and Randall 1994;Gardner 1997;Montanari et al. 2016;Tea et al. 2020), which added another seven journal articles and one book. For corals, we searched the Web of Science across all years for articles that contained the following terms in the title or abstract: ''coral AND hybrid*''. ...
... Future research will likely reveal that hybridisation has been significantly underestimated in poorly studied and cryptic groups. Indeed, the highest rates of hybridisation (15-50%) occur in well-studied groups of fishes (angelfish and butterflyfish; Pyle and Randall 1994;Tea et al. 2020), and corals (Psammocoridae, Pocilloporidae and Acroporidae: Richards et al., 2008;Stefani et al. 2008aStefani et al. , 2008bBenzoni et al. 2010;Schmidt-Roach et al. 2014; but see also Johnston et al. 2017). This finding supports the premise that there is a positive relationship between sampling effort and detection of hybrids (Mallet 2005). ...
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Hybridisation has traditionally been considered rare and unimportant in generating biodiversity in the marine environment, particularly in coral reefs ecosystems. Here we review the literature for evidence of hybridisation in coral reef organisms and find that hybridisation is prevalent in well-studied groups, namely fish and hermatypic corals. At least 183 fish and 81 scleractinian coral species are reported to hybridise, with the highest prevalence in angelfishes and psammocorid corals (hybridisation in 46–50% of species in each family). Mapping the geographic location of hybrids revealed that hybridisation was positively associated with proximity to biogeographic borders for fish but not corals, and not linked with areas of low species richness in either group. Molecular studies detected admixture, indicative of past hybridisation, in 30 of 53 cases for fish, and in 22 (possibly 39) out of 47 studies for corals. Patterns of introgression described in the fish studies supported a decrease in lineage diversity in nine cases and the formation of hybrid lineages in seven cases. In the other 14 cases, the two parent species remained genetically distinct despite evidence of introgression. For corals, the evidence of hybrid lineages was rare (5 species), rather there was more support indicating that hybridisation leads to a decrease (36 species), or no change (27 species), in lineage diversity. Collectively, these results show that hybridisation can influence the evolution of fishes and corals in multiple ways and suggests the prevalence of hybridisation is likely to be significantly underestimated for coral reef taxa. The application of new genomic tools will advance our knowledge of the prevalence and evolutionary importance of hybridisation in coral reef organisms. Furthermore, these tools will aid in identifying how natural and assisted hybridisation may help coral reef species adapt to increasing environmental changes.
... Hybridization is common in natural populations. Although it was previously thought that hybridization occurred exclusively between closely related species (Dasmahapatra et al. 2007), it is now recognized that hybridization occurs between deeply divergent lineages (Jasso-Martínez et al. 2018, Joseph et al. 2019, Tea et al. 2020. Questions remain about the processes that facilitate the events; for example does hybridization occur because species live in sympatry or does hybridization occur because species exhibit ecological or habitat similarities (Willis et al. 2014, Wood et al. 2016, Kyogoku and Kokko 2019? ...
... In their review of hybridization among birds, Gholamhosseini et al. (2013) found that hybridization was more frequent between sister species than non-sister species for 25 of the 29 genera reviewed, indicating hybridization is more common between sister species than non-sister species. That hybridization is common among non-sister species is not surprising, given the extensive evidence of hybridization between non-sister species across taxa (Dasmahapatra et al. 2007, Joseph et al. 2019, Tea et al. 2020. Although in our study admixture was higher between species from different clades, it is important to view these results in the context of the black-capped chickadee and Carolina chickadee hybrid zone (both of these species are part of the same clade; Harris et al. 2014), where hybridization is frequent and well studied (Taylor et al. 2014, Wagner et al. 2020). ...
