· high as 34.4 % has been reported at Sesoko Island, Japan (Loya et al. 2001). Some octocorals possess opportunistic life-history features such as rapid growth rates, high fecun-dity,
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Marine Biodiversity ISSN 1867-1616 Mar BiodivDOI 10.1007/s12526-013-0157-4
A taxonomic survey of Saudi Arabian RedSea octocorals (Cnidaria: Alcyonacea)
Roxanne D. Haverkort-Yeh, CatherineS. McFadden, Yehuda Benayahu,Michael Berumen, Anna Halász &Robert J. Toonen
1 23
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ORIGINAL PAPER
A taxonomic survey of Saudi Arabian Red Sea octocorals(Cnidaria: Alcyonacea)
Roxanne D. Haverkort-Yeh &
Catherine S. McFadden & Yehuda Benayahu &
Michael Berumen & Anna Halász & Robert J. Toonen
Received: 11 January 2013 /Revised: 3 April 2013 /Accepted: 12 April 2013# Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg 2013
Abstract A preliminary survey of Saudi Arabian Alcyonaceais presented, which combines classical taxonomy, multilocusmolecular barcodes, and in situ photographs. We explored 14locations along the west coast of the Kingdom of Saudi Arabiato assess the regional taxonomic diversity of non-gorgonianalcyonaceans. We collected samples from a total of 74 colo-nies, distributed among four families: 18 colonies ofAlcyoniidae, 14 of Nephtheidae, 9 of Tubiporidae, and 33 ofXeniidae. We sequenced the octocorals using multiple nuclear[ribosomal Internal Transcribed Spacers (ITS) and ATP Syn-thetase Subunit α (ATPSα)] and mitochondrial [MutS homo-log (mtMutS) and Cytochrome C Oxidase subunit one (COI)]loci, providingmolecular barcodes which will: (1) allow directcomparison of biodiversity from this location to others forwhich molecular data are available, and (2) facilitate futureidentifications of these taxa. Finally, this preliminary
phylogeny of sampled taxa provides insights on the resolutionof mitochondrial versus nuclear loci, and highlights octocoraltaxa that require further taxonomic attention.
Keywords Octocorallia . Systematics . Phylogenetics .
Taxonomy . Red Sea
Introduction
Octocorals of the order Alcyonacea are among the mostcommon members of many tropical coral reef habitats,including the Red Sea (Benayahu and Loya 1981), theIndo-West Pacific (Fabricius 1997), and the MediterraneanSea (e.g., Ros et al. 1985). They may also be conspicuousand ecologically important in many colder regions such asthe North Atlantic (e.g., Migné and Davoult 1997), southernAfrica (e.g., Benayahu 1993; Williams 1992), the PacificNorthwest coast of North America (McFadden andHochberg 2003), and Antarctica (e.g., Slattery et al. 1995).For example, in the Indo-West Pacific, best known for thehigh diversity of scleractinian corals and reef-associatedfishes, octocorals may occupy up to 25 % of primary space(Benayahu 1995; Fabricius 1997), and soft coral cover ashigh as 34.4 % has been reported at Sesoko Island, Japan(Loya et al. 2001). Some octocorals possess opportunisticlife-history features such as rapid growth rates, high fecun-dity, and extensive asexual reproduction. This opportunisticlife style, in combination with environmental changes thatare currently impacting scleractinians, may lead to a shiftfrom scleractinian-dominated coral reefs to ones in whichmuch of the available space is occupied by octocorals(Fabricius 1995; Tilot et al. 2008). For example, Tilot etal. (2008) documented total coral cover on two South Sinaireefs (Egyptian Red Sea) in 2002, compared findings to asimilar study in 1996 by Ormond, and found that thescleractinian cover had decreased by 5–25 % while the
Electronic supplementary material The online version of this article(doi:10.1007/s12526-013-0157-4) contains supplementary material,which is available to authorized users.
R. D. Haverkort-Yeh (*) :R. J. ToonenHawai‘i Institute of Marine Biology,University of Hawai‘i at Mānoa, Honolulu, HI, USAe-mail: rhaverkort@gmail.com
C. S. McFaddenDepartment of Biology, Harvey Mudd College,Claremont, CA, USA
Y. Benayahu :A. HalászDepartment of Zoology, George S. Wise Faculty of Life Sciences,Tel Aviv University, Ramat Aviv, Israel
M. BerumenRed Sea Research Center, King Abdullah University of Scienceand Technology, Thuwal, Kingdom of Saudi Arabia
M. BerumenBiology Department, Woods Hole Oceanographic Institute,Woods Hole, MA, USA
Mar BiodivDOI 10.1007/s12526-013-0157-4
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cover of octocorals had increased. Tilot et al. (2008)suggested that scleractinian cover at these sites likely de-creased because members of the genus Acropora were af-fected by a crown-of-thorns starfish outbreak between 1996and 1999 (De’ath and Moran 1998; Salem 1999). Thedecline in Acropora resulted in an increase in hard substrateavailable for settlement by octocorals of the family Xeniidaeand the genera Litophyton (Nephtheidae), Lobophytum andSinularia (Alcyoniidae), which were the primary taxa ac-counting for octocoral increase (Tilot et al. 2008).
