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Journal of Biogeography (J. Biogeogr.

) (2015) 42, 16011614

ORIGINAL
ARTICLE

When biogeographical provinces collide:


hybridization of reef fishes at the
crossroads of marine biogeographical
provinces in the Arabian Sea
Joseph D. DiBattista1,2*, Luiz A. Rocha3, Jean-Paul A. Hobbs2, Song He1,
Mark A. Priest1,4, Tane H. Sinclair-Taylor1, Brian W. Bowen5 and
Michael L. Berumen1

Red Sea Research Center, King Abdullah


University of Science and Technology, Thuwal,
23955-6900, Saudi Arabia, 2Department of
Environment and Agriculture, Curtin
University, PO Box U1987, Perth, WA 6845,
Australia, 3Section of Ichthyology, California
Academy of Sciences, San Francisco, CA 94118,
USA, 4Marine Spatial Ecology Laboratory,
University of Queensland, St. Lucia, Brisbane,
QLD 4072, Australia, 5Hawaii Institute of
Marine Biology, Kaneohe, HI 96744, USA

ABSTRACT

Aim Suture zones are areas where closely related species from different biogeographical regions come into contact and interbreed. This concept originated
from the study of terrestrial ecosystems but it remains unclear whether a similar phenomenon occurs in the marine environment. Here we investigate a
potential suture zone from a previously unknown hybrid hotspot at the Socotra Archipelago (Yemen), located in the Arabian Sea, where fauna from the
Red Sea, Gulf of Aden, Arabian Sea, western Indian Ocean and greater IndoPolynesian Province intersect.
Location Red Sea, Gulf of Aden, Arabian Sea and Indian Ocean.
Methods Putative hybrid reef fish were identified based on intermediate coloration and morphology. Underwater observations and collections were conducted to determine: (1) whether parent species form heterospecific social
groups or breeding pairs; (2) the sex and reproductive status of morphologically intermediate individuals; and (3) whether parent species were forming
mixed species associations owing to a dearth of conspecific partners. To support hybrid status, morphologically intermediate and parental individuals were
genotyped using mitochondrial DNA cytochrome c oxidase subunit I (COI),
nuclear recombination-activating gene 2 (RAG2) and the nuclear TMO-4C4
(TMO) gene.
Results We observed putative hybrids involving 14 species from four reef fish
families at Socotra. Most cases involved a parental species with a restricted distribution (e.g. Red Sea or Arabian Sea) and a broadly distributed Indo-Pacific
species. In most cases, at least one of the parent species was rare at Socotra.
Hybrid gene flow was largely unidirectional, and although introgression was
rare, we found evidence that some butterflyfish and surgeonfish hybrids were
fertile and formed breeding groups with parental species.

*Correspondence: Joseph D. DiBattista,


Department of Environment and Agriculture,
Curtin University, PO Box U1987, Perth, WA
6845, Australia.
E-mail: josephdibattista@gmail.com

2015 John Wiley & Sons Ltd

Main conclusions The rate of hybrid discovery at Socotra is much greater


than that recorded elsewhere in the marine environment and involved both
allopatric and sympatric species. This study highlights the importance of biogeographical location, reef habitat, environmental conditions and abundance
disparities at Socotra in potentially facilitating hybridization among reef fishes
at the edge of their distribution.
Keywords
Coral reef fish, Gulf of Aden, hybrid hotspot, introgression, mitochondrial
DNA, nuclear DNA, Socotra.

http://wileyonlinelibrary.com/journal/jbi
doi:10.1111/jbi.12526

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J. D. DiBattista et al.
INTRODUCTION
Hybridization has long been recognized as an important evolutionary process in freshwater and terrestrial systems, but is
considered rare or unimportant in marine systems, particularly in tropical seas (Hubbs, 1955; Arnold, 1997). However,
many recent studies have shown that hybridization is common in a wide range of marine organisms (Gardner, 1997),
including algae (Monteiro et al., 2012), corals (van Oppen et
al., 2001; Willis et al., 2006), echinoderms (Harper & Hart,
2007), mussels (Riginos & Cunningham, 2005), teleost fishes
(Schwenke, 2012), rays (Walter et al., 2013) and reptiles
(Sanders et al., 2014). Hybridization in terrestrial species is
concentrated at the boundaries of biogeographical provinces
where allopatric sister species come into secondary contact
(termed suture zones; Remington, 1968; Hewitt,
2000). These hybrid hotspots cover a small area of the globe,
but have disproportional importance in the evolution and
maintenance of biodiversity. In the marine environment,
hybridization is similarly prevalent at biogeographical borders
(Hobbs et al., 2009); however, initial findings indicate that
secondary contact between allopatric sister species may only
explain a minority of hybrid cases (Hobbs et al., 2013). Determining whether hybridization in the marine environment fits
the terrestrial model is important in order to identify the locations and processes that generate evolutionary novelty.
The prevalence of hybridization at suture zones has been
well documented in terrestrial and freshwater environments
(Mallet, 2005; Arnold, 2006). In contrast, the only known
marine suture zone occurs in the eastern Indian Ocean at
the Indo-Pacific biogeographical border (Christmas Island
and the Cocos-Keeling Islands; Hobbs & Salmond, 2008;
Hobbs et al., 2009; Hobbs & Allen, 2014). This border represents a narrow zone of contact between Indian and Pacific
Ocean fauna that diverged in allopatry following the formation of land bridges in the Indonesian archipelago during the
Pleistocene (Sunda Shelf Barrier; Rocha et al., 2007). Given
that at least six biogeographical provinces exist in the tropical Indo-West Pacific region (Spalding et al., 2007; Briggs &
Bowen, 2012), and that half of them intersect in the northwestern Indian Ocean (see Fig. 1), this region is ripe for
exploration to evaluate the existence, prevalence and generality of tropical marine suture zones.
The alternative to the suture zone model (secondary contact in allopatric species) is hybridization between sympatric
species. Under the sympatric model, hybridization can occur
anywhere two species co-occur. Preliminary research into
coral reef butterflyfishes indicates that the majority of
hybridization occurs between sympatric species (63% of
cases), but interestingly these cases are most common at biogeographical borders (Hobbs et al., 2013). Thus, conditions
at biogeographical borders appear to promote hybridization
among both sympatric and allopatric species. A lack of conspecific partners may facilitate the breakdown in assortative
mating and drive species to hybridize (Arnold, 1997). Given
that a species abundance is expected to decrease towards its
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range edge (Brown, 1984), hybridization may be prevalent at