... By comparison Randler (2006) found that hybridization was more frequent between parapatric than sympatric species, although Randler notes that there are exceptions to this pattern and similar to Willis et al. (2014) states that sympatry may increase the potential for individuals to find mates (Tubaro and Lijtmaer 2002). Across taxa there is growing evidence that hybridization is common in natural populations among deeply divergent lineages and that sympatry may help to facilitate these events (Jasso-Martínez et al. 2018, Tea et al. 2020. ...
Hybridization is an important aspect of speciation, yet questions remain about the ecological and environmental factors that influence hybridization among wild populations. We used microsatellite genotyping data and collected land cover and environmental data for four North American chickadee species: black‐capped Poecile atricapillus, mountain P. gambeli, chestnut‐backed P. rufescens and boreal P. hudsonicus chickadees. Combining these datasets, we sought to examine whether there is evidence of admixture between four widely distributed North American chickadee species; whether admixture takes place more often between more closely related species pairs or between species pairs with more similar ecological preferences; and whether certain habitat types have higher rates of admixture than others. We detected admixture for five of the six species pairs analyzed (chestnut‐backed–mountain chickadee pair showed no evidence of admixture), and found rates of admixture varied geographically, and within taxa pairs. Admixture was higher among less closely related species than more closely related species, although habitat similarity was not a significant predictor. Finally, rates of admixture were higher in urban parkland habitats than deciduous, mixed or coniferous forest habitats. Our work indicates admixture occurs frequently among North American parids, and habitat and environmental variation may play an important role in the frequency and geographic distribution of hybridization.
... Coral reef fishes represent the most species rich vertebrate communities in the world. Hybridisation is common in these fishes and we focus our study on angelfishes (family Pomacanthidae), which have the highest proportion of recorded hybrids for any coral reef fish family 18,19 . Hybridisation is particularly common among pygmy angelfishes (genus Centropyge), which appear to have recently diverged and are hybridising in narrow regions of secondary contact 18,20,21 . ...
... Hybridisation is common in these fishes and we focus our study on angelfishes (family Pomacanthidae), which have the highest proportion of recorded hybrids for any coral reef fish family 18,19 . Hybridisation is particularly common among pygmy angelfishes (genus Centropyge), which appear to have recently diverged and are hybridising in narrow regions of secondary contact 18,20,21 . A well-studied case of angelfish hybridisation is that between the Lemonpeel Angelfish Centropyge flavissima and Eibli's Angelfish Centropyge eibli. ...
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Hybridisation and introgression of eukaryotic genomes can generate new species or subsume existing ones, with direct and indirect consequences for biodiversity. An understudied component of these evolutionary forces is their potentially rapid effect on host gut micro-biomes, and whether these pliable microcosms may serve as early biological indicators of speciation. We address this hypothesis in a field study of angelfishes (genus Centropyge), which have one of the highest prevalence of hybridisation within coral reef fish. In our study region of the Eastern Indian Ocean, the parent fish species and their hybrids cohabit and display no differences in their diet, behaviour, and reproduction, often interbreeding in mixed harems. Despite this ecological overlap, we show that microbiomes of the parent species are significantly different from each other in form and function based on total community composition, supporting the division of parents into distinct species, despite the confounding effects of introgression acting to homogenize parent species identity at other molecular markers. The microbiome of hybrid individuals, on the other hand, are not significantly different to each of the parents, instead harbouring an intermediate community composition. These findings suggest that shifts in gut microbiomes may be an early indicator of speciation in hybridising species.
... Although hybridisation has long been recognised and is common among marine and freshwater fishes (Scribner et al. 2001;Montanari et al. 2016), its discovery in chondrichthyans only occurred in the last decade. In fishes, hybrid individuals often display colour patterns or morphologies intermediate to the parental species and are therefore relatively easy to identify (Montanari et al.2016;Tea et al. 2020). However, chondrichthyans often have conserved morphology between species and this makes the recognition of hybrids more difficult. ...