There is a long history of octocoral collections from the RedSea, with early publications such as Lamarck (1816),Ehrenberg (1834), Klunzinger (1877), and Kükenthal (1913).Numerous ecological studies on octocorals have also beenconducted there (e.g., Benayahu and Loya 1981; Benayahu etal. 1989; Benayahu 1995; Zeevi-Ben-Yosef and Benayahu2008). Many of these studies laid the foundation for similarstudies worldwide (e.g., references in Benayahu 2002; Kahnget al. 2011). Despite the large size of the Saudi Arabian reefsystem (approximately 1,800 km of coastline), surveys ofoctocorals there remain scarce. Klunzinger (1877) andKükenthal (1913) reported some octocorals from Saudi Arabia,but since these early studies no survey has been conducted onthe octocoral fauna of this region of the Red Sea. Sheppard andSheppard (1985, 1991) and Sheppard (1985) thoroughly ex-amined the scleractinian fauna of the Saudi Arabian coast;however, their study did not include octocorals with the soleexception of the organ pipe coral, Tubipora musica.
With myriad global climate changes impacting coral reefs(Hughes et al. 2003; Hoegh-Guldberg et al. 2007), understand-ing the evolutionary relations among octocoral taxa, which areincreasing in abundance on many reefs, becomes an issue ofsignificant conservation relevance. Despite the capacity of clas-sical taxonomy to provide reliable identifications for someoctocoral taxa, the taxonomy and systematic relationships ofoctocorals remain subjects of considerable uncertainty(McFadden et al. 2010). This uncertainty is caused by a paucityof taxonomic studies on several octocoral families. Poor orig-inal descriptions, uncertainty concerning morphological fea-tures to be used for identification, possible polymorphismwithin species, and in some cases even overlapping morpholo-gy between species have raised the need for taxonomic revisionof this sub-class. Several phylogenetic studies on Alcyonaceahave found little correspondence between molecular clades andclassical taxonomic groups, even at taxonomic levels as high asthe sub-ordinal level (Berntson et al. 2001; Daly et al. 2007;McFadden et al. 2010). However, in other studies, correspon-dence betweenmolecular clades and classical taxonomic groupsis found at the genus level (Concepcion et al. 2008). Fortaxonomic convenience, Alcyonacea is often sub-divided intosix sub-ordinal groups (Alcyoniina, Calcaxonia, Holaxonia,Protoalcyonaria, Scleraxonia, Stolonifera), representing differ-ent grades of colony form and skeletal composition (Fabricius
and Alderslade 2001; Daly et al. 2007). However, it is widelyacknowledged that these groups do not reflect phylogeneticrelationships (Berntson et al. 2001; Fabricius and Alderslade2001; McFadden et al. 2006a). For example, in a phylogeneticanalysis of Octocorallia based on the mitochondrial genes ND2and mtMutS, McFadden et al. (2006a) found that topology testsrejected the monophyly of the sub-ordinal groups, Alcyoniina,Scleraxonia, and Stolonifera. Thismolecular andmorphologicaldiscord has led to questions about the taxonomic value of someof the currently-used morphological characters (Fabricius andAlderslade 2001), and the evolutionary relationships inferredamong families (McFadden et al. 2006a).