biogeographical borders because many species are at their
range edge and have low abundance. Although, the abundant centre hypothesis does not appear to hold true for
many marine species (Sagarin & Gaines, 2002), rarity at the
range edge may still be important given that the majority of
hybrid cases involve parent species that are rare (Hobbs
et al., 2009, 2013; Hobbs & Allen, 2014).
The Socotra Archipelago (see Fig. 1) is located at the
boundaries of three marine biogeographical provinces: the Red
Sea and Gulf of Aden, Western Indian Ocean, and Indo-Polynesian provinces as defined by Briggs & Bowen (2012, 2013).
The archipelago lies 380 km south-east of the coast of mainland Yemen, 100 km east of the Horn of Africa and is the most
biologically diverse island group in the Arabian Region. Socotra is internationally renowned for its unique terrestrial biodiversity (van Damme, 2009); however, much less is known
about the marine life (but see Kemp, 1998; Zajonz et al., 2000;
DeVantier et al., 2004; Baird et al., 2014). Although the
marine community is considered part of the Western Arabian
Sea Ecoregion (Spalding et al., 2007) it represents a transition
zone where distinct but related faunal communities overlap
and create the potential for species to hybridize.
Hybridization may also be facilitated by the unique environmental conditions that shaped Socotra. The archipelago
lies at the epicentre of one of the most geologically and
oceanographically variable regions of the worlds tropical
oceans. The Red Sea, a region severely affected by fluctuations
in sea level, temperature and salinity over the last 2 Myr, lies
1135 km to the west (Siddall et al., 2003). The southern coast
of Oman, with major fluctuations in annual temperature and
nutrient regimes, lies 630 km to the north-east (McIlwain
et al., 2011). During the South-West Monsoon (May to September), the northward flowing Somali Current causes an
upwelling of cold nutrient-rich water along the south coast of
the Socotra Archipelago (min. temp. 13 C off the Somali
coast; Currie et al., 1973). During the North-East Monsoon
(October to February), current direction is reversed and
results in elevated water temperatures (c. 27 C) and higher
wave energy on the north coast of the archipelago (Kemp,
1998). Socotra may therefore represent a site where larvae
from ecologically diverse sources are settling; populations will
persist or decline based on their ability to deal with the local
conditions. The importance of Socotra as a natural ecological
and evolutionary laboratory therefore reflects three properties:
(1) the dramatically different environments that characterize
the surrounding regions; (2) the small geographical scale over
which these environmental differences occur and currents
capable of transporting pelagic larvae from adjacent regions;
and (3) the location at the biogeographical crossroads of
these regions (Kemp, 1998; DeVantier et al., 2004).
In this study, we outline findings from our investigation
of putative hybrid coral reef fishes at the Socotra Archipelago. We determine whether hybridization has occurred as a
result of secondary contact between previously isolated
species from the three distinct biogeographical provinces,
Journal of Biogeography 42, 16011614
2015 John Wiley & Sons Ltd

Marine hybrid hotspot at Socotra

Figure 1 Map indicating collection sites for all reef fish considered in this study. Putative hybrids were sampled exclusively at Socotra,
whereas the parental species, in addition to Socotra, were sourced from reference populations in the adjacent Red Sea, Gulf of Aden,
Arabian Sea and Indian Ocean. Site-specific samples sizes are provided in Table S1 in Appendix S1. The Socotra Archipelago is located
in the Western Arabian Sea Ecoregion, at the boundary of three major biogeographical provinces: the Red Sea and Gulf of Aden (red
shading), Western Indian Ocean (green shading) and Indo-Polynesian (blue shading). The inset shows the seven survey sites on the
north-eastern coast of the main island of Socotra.

examine the role of ecological and behavioural factors in


facilitating hybridization and explain how these factors may
be influenced by variable environmental conditions at this
isolated archipelago.
MATERIALS AND METHODS
Sample collection and ecological data
Putative hybrid coral reef fishes were first identified based on
intermediate coloration and morphology during underwater
surveys at seven sites along the north-eastern coast of the
main island of Socotra in October 2013 (Fig. 1; see Table S1
in Appendix S1 for sample size information). The taxonomy
and coloration of reef fishes is well known, particularly for
conspicuous groups (e.g. butterflyfishes), and identification of
intermediate coloration was based on the authors extensive
experience in observing, surveying and collecting coral reef
fishes throughout the Indo-Pacific, including the Red Sea.
Intermediate coloration has proven to be an accurate and
reliable predictor of reef fish hybrids (Marie et al., 2007; YaJournal of Biogeography 42, 16011614
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akub et al., 2006; Hobbs et al., 2009; DiBattista et al., 2012a;