Context Knowledge of sawshark reproductive biology is limited to general parameters such as reproductive mode and litter size. The mating system is currently unknown. Aim To test for multiple paternity in the common (Pristiophorus cirratus) and southern (Pristiophorus nudipinnis) sawshark and investigate the occurrence of hybridisation between these two species. Methods Pups from a single litter of each species and an adult P. nudipinnis displaying mismatches in its morphology and mitochondrial DNA were genotyped with nuclear single-nucleotide polymorphisms (SNPs). Multiple paternity was assessed using pairwise relatedness and sibship analysis, and hybridisation was examined using three approaches (principal-component analysis, admixture analysis and clustering with NewHybrids). Key results Multiple paternity was detected in both species, with two males siring the seven-pup litter in P. cirratus and two males siring the two-pup litter in P. nudipinnis. Hybridisation between the two species was also confirmed, with the mismatched adult identified as a first-generation hybrid. Conclusions The mating system of sawsharks involves polyandry, and hybridisation between the two co-occurring Australian species is possible. Implications These results provide new information on sawshark reproductive biology and highlight the need for combined use of mitochondrial and nuclear markers in future genetic studies involving these species.
... Hybridization between species is a potential source of morphological [1], functional [2] and communication signal novelty [3,4]. Novel phenotypes can arise in hybrids whenever recombination is heterogeneous across the genome [5]. ...
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Hybridization is a known source of morphological, functional and communicative signal novelty in many organisms. Although diverse mechanisms of established novel ornamentation have been identified in natural populations, we lack an understanding of hybridization effects across levels of biological scales and upon phylogenies. Hummingbirds display diverse structural colours resulting from coherent light scattering by feather nanostructures. Given the complex relationship between feather nanostructures and the colours they produce, intermediate coloration does not necessarily imply intermediate nanostructures. Here, we characterize nanostructural, ecological and genetic inputs in a distinctive Heliodoxa hummingbird from the foothills of eastern Peru. Genetically, this individual is closely allied with Heliodoxa branickii and Heliodoxa gularis, but it is not identical to either when nuclear data are assessed. Elevated interspecific heterozygosity further suggests it is a hybrid backcross to H. branickii. Electron microscopy and spectrophotometry of this unique individual reveal key nanostructural differences underlying its distinct gorget colour, confirmed by optical modelling. Phylogenetic comparative analysis suggests that the observed gorget coloration divergence from both parentals to this individual would take 6.6–10 My to evolve at the current rate within a single hummingbird lineage. These results emphasize the mosaic nature of hybridization and suggest that hybridization may contribute to the structural colour diversity found across hummingbirds.
... Species identification using the UVC method is reliant only on the expertise of the observer based on morphological features, and species identified cannot be reviewed and validated after each survey without obtaining good underwater photos. Another limitation is the detection of smaller species, and the differentiation of hybrids and introgressed individuals from their parental species, a scenario common in the coral reefs (Tea et al., 2020;Huang et al., 2022). Recognizing these limitations prompt the importance of obtaining samples or photographs at the least. ...
The Palawan archipelago is considered as the Philippines’ last ecological frontier, a Man and Biosphere Reserve, and a priority conservation area because of its high marine biodiversity. However, a comprehensive checklist of current marine fishes occurring in the province that can be used for diversity conservation priorities is still lacking. This paper aims to present an updated checklist of marine fishes recorded in Palawan waters (including new records) and their current conservation status. Underwater visual census (UVC) surveys (2003 – 2021), and published literature from 156 sites were used to compile the checklist, and the species’ updated nomenclatures were also presented. Currently, 1,056 marine fishes (32 elasmobranchs, 1,024 bony fishes) are included in the list, classified under two classes, 42 orders, 115 families, and 366 genera. More species were recorded in the Sulu Sea (904) than in the West Philippine Sea (557) which could be attributed to more studies being conducted in the former than in the latter. We also included 299 newly recorded species in this province, mostly from UVC surveys. Most of the species have Least Concern and Not Evaluated IUCN status, but a few species are already threatened, comprised mostly of elasmobranchs. Necessary information from this checklist is crucial in crafting a management plan for the protection and conservation of marine fishes in Palawan and nearby areas, especially those that are already declared as threatened species.