In this paper, we report on a study of octocorals, using acombination of classical morphological taxonomy, multilocusmolecular barcodes [(from nuclear (ITS and ATPSα), and mito-chondrial (mtMutS and COI) loci] and photography. The studyincluded 13 genera within four families. Of the familyAlcyoniidae (Lamouroux, 1812) we included three genera:(1) Rhytisma Alderslade, 2000, (2) Sarcophyton Lesson,1834, and (3) Sinularia May, 1898. Within the familyNephtheidae (Gray, 1862), we included four genera: (1)Dendronephthya Kükenthal, 1905, (2) Litophyton Forskål,1775, (3) Paralemnalia Kükenthal, 1913, and (4)Stereonephthya Kükenthal, 1905. The family Tubiporidaecomprises only Tubipora (Linnaeus, 1758). Finally, the familyXeniidae (Wright and Studer 1889) included five genera: (1)Anthelia Lamarck, 1816, (2) Heteroxenia Kölliker, 1874, (3)Ovabunda Alderslade, 2001, (4) Sympodium Ehrenberg,1834, and (5) Xenia Lamarck, 1816. Our goal was to providea taxonomic survey of the Saudi Arabian octocorals, utilizinga combination of classical taxonomy, molecular data, and insitu photographs. Gorgonians were excluded due to limitedcollection time. The results of the survey provide a foundationfor studies on the ecology and biodiversity of Saudi ArabianAlcyonacea, and will support conservation management de-cisions in this region. This survey also represents an importantcontribution to understanding regional (within the Red Sea)and broad-scale (Indo-West Pacific) octocoral biogeographicpatterns. Our molecular data highlight several taxa for whichfurther taxonomic study is warranted and will be useful giventhe increasing utilization of DNA barcoding as a means offacilitating genetic biodiversity surveys. Finally, the availabil-ity of molecular barcodes allows us to directly compare bio-diversity of this location to others for whichmolecular data areavailable.
Materials and methods
Sample collection
Over the course of 8 days in April 2011, a total of 74 collec-tions were made at 1–15 m depth from 14 reef sites in four
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main areas along the western coast of Saudi Arabia: Thuwal,Jeddah, Al Lith, and the Farasan Islands (arranged north tosouth) (Fig. 1; Table 1). We focused primarily on collecting allmorphotypes of members of the family Xeniidae, but alsocollected other non-gorgonian alcyonacean octocoralmorphotypes by the roving diver technique (Munro 2005;Hoeksema and Koh 2009). Depending on abundance ofoctocoral colonies, collections were made of up to four colo-nies for each (visible) morphotype, such as different colormorphs. Each sampled colony was measured and a smallpiece (1–5 cm) was sampled using scissors or dive knife.Multiple photos and live videos (∼1 min) were taken of eachcolony before and during disturbance (upon hand-touch) torecord colony response as well as of the surrounding environ-ment (Canon G11 camera with a Canon underwater housing).Morphological features and behavior of the colonies derivedfrom the photographs aided the classical taxonomic identifi-cation (see below). Colony size and color, polyp retraction(process of invagination of the anthocodia within the upperpart of the anthostele, see Fabricius and Alderslade 2001),tentacle contraction (process of deflation without invagina-tion, see Fabricius and Alderslade 2001), stalk branching, andpolyp pulsation (applicable to Xeniidae, see Reinicke 1997)were recorded for each colony (see Electronic SupplementaryMaterial). In addition, the depth of collection and type ofsubstrate (hard vs. sand) were recorded for each colony sam-pled. Paired tissue samples from each colony were stored in95 % ethanol and saturated salt DMSO (SSD) buffer formolecular analyses (following Gaither et al. 2011), and anadditional sample was stored in 70 % ethanol for morpholog-ical taxonomic study.
Classical taxonomy
Generic field identifications based on colony features wereverified by subsequent expert taxonomic identificationbased on colony morphology, sclerites, presence of dimor-phic polyps, and zooxanthellae (Fabricius and Alderslade2001). Additionally, examination of xeniid octocorals in-cluded the number of pinnule rows on the polyp tentaclesand the number of pinnules on the aboral side of the polyptentacles (Reinicke 1997). For species identification, scler-ites were obtained by dissolving small tissue samples in10 % sodium hypochlorite, rinsed in distilled water andexamined under a light microscope. Identifications werefacilitated in part by comparisons with permanent scleritepreparations of type material kept at the Zoological Muse-um, Department of Zoology, Tel Aviv University, Israel(ZMTAU). Tentative field identifications of Sarcophytoncolonies could not be confirmed due to lack of the base ofthe colony for those samples (see below: SA053, SA066,SA067, and SA077). All samples are deposited at the U.S.National Museum of Natural History, Washington, D.C.(USNM) (USNM 1201935–1202016) (Table 1).