Montanari et al., 2014). Individuals with intermediate coloration were collected from depths of 1 to 26 m, photographed,
morphologically examined and sequenced at one mitochondrial DNA (mtDNA) and two nuclear markers to test hybrid
status. Genetic analysis of putative hybrids and parental fish
from Socotra were supplemented with DNA sequences of
pure parental populations previously sampled in the adjacent Red Sea, Arabian Sea or Indian Ocean. The parental species examined in this study represent distinct lineages based
on established phylogenetic analyses (Fessler & Westneat,
2007; Hsu et al., 2007; Bellwood et al., 2010; Litsios et al.,
2012; Sorenson et al., 2013; M.R. Gaither & J.E. Randall, unpubl. data). Select fish specimens were subsequently preserved
in formalin and vouchered at the California Academy of Sciences (see Table S2 in Appendix S1).
To determine whether low conspecific abundance is linked
to putative hybridization at Socotra, average abundances for
each parental species (and putative hybrid individuals) were
recorded during underwater visual censuses consisting of
three replicate (50 m 9 5 m) transects per site. Species were
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J. D. DiBattista et al.
considered rare if their mean abundance was less than one
individual per 250 m2 and if their mean abundance did not
differ significantly from zero (one-sample, one-tailed t-test;
Zar, 1999). Owing to their small size or cryptic behaviour,
Cirrhitichthys and Dascyllus species were excluded from these
surveys. To determine whether there was a breakdown in
assortative mating, heterospecific social groups and breeding
pairs were collected following observations of group members. The sex and maturity of some captured individuals
were determined through macroscopic or microscopic examination of gonads (Hobbs et al., 2004; Mackie et al., 2005).
DNA sequencing
Tissue samples were preserved in salt-saturated (NaCl) solution with 20% dimethyl sulfoxide (Seutin et al., 1991). Total
genomic DNA was extracted using the HotSHOT protocol
(Meeker et al., 2007) and subsequently stored at 20 C.
Fragments of mtDNA from the cytochrome c oxidase subunit
I gene (COI), or nuclear DNA from the recombination-activating gene 2 (RAG2) and TMO-4C4 (TMO) (for a subset of
samples, see Table S1), were amplified using the primers
FishF2 and FishR2 (Ward et al., 2005) and modified primers
of DiBattista et al. (2012a), respectively. Alternative variants
of RAG2 and TMO primers were tested (Lovejoy, 1999; Streelman et al., 2002), in addition to primers for nuclear genes
commonly used for phylogenetics [S7 ribosomal protein gene
(Chow & Hazama, 1998) and bone morphogenetic protein 4
(BMP4; Cooper et al., 2009)], but none of these provided
consistent amplification across species (data not shown). The
more variable mtDNA control region was amplified for select
species (where COI data was ambiguous) using the primers
CRA and CRE developed by Lee et al. (1995).
Polymerase chain reaction (PCR) mixes contained BioMix
Red (Bioline Inc., Springfield, NJ, USA), 0.26 lm of each primer and 550 ng template DNA in 15 lL total volume. PCR
cycling parameters were as follows: initial 95 C denaturation
for 3 min, followed by 35 cycles of 94 C for 30 s, species- and
marker-specific annealing temperature for 60 s (see Table S1),
and 72 C for 60 s, with a final elongation step of 72 C for
10 min. All PCR products were cleaned by incubating with
exonuclease I and FastAPtm Thermosensitive Alkaline Phosphatase (ExoFAP; USB, Cleveland, OH, USA) at 37 C for
60 min, followed by 85 C for 15 min. DNA was sequenced in
the forward direction (and reverse direction for questionable
haplotypes or genotypes) with fluorescently labelled dye terminators following manufacturers protocols (BigDye; Applied
Biosystems Inc., Foster City, CA, USA), and analysed using an
ABI 3130XL Genetic Analyzer (Applied Biosystems). The
sequences were aligned, edited and trimmed to a uniform
length using Geneious Pro 5.6.7 (Drummond et al., 2009). In
all cases, the alignment was unambiguous with no frameshift
mutations or indels; individual mtDNA and nuclear sequences
are deposited in GenBank [accession numbers: KR023187
KR023620 (COI); KR022205KR022636 (RAG2); KR022637
KR023186 (TMO)].
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Allelic states of nuclear sequences trimmed to a uniform


length with more than one heterozygous site (three or four
out of seven species datasets for RAG2 or TMO, respectively)
were estimated using the Bayesian program phase 2.1 (Stephens & Donnelly, 2003) as implemented in the software
DnaSP 5.0 (Librado & Rozas, 2009). We conducted three
runs in phase for each dataset with a burn-in of 10,000 and
200,000 iterations. All runs returned consistent allele identities and phase was able to differentiate most alleles with >
95% probability except for a few Chaetodon melapterus
(RAG2, n = 3), C. trifasciatus (RAG2, n = 2), Cirrhitichthys
calliurus (TMO, n = 2), C. oxycephalus (TMO, n = 3), Dascyllus carneus (RAG2, n = 11) and D. marginatus (RAG2,
n = 5; TMO, n = 2), which were all excluded from further
nuclear DNA analysis.
The evolutionary relationship among COI haplotypes,
RAG2 alleles or TMO alleles was resolved for each pair of
potentially hybridizing species with unrooted networks constructed with the program Network 4.5.1.0 (http://www.flux
us-engineering.com/sharenet.htm) using a median-joining
algorithm. We deemed evidence of introgression among interbreeding species to represent genetic support for hybrid status.
RESULTS
Our field survey of reef fishes at the Socotra Archipelago
identified seven putative hybrids from four reef fish families
(Table 1, Fig. 2, Table S1). In four of these cases, the parental species were at the periphery of their geographical range,
most often Red Sea to Gulf of Aden endemics interacting
with widespread Indian Ocean species (Table 1). Our underwater surveys and previous data (Zajonz et al., 2000) reveal
that in six of the seven cases, either one or both parental
species were rare (i.e. abundance not significantly different
from zero; one-sample t-test t0.05 (1), 8 < 1.86, P > 0.05; see
Table 1). Heterospecific social groups were documented with
a combination of behavioural, reproductive or morphological
data. Genetic analyses further supported hybrid status in four
of seven cases based on introgression of mtDNA or separation among nuclear alleles, but were inconclusive for the
remaining three cases, which should still be regarded as putative hybrids (based on intermediate coloration).
Chaetodon collare Bloch, 1787 and Chaetodon lunula
de, 1802)
(Lacepe
These butterflyfish species overlap across most of their broad
range in the Indian Ocean, and Socotra represents the northwestern range edge for both species. The two species group
together in the same clade based on multi-gene phylogenies
of the family Chaetodontidae (Fessler & Westneat, 2007; Hsu
et al., 2007; Bellwood et al., 2010), but no hybrids have previously been reported (Hobbs et al., 2013). We collected one
solitary mature female, which had intermediate coloration
(Fig. 2c). A second putative hybrid was observed paired with
C. collare. Both parental species were rare at Socotra and
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Journal of Biogeography 42, 16011614