... This 45-mm-long (standard length) museum specimen is labeled as a juvenile Cephalopholis formosa. It was Tea et al. 2020). Based on the intermediate color patterns of the individuals we observed in Djibouti, they may be hybrids of Cephalopholis formosa and C. oligosticta (Fig. 2). ...
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We observed groupers with an unusual color pattern in Ghoubet-Al-Kharab Bay, Djibouti, on two occasions in 2014 and 2020. It matches the field observation of a juvenile Cephalopholis formosa in India in 1980. If this is the case, our observation represents a major range extension for C. formosa . Alternatively, based on the intermediate coloration of the specimens and sightings, we propose that they may be hybrids of C. formosa and C. oligosticta. We present two possible scenarios that may have facilitated hybridization: rarity and overlapping ranges in Djibouti with self-recruitment or rarity and overlapping ranges in another location (e.g., Socotra or the Arabian Sea) with long-distance larval dispersal to Djibouti. This hybridization is possible given the genetic similarity between the two putative parent species and because similar hybridization cases have been recorded within this genus and family elsewhere. However, both of these scenarios would require a range extension for one or both parent species as they are not previously known to overlap . Nevertheless, further field observations and genetic studies are required to verify the proposed identification of the putative hybrid and test the presented scenarios .
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Although commonly reported among widespread marine taxa, hybridization has only been recorded for one species of marine sponge to date. Sponges have evolved an array of sexual and asexual reproductive strategies, but it remains unclear if the lack of reported hybrids in this phylum is due to their reproductive diversity (which promotes reproductive isolation) or instead due to a lack of data. In this study, we aimed to determine whether hybridization occurs between two cryptic species of Tethya burtoni (T. burtoni sp. A and T. burtoni sp. B) that live sympatrically in central New Zealand. We also examined how both small-scale population structure (for five locations) and asexual reproduction contributed to instances (or lack thereof) of hybridization for these sponges. Using 11 microsatellite markers, we found no evidence of hybridization between species. Both species exhibited differences in distribution, where one species was present at all five locations, but the other was only detected at three locations. For these three locations (all within 20 km of each other), both species exhibited high levels of gene flow. Asexual buds did not appear to disperse far, and groups of clonal individuals were found within areas of < 900 cm². Asexual reproduction, therefore, did not play an obvious role in connectivity between populations, but instead appeared to be important for population maintenance. While specific mechanisms for reproductive isolation, such as gamete recognition, between both T. burtoni species remain unknown, we suggest that such mechanisms likely exist due to the strong differentiation found between both species (FST = 0.191, P < 0.0001). Further, the observed high levels of both gene flow and asexual reproduction may also act to reduce the potential for hybridization by maintaining genetic diversity and increasing the chance of mating with a conspecific individual.
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Hybridisation among species of coral-reef fish was previously considered to be rare. However, recent studies have revealed that hybridisation is prevalent in coral-reef fish, highlighting the need to understand the causes of this process. The angelfishes (family Pomacanthidae) have the greatest proportion (~ 30%) of hybridising species to date, with 26 species reported to hybridise. The aim of this study was to examine ecological factors (rarity of parent species and niche overlap) that promote hybridisation in terrestrial environments and to test these factors in the marine environment by examining hybridising angelfishes at Christmas Island (Indian Ocean). Three species of pygmy angelfish (Centropyge flavissima, C. eibli, and C. vrolikii) and their hybrids were investigated to address three objectives: (1) to determine temporal and spatial patterns in abundance and whether these patterns are linked to environmental factors; (2) to test for overlapping patterns in habitat use; and (3) to test for overlapping patterns in diet. Based on 14 yr of surveys, C. flavissima was abundant (4.53 individuals per 250 m² ± 0.66), whereas C. eibli, C. vrolikii, and all hybrid combinations were consistently rare (average abundance < 0.3 per 250 m² ± 0.03). Parent species and their hybrids were more abundant at 20 m depth compared to 5 m. All species and their hybrids had similar patterns of abundance around Christmas Island, with significantly high abundances evident at the most sheltered sites. Parent species and their hybrids also had similar diets that comprised a mix of green, red, and brown algae. The rarity of parent species, their niche overlap, and the haremic reproductive strategy likely promote hybridisation in angelfishes at Christmas Island. This study provides empirical evidence that hybridisation in reef fishes conforms to terrestrial-based hypothesis, and thus advances our understanding of the processes underlying hybridisation in coral-reef systems.
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Bayesian inference of phylogeny using Markov chain Monte Carlo (MCMC) (Drummond et al., 2002; Mau et al., 1999; Rannala and Yang, 1996) flourishes as a popular approach to uncover the evolutionary relationships among taxa, such as genes, genomes, individuals or species. MCMC approaches generate samples of model parameter values - including the phylogenetic tree -drawn from their posterior distribution given molecular sequence data and a selection of evolutionary models. Visualising, tabulating and marginalising these samples is critical for approximating the posterior quantities of interest that one reports as the outcome of a Bayesian phylogenetic analysis. To facilitate this task, we have developed the Tracer (version 1.7) software package to process MCMC trace files containing parameter samples and to interactively explore the high-dimensional posterior distribution. Tracer works automatically with sample output from BEAST (Drummond et al., 2012), BEAST2 (Bouckaert et al., 2014), LAMARC (Kuhner, 2006), Migrate (Beerli, 2006), MrBayes (Ronquist et al., 2012), RevBayes (Höhna et al., 2016) and possibly other MCMC programs from other domains.
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For many animals, affiliative relationships such as pair bonds form the foundation of society and are highly adaptive. Animal systems amenable for comparatively studying pair bonding are important for identifying underlying biological mechanisms, but mostly exist in mammals. Better establishing fish systems will enable comparison of pair bonding mechanisms across taxonomically distant lineages that may reveal general underlying mechanistic principles. We examined the utility of wild butterflyfishes (f: Chaetodontidae; g: Chaetodon) for comparatively studying pair bonding. Using stochastic character mapping, we provide the first analysis of the evolutionary history of butterflyfish sociality, revealing that pairing is ancestral, with at least seven independent transitions to gregarious grouping and solitary behavior since the late Miocene. We then formally verified social systems in six sympatric and wide-spread species representing a clade with one ancestrally reconstructed transition from paired to solitary grouping at Lizard Island, Australia. In situ observations of the size, selective affiliation and aggression, fidelity, and sex composition of social groups confirmed that Chaetodon baronessa, C. lunulatus, and C. vagabundus are predominantly pair bonding, whereas C. rainfordi, C. plebeius, and C. trifascialis are predominantly solitary. Even in the predominantly pair bonding species, C. lunulatus, a proportion of adults (15%) are solitary. Importantly, inter- and intra-specific differences in social systems do not co-vary with other previously established attributes, including parental care. Hence, the proposed butterflyfish populations are promising for inter- and intra-species comparative analyses of pair bonding and its mechanistic underpinnings. Avenues for further developing the system are proposed, including determining whether the aforementioned utility of these species applies across their geographic disruptions.