2015 John Wiley & Sons Ltd
L,S,H

Acanthuridae
Acanthurus lineatus (species 1)
Acanthurus sohal (species 2)
16
Indo-Pacific

0.14*,2
NW Indian Ocean

0*
Red Sea & Gulf of Aden
0.06*,2
Indian Ocean

4.67
Red Sea to Arabian Gulf

0.14*,2
Indo-Pacific

0*
NW Indian Ocean
133.40*,2
Red Sea

0*
Indian Ocean

0*
NW Indian Ocean

0.67*
Indo-Pacific

species 2

species 1 (0.09)
species 2 (0.91)
introgression (yes)

species 1 (0)
species 2 (1.0)
introgression (no)

species 1 (0)
species 2 (1.0)
introgression (no)

no mtDNA separation

species 1 (0)
species 2 (1.0)
introgression (no)
species 1 (0)
species 2 (1.0)
introgression (n/a)
species 1 (0)
species 2 (1.0)
introgression (yes)

mtDNA
(introgression?)

Indicates species that were not observed during our surveys, but have been observed previously at Socotra (Kemp, 1998; Zajonz et al., 2000).
Indicates densities for species that were not surveyed in this study but have been surveyed previously (Zajonz et al., 2000).
*Denotes rare species whose abundance was not significantly different from zero (one-sample t-test: t0.05 (1),8 < 1.86, P > 0.05).

C,O1,H

Cirrhitidae
Cirrhitichthys calliurus (species 1)
Cirrhitichthys oxycephalus (species 2)

C1,M,H

B1,O1,H

M,T,H

Chaetodon melapterus (species 1)


Chaetodon trifasciatus (species 2)

Pomacentridae
Amphiprion bicinctus (species 1)
Amphiprion omanensis (species 2)
Dascyllus carneus (species 1)
Dascyllus marginatus (species 2)

0*
W Indian Ocean

G1,L,H

Chaetodon gardineri (species 1)


Chaetodon leucopleura (species 2)
12.78
W Indian Ocean

0*
Indo-West Pacific

C,L,H

Chaetodontidae
Chaetodon collare (species 1)
Chaetodon lunula (species 2)

species 1

Observed

Putative hybrid

Mean density per 250 m2 and geographical range

distinct (RAG2 only)

yes (TMO only)

yes (TMO only)

HS,C,MG

M,C

M,C

HP,C,MG

no

no

HS,C,MG

C,MG

Hybrid support

n/a

yes (RAG2 & TMO)

nuclear DNA
(parents distinct?)

Table 1 Abundance, geographical distribution, and genetic composition of species potentially hybridizing at the Socotra Archipelago. Observations are based on the occurrence of parental
species (denoted by the first letter of the species name) and putative hybrids (H) during 5 days of diving or snorkelling on reefs between 1 and 26 m depth. The estimated mean densities
(individuals per 250 m2) of parent species are where parent species 1 is the species listed first in the putative hybrid column and parent species 2 is listed second. Geographical range of
parent species is based on published reports (Froese & Pauly, 2013). Mitochondrial DNA (mtDNA) indicates the proportion of putative hybrids grouping with each parent species, and in
parentheses, whether there is evidence for introgression (i.e. mismatch between morphology and mtDNA) based on the cytochrome c oxidase subunit I (COI) gene (see also Fig. 3 and
Appendix S3). Nuclear DNA indicates the proportion of putative hybrids grouping with each parent species, and in parentheses, whether the two parental species are genetically distinct
based on the recombination-activating gene 2 (RAG2) or TMO-4C4 (TMO) gene (see also Fig. 4). Additional support for each putative hybrid is listed as: heterospecific social groups (HS)
or heterospecific breeding pair (HP) of the parent species, intermediate coloration (C), intermediate morphology (M; traits other than colour), and mature gonads (MG) of the
interbreeding species.

Marine hybrid hotspot at Socotra

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J. D. DiBattista et al.
(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)

(p)

(q)

(r)

(s)

(t)

individuals seen outside of transects were either single or in


homospecific pairs or groups. The putative hybrids were not
recorded in surveys but observations outside of transects
indicated that they were rarer than both parental species.
The sampled putative hybrid had an mtDNA signature characteristic of C. lunula and shared nuclear alleles among the
parental lineages (see Appendix S2), which were distinct at
RAG2 and TMO (Figs 3a & 4a).
1606

Figure 2 The Socotra Archipelago


constitutes a marine suture zone where
closely related reef fish species come into
secondary contact and interbreed to
potentially produce hybrids. The photos in
the panel represent the following: (a)
Chaetodon collare; (b) Chaetodon lunula; (c)
C. collare 9 C. lunula putative hybrid; (d)
Chaetodon gardineri; (e) Chaetodon
leucopleura; (f) C. gardineri 9 C. leucopleura
putative hybrid; (g) heterospecific pair of
Chaetodon melapterus (left) and Chaetodon
trifasciatus (right); (h) C. melapterus 9 C.
trifasciatus putative hybrids; (i) Amphiprion
bicinctus; (j) Amphiprion omanensis; (k) A.
bicinctus 9 A. omanensis putative hybrid; (l)
Dascyllus carneus; (m) Dascyllus marginatus;
(n) D. carneus 9 D. marginatus putative
hybrid; (o) Cirrhitichthys calliurus; (p)
Cirrhitichthys oxycephalus; (q) C. calliurus 9
C. oxycephalus putative hybrid; (r)
Acanthurus lineatus; (s) Acanthurus sohal; (t)
A. lineatus 9 A. sohal putative hybrid. Note
that photo panels (d), (i), (j), (l), (m) and
(p) were not taken at Socotra but at
adjacent reference sites. Photo credits: (d)
J.E. Randall; (c), (e), (f), (h), (k), (n), (o),
(q) J-P.A.H.; (a), (b), (g), (i), (j), (l), (m),
(p), (r), (s), (t) T.H.S-T.