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Coral reefs are highly diverse ecosystems, where numerous closely related species often coexist. How new species arise and are maintained in these high geneflow environments have been long-standing conundrums. Hybridization and patterns of introgression between sympatric species provide a unique insight into the mechanisms of speciation and the maintenance of species boundaries. In this study, we investigate the extent of hybridization between two closely related species of coral reef fish: the common coral trout (Plectropomus leopardus) and the bar-cheek coral trout (Plectropomus maculatus). Using a complementary set of 25 microsatellite loci, we distinguish pure genotype classes from first- and later-generation hybrids, identifying 124 interspecific hybrids from a collection of 2,991 coral trout sampled in inshore and mid-shelf reefs of the southern Great Barrier Reef. Hybrids were ubiquitous among reefs, fertile and spanned multiple generations suggesting both ecological and evolutionary processes are acting to maintain species barriers. We elaborate on these finding to investigate the extent of genomic introgression and admixture from 2,271 SNP loci recovered from a ddRAD library of pure and hybrid individuals. An analysis of genomic clines on recovered loci indicates that 261 SNP loci deviate from a model of neutral introgression, of which 132 indicate a pattern of introgression consistent with selection favouring both hybrid and parental genotypes. Our findings indicate genome-wide, bidirectional introgression between two sympatric species of coral reef fishes and provide further support to a growing body of evidence for the role of hybridization in the evolution of coral reef fishes.
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The pygmy angelfishes (genus Centropyge) provide numerous examples of discordance between color morphology, taxonomy and evolutionary genetic lineages. This discordance is especially evident in the Centropyge flavissima complex, which includes three primary color morphs, three previously recognized species (C. flavissima, C. eibli and C. vrolikii) and three distinct mitochondrial (mtDNA) lineages that do not align with species designations. Our previous research showed that the putative C. flavissima arose independently in the Indian and Pacific Oceans, and the three mtDNA lineages align with geography rather than species assignments. Here we add 157 specimens to the previous dataset of 291 specimens, spread across a greater geographic range, to pinpoint the distribution of mtDNA lineages and color morphs. We found that the mtDNA lineages show remarkably strong geographic boundaries corresponding to the Indian Ocean, Central-West Pacific and Central-South Pacific. We also test the validity of the ‘Black Tiger Centropyge’ in the C. flavissima species complex, a taxonomic oddity that is restricted to shoals and atolls off the coast of northwestern Australia, and the newly named C. cocosensis (Shen et al. sp. nov. 2016) assigned to the C. flavissima lineage in the Indian Ocean. We conclude that the Black Tiger Centropyge is not a valid species but an intermediate between sympatric color morphs that correspond to the putative species C. eibli and C. vrolikii. Our greater sampling effort also do not support the genetic distinctiveness of C. cocosensis given shared mtDNA haplotypes with the sympatric C. eibli and C. vrolikii, but instead we find conflicting lines of evidence concerning the taxonomy of this group. We urge caution and taxonomic restraint until the true nature of this species complex can be revealed.
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Hybridisation is a significant evolutionary process that until recently was considered rare in the marine environment. A suture zone in the eastern Indian Ocean is home to numerous hybridising sister species, providing an ideal opportunity to determine how hybridisation affects speciation and biodiversity in coral reef fishes. At this location, hybridisation between two grouper (Epinephelidae) species: Cephalopholis urodeta (Pacific Ocean) and C. nigripinnis (Indian Ocean) was investigated to determine the genetic basis of hybridisation and to compare the ecology and life history of hybrids and their parent species. This approach aimed to provide insights into the taxonomic and evolutionary consequences of hybridisation. Despite clear phenotypic differences, multiple molecular markers revealed hybrids, and their parent species were genetically homogenous within and (thousands of kilometres) outside of the hybrid zone. Hybrids were at least as fit as their parent species (in terms of growth, reproduction, and abundance) and were observed in a broad range of intermediate phenotypes. The two species appear to be interbreeding at Christmas Island due to inherent biological and ecological compatibilities, and the lack of genetic structure may be explained by three potential scenarios: (1) hybridisation and introgression; (2) discordance between morphology and genetics; and (3) incomplete lineage sorting. Further molecular analyses are necessary to discriminate these scenarios. Regardless of which applies, C. urodeta and C. nigripinnis are unlikely to evolve in reproductive isolation as they cohabit where they are common (Christmas Island) and will source congeneric mates where they are rare (Cocos Keeling Islands). Our results add to the growing body of evidence that hybridisation among coral reef fishes is a dynamic evolutionary factor.