Chaetodon gardineri Norman, 1939 and Chaetodon


leucopleura Playfair, 1867
These two species are from different biogeographical regions
(Western Indian Ocean Province and Western Arabian Sea
Bioregion), but both reach their range edge at Socotra. The
parental species were not observed during surveys, however
we did note eight C. leucopleura and one putative hybrid
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Newman, S.J., Frisch, A.J. & van Oppen, M. (2006)
Contrasting patterns of genetic structure in two species of
the coral trout Plectropomus (Serranidae) from east and
west Australia: introgressive hybridisation or ancestral
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Hewitt, G.M. (2000) The genetic legacy of the Quaternary
ice ages. Nature, 405, 907913.
Hobbs, J.-P.A. & Allen, G.R. (2014) Hybridisation among
coral reef fishes at Christmas Island and the Cocos (Keeling) Islands. Raffles Bulletin of Zoology, Supplement, 30,
220226.
Hobbs, J.-P.A. & Salmond, J. (2008) Cohabitation of Indian
and Pacific Ocean species at Christmas and Cocos (Keeling) islands. Coral Reefs, 27, 933933.
Hobbs, J.-P.A., Munday, P.L. & Jones, G.P. (2004) Social
induction of maturation and sex determination in a coral
reef fish. Proceedings of the Royal Society B: Biological Sciences, 271, 21092114.
Hobbs, J.-P.A., Frisch, A.J., Allen, G.R. & van Herwerden, L.
(2009) Marine hybrid hotspot at Indo-Pacific biogeographic border. Biology Letters, 5, 258261.
Hobbs, J.-P.A., van Herwerden, L., Pratchett, M.S. & Allen,
G.R. (2013) Hybridization among butterflyfishes. Biology
of butterflyfishes (ed. by M.S. Pratchett, M.L. Berumen and
B. Kapoor), pp. 4869. CRC Press, Boca Raton, FL.
Hsu, K.C., Chen, J.P. & Shao, K.T. (2007) Molecular phylogeny of Chaetodon (Teleostei: Chaetodontidae) in the
Indo-West Pacific: evolution in geminate species pairs
and species groups. The Raffles Bulletin of Zoology, 14,
7786.
Hubbs, C.L. (1955) Hybridization between fish species in
nature. Systematic Zoology, 4, 120.
Johannesson, K. & Andre, C. (2006) Life on the margin:
genetic isolation and diversity loss in a peripheral marine
ecosystem, the Baltic Sea. Molecular Ecology, 15, 2013
2029.
Karl, S.A. & Avise, J.C. (1992) Balancing selection at allozyme loci in oysters: implications from nuclear RFLPs. Science, 256, 100102.
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50 species of the northeastern Pacific rocky intertidal community. PLoS ONE, 5, e8594.
Kemp, J. (1998) Zoogeography of the coral reef fishes of
the Socotra Archipelago. Journal of Biogeography, 25,
919933.
Journal of Biogeography 42, 16011614
2015 John Wiley & Sons Ltd

J. D. DiBattista et al.

Species 1, ref. pop.


Species 2, ref. pop.
Species 1, Socotra
Species 2, Socotra
Hybrid, Socotra

RAG2

TMO

(a)

(b)

(c)

(d)

(e)

(f)

10 mutations

(g)

tive hybrids were even rarer and not observed in transects


(Table 1). In addition to the putative hybrids, six heterospecific pairs were observed in the field. Two of these pairs
were collected, and in both cases C. trifasciatus was a
mature female and C. melapterus was a mature male
(Table 1). The mtDNA from C. trifasciatus females was
1608

Figure 4 Median-joining networks showing


relationships among nuclear DNA alleles
[recombination-activating gene 2 (RAG2)
and TMO-4C4 (TMO) gene] based on a
subset of each pair of study species that
appear to hybridize at Socotra [(a)
Chaetodon collare (species 1) and Chaetodon
lunula (species 2), n = 82 RAG2 and n = 86
TMO alleles; (b) Chaetodon gardineri
(species 1) and Chaetodon leucopleura
(species 2), n = 10 RAG2 and n = 20 TMO
alleles; (c) Chaetodon melapterus (species 1)
and Chaetodon trifasciatus (species 2),
n = 144 RAG2 and n = 140 TMO alleles;
(d) Amphiprion bicinctus (species 1) and
Amphiprion omanensis (species 2), n = 82
RAG2 and n = 80 TMO alleles; (e) Dascyllus
carneus (species 1) and Dascyllus marginatus
(species 2), n = 48 RAG2 and n = 96 TMO
alleles; (f) Cirrhitichthys calliurus (species 1)
and Cirrhitichthys oxycephalus (species 2),
n = 12 RAG2 and n = 36 TMO alleles; (g)
Acanthurus lineatus (species 1) and
Acanthurus sohal (species 2), n = 54 RAG2
and n = 92 TMO alleles], but were also
collected at reference populations (ref. pop.)
in the Saudi Arabian Red Sea, Gulf of Aden
(Djibouti), Arabian Sea (Oman), or Indian
Ocean [Christmas Island (Australia), CocosKeeling Islands (Australia), Madagascar,
Maldives or Mayotte]. Site-specific samples
sizes and RAG2 or TMO fragment length
are provided in Table S1 in Appendix S1.
Each circle represents a unique haplotype
and its size is proportional to its total
frequency. Thin branches and black crossbars represent a single nucleotide change,
thick black bars represent greater than one
nucleotide change (as indicated), small open
circles represent missing haplotypes and
colours denote collection location as
indicated by the embedded key.

detected in the putative hybrids, thus C. melapterus females


either do not interbreed, their hybrid offspring are unfit or
their potential hybrid progeny eluded detection based on
our small sample size (Figs 2 & 3c). Interestingly, one specimen identified as C. melapterus had C. trifasciatus mtDNA,
indicating introgression. This pattern was confirmed at
Journal of Biogeography 42, 16011614
2015 John Wiley & Sons Ltd

Marine hybrid hotspot at Socotra


non-coding mtDNA (control region), which experiences
higher polymorphisms (see Appendix S3). RAG2 and TMO
however, could not resolve the closely related parentals,
which suggests a recent separation of the species (Fig. 4c,
Appendix S2).
ppell, 1830 and Amphiprion
Amphiprion bicinctus Ru
omanensis Allen & Mee, 1991
Seven clownfish individuals were collected from Socotra for
genetic examination because of intermediate coloration relative to parental species (Fig. 2, Table 1). These two species
are from different biogeographical provinces and reach the
edge of their ranges at Socotra. Anemonefishes are very rare
at Socotra and no species were observed in our transects,
although A. bincincuts, A. omanensis, A. akallopisos, Amphiprion cf. chagosensis and an unknown clownfish classified as
Amphiprion sp. have previously been reported from Socotra
(Kemp, 1998; Zajonz et al., 2000). All putative hybrids and
parentals had nearly identical COI sequences (Fig. 3d), as
well as shared mtDNA control region haplotypes (Appendix
S3), but the species were resolved by a single nucleotide difference at RAG2 (Fig. 4d); putative hybrids grouped with A.
bicinctus based on this nuclear gene (see also Appendix S2).
Dascyllus carneus Fischer, 1885 and Dascyllus
ppell, 1829)
marginatus (Ru
One putative hybrid individual was identified based on intermediate coloration (Fig. 2, Table 1). Dascyllus carneus is
widely distributed in the Indian Ocean whereas D. marginatus
is endemic in the Red Sea to Gulf of Oman; Socotra represents
the edge of both species geographical ranges. Although we did
not count this species in the field, previous surveys indicate
that D. carneus is rare and D. marginatus is common (Zajonz
et al., 2000; Table 1). The putative hybrid had mtDNA characteristic of D. carneus, which was 8.8% divergent from the D.
marginatus sampled in Socotra and the southern Red Sea
(Fig. 3e). TMO was able to resolve this species complex
(Fig. 4e) and the putative hybrid grouped with D. marginatus
based on this nuclear gene (see also Appendix S2).
Cirrhitichthys calliurus Regan, 1905 and
Cirrhitichthys oxycephalus Bleeker, 1855
One putative hybrid was collected from Socotra that
appeared intermediate in size and coloration between the
single C. calliurus we collected in Socotra and C. oxycephalus
collected at sites in the Red Sea and Indian Ocean (Fig. 2,
Table 1). Cirrhitichthys calliurus occurs in the western Indian
Ocean and western Arabian Sea, and C. oxycephalus occupies
the Indo-Polynesian Province, so Socotra is located at the
edge of their geographical ranges (see Fig. 1). Hawkfishes
were not included in our surveys, but Zajonz et al. (2000)
reported that both species are rare at Socotra. Both mtDNA
and TMO sequences of the putative hybrid allied with C.
Journal of Biogeography 42, 16011614
2015 John Wiley & Sons Ltd

oxycephalus reference sequences (Fig. 3f,4f, Appendix S2),


but its colour was much closer to that of C. calliurus.
Acanthurus lineatus Linnaeus, 1758 and Acanthurus
sohal (Forssk
al, 1775)
These two surgeonfish species are closely related based on
phylogenies of the family Acanthuridae (along with A. fowleri
from the Pacific; S. Klanten et al., unpubl. data). Acanthurus
lineatus occurs from East Africa to the central West Pacific,
and A. sohal is a Red Sea to Arabian Gulf endemic, with
both of their range edges overlapping at Socotra. Both species were very common in shallow areas (1 to 5 m) at two of
the survey sites and although individuals with intermediate
coloration were observed in transects (mean density 1.67
individuals per 250 m2) they were rarer than both parent
species. Heterospecific groups were regularly observed and
included putative hybrid individuals. Sampled putative
hybrids included mature females and males, and consisted of
individuals with intermediate coloration, but also every
degree of shading in between the parental colorations (Fig. 2,
Table 1). The mtDNA haplotype network indicates that A.
sohal and A. lineatus are distinguished by 40 fixed mutations
at COI (d = 0.073). Genetic analyses indicate a bias in the
direction of mtDNA geneflow with A. lineatus mtDNA predominating in putative hybrids (92%; Table 1, Fig. 3g). The
presence of A. sohal morphotypes with A. lineatus mtDNA
indicates introgression among parental types. RAG2 resolved
the parentals in this case; putative hybrid alleles within most
individuals grouped with both parental types (Fig. 4g,
Appendix S2).
DISCUSSION
Our study provides the first evidence of a marine hybrid hotspot at the Socotra Archipelago in the Arabian Sea. Multiple
cases of putative hybridization between closely related species
from adjacent biogeographical provinces provide support for
Socotra as the second suture zone described from tropical
seas (in addition to Christmas Island and the Cocos-Keeling
Islands in the eastern Indian Ocean; Hobbs et al., 2009).
However, hybridization also occurred between sympatric species, indicating that conditions other than geography are
potentially contributing to this hybrid hotspot. This study
follows several recent publications in demonstrating that isolated peripheral reef habitats may not be evolutionary deadends, but rather sources of evolutionary novelty contributing
to the broader Indo-Pacific (reviewed in Bowen et al., 2013;
see also DiBattista et al., 2013).
Putative hybridization was first detected at Socotra by
observing individuals with intermediate coloration. Members
of the family with the most recorded putative hybrids (Chaetodontidae) are among the most conspicuous reef inhabitants, and most species exhibit monogamous pair bonding
(Yabuta & Berumen, 2013). Chaetodontidae also has the
highest recorded rate of natural hybridization among reef
1609