Marine angelfishes (F: Pomacanthidae) are amongst the most conspicuous reef fish families inhabiting reefs on tropical and subtropical latitudes. While being disproportionately represented in the marine ornamental fish trade, only a handful of taxonomically restricted studies explored their biogeographic history and the evolution body size and trophic guilds. Here, we reconstruct the phylogenetic history for 70 pomacanthid species (85% of nominal species), based on previously published data for three nuclear and four mitochondrial markers. We use the resulting phylogenetic framework to explore the ancestral biogeography and ecological diversification of the family. Divergence times and ancestral range estimation highlight the origins of the family most likely lie in the Central Pacific region. Vicariance among ocean basins reflects the impact of the Terminal Tethyan Event and the closure of the Isthmus of Panama in the historical biogeography of Pomacanthus and Holacanthus genera. The reconstruction also uncovers ancestral colonization pathways via the Pacific Ocean into the western Atlantic waters for Holacanthus. We confirm the Indian Ocean invasion scenario previously proposed for the “acanthops” complex (genus: Centropyge). Finally, interspecific variation in body size among clades appeared to be correlated to some degree with trophic guilds, whereby 15% of variance in body size was explained by trophic modes. This suggests that the higher ecological diversification observed in the Centropyge clade might be promoted by smaller body sizes acting as an ecological novelty allowing the expansion of the genus within available niches.
Diagnostic molecular markers are an essential tool in the study of species' ecology and evolution, particularly in closely related and sympatric species. Furthermore, the increasing awareness of wild-hybrids has led to a renewed interest in rapid diagnostic assays. Here, we test the ability of two mitochondrial (Cytb and COI) and two nuclear markers (ETS2 and TMO-4c4) to confidently discriminate purebred P. leopardus and P. maculatus and their first-generation hybrids. A sample of 48 purebred individuals and 91 interspecific hybrids were used in this study and their delineation confirmed using a set of microsatellite markers. Our results indicate mitochondrial markers could not distinguish even between species but both nuclear markers confidently identified species and first-generation hybrids. However, later-generation hybrids could not always be confidently identified due to ongoing introgression between species. Our findings provide a robust tool to distinguish purebred individuals and interspecific hybrids in a pair of species with an unexpectedly high incidence of hybridization. The quick species discrimination abilities provided by these diagnostic markers are important for stock assessment and recruitment studies of these important fishery species.
Species identification based on the DNA sequence of a fragment of the cytochrome c oxidase subunit I (COI) gene in the mitochondrial genome, DNA barcoding, is widely applied to assist in sustainable exploitation of fish resources and the protection of fish biodiversity. The aim of this study was to establish a reliable barcoding reference database of the native ray-finned fishes in Taiwan. A total of 2,993 individuals, belonging to 1,245 species within 637 genera, 184 families, and 29 orders of ray-finned fishes and representing approximately 40% of the recorded ray-finned fishes in Taiwan, were PCR amplified at the barcode region and bidirectionally sequenced. The mean length of the 2,993 barcodes is 549 bp. Mean congeneric K2P distance (15.24%) is approximately 10-fold higher than the mean conspecific one (1.51%), but roughly 1.4-fold less than the mean genetic distance between families (20.80%). The Barcode Index Number (BIN) discordance report shows that 2,993 specimens represent 1,275 BINs and, among them, 86 BINs are singletons, 570 BINs are taxonomically concordant, and the other 619 BINs are taxonomically discordant. Barcode gap analysis also revealed that more than 90% of the collected fishes in this study can be discriminated by DNA barcoding. Overall, the barcoding reference database established by this study reveals the need for taxonomic revisions and voucher specimen rechecks, in addition to assisting in the management of Taiwan's fish resources and diversity. This article is protected by copyright. All rights reserved.