J. D. DiBattista et al.
fishes, although this is likely to be partly a function of their
vivid coloration and popularity in the aquarium trade, which
makes intermediate colours easy to detect (Hobbs et al.,
2013). Putative hybrids between cryptic or drab coloured
species in other families are likely to have gone unnoticed,
which may have resulted in an underestimate of hybridization.
While the detection of an intermediate colour morph is
not conclusive evidence of hybridization, it has proven a reliable indicator. For example, 11 putative hybrids were first
identified at the CocosChristmas hybrid hotspot based on
intermediate coloration (Hobbs et al., 2009). Additional support included observations of heterospecific breeding pairs
(butterflyfishes) and harems (angelfishes), as well as interbreeding within these social groups (Hobbs et al., 2009).
Subsequent genetic analyses supported hybridization in eight
of these cases (Marie et al., 2007; Yaakub et al., 2006; DiBattista et al., 2012a; Montanari et al., 2014; S. Klanten et al.,
unpubl. data), and the remaining three cases are untested.
Therefore available evidence indicates that intermediate coloration is a reliable predicator of hybrid reef fishes. Indeed,
the conspicuous colour patterns of many reef fish taxa make
hybrids stand out amongst their parentals. Such was the case
at Socotra, where putative hybrid individuals were instantly
recognized based on the authors experience throughout the
Red Sea and greater Indian Ocean.
Hybrid status was supported using mtDNA and nuclear
DNA in most (but not all) cases (Table 1). For Chaetodon
melapterus and C. trifasciatus, the putative hybrids could not
be resolved at nuclear loci, which is not necessarily evidence
against hybridization but may instead reflect the recent
divergence between parental species (e.g. DiBattista et al.,
2012a). We believe that hybridization is still the most likely
explanation in this case given the intermediate coloration
and the presence of multiple heterospecific pairs that contained mature heterosexual individuals (and the parental species are noted for forming monogamous breeding pairs), but
further genetic testing is needed. Genetic confirmation is particularly important for species that exhibit a range of colour
variants or change colour throughout their life, however the
putative hybrids were from parentals with almost no intraspecific colour variation. Our genetic markers also detected
evidence of introgression in species (Chaetodon melapterus 9
C. trifasciatus; Acanthurus lineatus 9 A. sohal) based on mixing of mtDNA, further supported by the finding of putative
F1 hybrids with mature gonads in each case (along with
Chaetodon collare 9 C. lunula). As in terrestrial systems
(Mallet, 2005, 2007), cryptic hybridization in reef fishes may
frequently go undetected given that backcrossed individuals
can be morphologically indistinguishable from parental species (McMillan et al., 1999; Yaakub et al., 2006; Marie et al.,
2007). Cryptic hybridization (as revealed by genetic
approaches) appears elsewhere in Indo-Pacific fishes (Yaakub
et al., 2006; Kuriiwa et al., 2007). Evidence for bi-directional
introgression in reef fish may also be influenced by sample
size, given that studies with few samples do not detect intro1610

gression (DiBattista et al., 2012b), or do so only in one


direction (van Herwerden et al., 2006).
All of our study cases reinforce the general observation
that hybridization is most frequent among close relatives
(Mallet, 2005). The most plausible explanation for putative
hybridization in four of the seven cases at Socotra is that closely related fauna from different biogeographical provinces
come into contact at the edges of their ranges, as observed
for most hybrids at the CocosChristmas suture zone (Hobbs
et al., 2009). In three of these cases, one or both parent species is rare, and the lack of conspecific partners may promote
hybridization.
The remaining three interspecific pairs at Socotra occur in
sympatry (Chaetodon collare 9 C. lunula; Chaetodon gardineri 9 C. leucopleura; Cirrhitichthys calliurus 9 C. oxycephalus); however, none of these putative hybrids have been
found outside of Socotra despite greater sampling intensity
(Hobbs et al., 2013). This indicates that conditions unique
to Socotra may facilitate hybridization. All six parent species
are at the edge of their range and have low abundance at
Socotra (Table 1). Thus, it appears that conditions at peripheral locations on the range edge influence the abundance of
a species, which in turn may increase its likelihood to
hybridize. In butterflyfishes across the Indo-Pacific, hybridization is most common (63% of cases) among sympatric
species, particularly at biogeographical borders, indicating
that it is not the location per se, but rather the conditions at
that location that promote hybridization among sympatric
fishes (Hobbs et al., 2013). Fluctuations in environmental
conditions around Socotra, and the potential supply of larvae
from distant biogeographical provinces means that there is
considerable mismatch between the environmental conditions experienced by the parents and the offspring. This discrepancy may influence abundance, biological traits (e.g.
growth, reproduction, diet and habitat use), and ultimately
survivorship. Therefore, we forward the hypothesis that conditions (or variability in conditions) at peripheral locations
influence the presence and abundance of species, and in turn
facilitate hybridization. The fact that both hybrid hotspots
(Socotra and CocosChristmas) are peripheral islands at biogeographical borders indicates that there may be specific factors that promote hybridization in reef fishes.
While our focus has been on the discovery of a second
marine suture zone in the tropical Indo-Pacific, there is evidence that additional suture zones exist in temperate oceans.
A broad contact zone of biogeographical regions exists along
the southern Atlantic coast of Florida, where divergent
mtDNA lineages cohabit (e.g. Bowen & Avise, 1990; Karl &
Avise, 1992). The Baltic Sea exhibits hybridization and a
sharp genetic cline around the entrance, possibly indicating
an emerging suture zone (Riginos & Cunningham, 2005; Johannesson & Andre, 2006). Finally, a faunal transition on
the west coast of North America, between the Oregon and
California biogeographical provinces, corresponds to genetic
breaks in coastal marine fauna (e.g. Kelly & Palumbi, 2010;
Sivasundar & Palumbi, 2010), possibly signalling another
Journal of Biogeography 42, 16011614
2015 John Wiley & Sons Ltd

Marine hybrid hotspot at Socotra


suture zone. Taken together, these studies suggest that
suture zones may be more common than previously thought
in the marine environment. Difficulties in recognizing or
collecting hybrid individuals of some marine species, limited
access to these contact zones and imperfectly resolved
genetic markers combine to hinder our ability to confirm
the existence of suture zones as a general feature of marine
biogeography.
In contrast to terrestrial systems, preliminary findings
indicate that hybridization between sympatric species may
play a much larger role in marine systems (Hobbs & Allen,
2014). Given the characteristics of the marine environment
and marine species in general (e.g. typically ectothermic,
large geographical ranges with many sympatric congeners,
highly fecund, external fertilization and a highly dispersive
larval stage), models developed from terrestrial and freshwater systems may not adequately explain hybridization in marine species. Initial hypotheses suggested that strong selection
for intrinsic reproductive barriers in a high-dispersal medium would minimize hybridization in marine species
(reviewed in Palumbi, 1992, 1994); the recent surge in
reported hybrids has shifted this view. In marine systems, we
instead hypothesize that hybridization is most likely among
species under the following set of conditions: (1) isolated
locations at biogeographical borders, particularly where environmental conditions differ from neighbouring larval source
locations; (2) where abundance of one or both parent species
is rare; and (3) between closely related taxa. Borders between
marine biogeographical provinces therefore provide fertile
zones to test these predictions and determine whether
hybridization among allopatric or sympatric species predominates, as well as the conditions that facilitate interbreeding.
CONCLUSIONS
This study provides evidence for seven cases of interspecific
breeding at the Socotra Archipelago. This is particularly
striking because our preliminary surveys covered less than
1% of Socotras coral reef habitat on only one of four
islands, did not sample habitat below 26 m and only examined 83 of the 620 species of Socotra marine fishes. The rate
of discovery of putative hybrids at Socotra (seven different
cases in 6 days) is unprecedented in the marine environment
and is much greater than the rate of discovery at the more
thoroughly investigated ChristmasCocos hybrid hotspot (11
cases over 30 years: Hobbs et al., 2009). The putative hybrids
found at Socotra follow a similar path of discovery to those
at Christmas Island and the Cocos-Keeling Islands in that
individuals were first identified by intermediate coloration
and were later supported with genetic analyses. Underwater
surveys at the ChristmasCocos hybrid hotspot continue to
identify new hybrids (Hobbs & Allen, 2014), and we suspect
that our study has underestimated the breadth and frequency
of interspecific breeding at Socotra. Our observations supporting a significant marine hybrid hotspot at Socotra, which
suggests that this archipelago may provide a rich system to
Journal of Biogeography 42, 16011614
2015 John Wiley & Sons Ltd

investigate predictions related to reproductive isolation and


speciation across a broad range of marine taxa.
ACKNOWLEDGEMENTS
This research was supported by the KAUST Office of Competitive Research Funds (OCRF) under Award No. CRG-12012-BER-002 and baseline research funds to M.L.B.,
National Geographic Society Grant 9024-11 to J.D.D.,
National Science Foundation grant OCE-0929031 to B.W.B.
and California Academy of Sciences funding to L.A.R. For
support in Socotra, we kindly thank the Ministry of Water
and Environment of Yemen, staff at the Environment Protection Authority (EPA) Socotra, and especially Salah Saeed Ahmed, Fouad Naseeb and Thabet Abdullah Khamis as well as
Ahmed Issa Ali Affrar from Socotra Specialist Tour for handling general logistics. For logistic support elsewhere, we
thank Eric Mason at Dream Divers, Nicolas Prevot at Dolphin Divers and the crew of the M/V Deli in Djibouti, David
Pence, the KAUST Coastal and Marine Resources Core Lab
and Amr Gusti, the Administration of the British Indian
Ocean Territory, the Ministry of Agriculture and Fisheries in
Oman including Abdul Karim, the Ministere de la P^eche et
des Resources Halieutiques Madagascar, the Western Australia Department of Fisheries, Parks Australia as well as the
University of Milano-Bicocca Marine Research and High
Education Centre in Magoodhoo, the Ministry of Fisheries
and Agriculture, Republic of Maldives and the community of
Maghoodhoo, Faafu Atoll. For specimen collections, we
thank Tilman Alpermann, Giacomo Bernardi, Matthew
Craig, Michelle Gaither, Hugo Harrison, Jennifer McIlwain,
Gerrit Nanninga, Samuel Payet, Frederic Ramahatratra, Pablo
Saenz-Agudelo and members of the Reef Ecology Lab at
KAUST. We also acknowledge important contributions from
Robert J. Toonen, John E. Randall, Jo-Ann C. Leong, David
Catania for assistance with specimen archiving and the
KAUST Bioscience Core Laboratory for their assistance with
DNA sequencing.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Appendix S1 Study species, mitochondrial and nuclear
DNA loci, sample size and sampling sites (Table S1). Reef
fish specimens from this study vouchered at the California
Academy of Sciences (Table S2).
Appendix S2 Individual-level information (where available)
for all fish sampled at the Socotra Archipelago.
Appendix S3 Median-joining networks showing relationships among mitochondrial DNA control region haplotypes.
BIOSKETCH
The authors interests are focused on elucidating the
evolutionary processes that generate marine biodiversity.
They have carried out phylogeographical surveys of over
20 reef fish species in the Red Sea, Arabian Sea and
greater Indo-Pacific to test existing evolutionary models,
resolve the life history traits that influence dispersal and
population separations in reef organisms and inform marine conservation (e.g. defining the boundaries of marine
protected areas).
Author contributions: J.D.D. conceived the idea for this
study, collected tissue samples and produced DNA
sequences, analysed the data and led the writing. In addition to writing, B.W.B., L.A.R., J-P.A.H., M.A.P., T.H.S-T.
and M.L.B collected tissue samples or conducted abundance
surveys. S.H. collected tissue samples and produced DNA
sequences.

Editor: David Bellwood

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2015 John Wiley & Sons Ltd