DNA isolation and amplification
The mitochondrial markers mtMutS (previously namedmsh1, ∼730 bp, n=64) and COI (∼770 bp, n=69) wereamplified and sequenced using published primers and pro-tocols (McFadden et al. 2011). PCR products from themitochondrial markers were purified and sent to the Univer-sity of Washington’s High-Throughout Genomics Center
Fig. 1 Sites along the westcoast of the Kingdom of SaudiArabia where collections ofoctocorals were made: Jeddah(21°43′N, 39°06′E), Thuwal(22°15′N, 38°57′E ), Al Lith(19°49′N′, 40°07′E), and theFarasan Islands (16°47′N,42°11′E). (The map wasdesigned with SimpleMappr;Shorthouse 2010)
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Tab
le1
Specimen
listwith
correspo
ndingGenBankaccessionnu
mbers
(*means
heterozygo
tecolonies),
correspo
ndingUSNM
numbers
(accession
numbers
attheSmith
sonian
Museum,
Washing
ton,
D.C.),andcorrespo
ndingsamplinglocatio
nsandcoordinates.Onlyasing
lesamplewas
collected
foreach
individu
allistedhere
Fam
ilyGenus
Species
Site
Coo
rdinates
Depth
USNM
ATPSα
17ITS
MtM
utS
COI71
SA00
1Xeniid
aeOvabu
nda
ainex
Thu
wal,PalaceNorth
22°18′N,
38°58′E
13m
1201
935
KC86
5037
KC86
4797
KC86
4858
KC86
4922
SA00
2Xeniid
aeOvabu
nda
goha
riThu
wal,Shark
Reef
22°25′N,
38°59′E
13m
1201
936
KC86
5042
KC86
4798
KC86
4859
KC86
4923
SA00
3Xeniid
aeXenia
sp.
Thu
wal,Shark
Reef
22°25′N,
38°59′E
9m
1201
937
KC86
4994
KC86
4860
KC86
4924
SA00
4Xeniid
aeOvabu
nda
biseriata
Thu
wal,PalaceNorth
22°18′N,
38°58′E
13m
1201
938
KC86
5013
KC86
4799
KC86
4861
KC86
4925
SA00
5Xeniid
aeOvabu
nda
arab
ica
Thu
wal,PalaceNorth
22°18′N,
38°58′E
13m
1201
939
KC86
5041
KC86
4800
KC86
4862
KC86
4926
SA00
6Xeniid
aeXenia
sp.
Thu
wal,Shark
Reef
22°25′N,
38°59′E
9m
1201
940
KC86
4995
*KC86
4801
KC86
4863
KC86
4927
SA00
7Xeniid
aeOvabu
nda
macrospiculata
Thu
wal,PalaceSou
th22
°15′N,
38°57′E
6m
1201
941
KC86
5029
KC86
4802
KC86
4864
KC86
4928
SA00
8Xeniid
aeXenia
sp.
Thu
wal,Shark
Reef
22°25′N,
38°59′E
9m
1201
942
KC86
4803
KC86
4865
KC86
4929
SA00
9Xeniid
aeOvabu
nda
biseriata
Thu
wal,Shi’b
Nazar
22°19′N,
38°51′E
12m
1201
943
KC86
5028
*KC86
4866
KC86
4930
SA01
0Xeniid
aeOvabu
nda
biseriata
Thu
wal,PalaceNorth
22°18′N,
38°58′E
13m
1201
944
KC86
5040
*KC86
4804
KC86
4867
KC86
4931
SA011
Xeniid
aeXenia
actuosa
AlLith
,North
BrownReef
19°52′N,
40°06′E
7m
1201
945
KC86
4993
KC86
4805
KC86
4868
KC86
4932
SA01
3Xeniid
aeHeteroxenia
fuscescens
AlLith
,North
BrownReef
19°52′N,
40°06′E
9m
1201
947
KC86
5008
KC86
4806
KC86
4869
KC86
4933
SA01
4Nephtheidae
Lito
phyton
sp.
AlLith
,Sou
thBrownReef
19°49′N,
40°07′E
15m
1201
948
KC86
4870
KC86
4934
SA01
6Alcyo
niidae
Rhytisma
fulvum
fulvum
AlLith
,Sou
thBrownReef
19°49′N,
40°07′E
13m
1201
950
KC86
4807
KC86
4935
SA01
7Alcyo
niidae
Rhytisma
fulvum
fulvum
AlLith
,North
BrownReef
19°52′N,
40°06′E
9m
1201
951
KC86
4808
SA01
9Tub
iporidae
Tubipo
ramusica
AlLith
,Sou
thBrownReef
19°49′N,
40°07′E
13m
1201
953
KC86
4871
KC86
4936
SA02
0Xeniid
aeOvabu
nda
ainex
AlLith
,North
BrownReef
19°52′N,
40°06′E
8m
1201
954
KC86
5036
KC86
4809
KC86
4872
KC86
4937
SA02
1Xeniid
aeHeteroxenia
fuscescens
Thu
wal,PalaceSou
th22
°15′N,
38°57′E
4m
1201
955
KC86
5010
KC86
4810
KC86
4873
KC86
4938
SA02
2Xeniid
aeOvabu
nda
ainex
Thu
wal,Shi’b
Nazar
22°19′N,
38°51′E
8m
1201
956
KC86
5038
KC86
4811
KC86
4874
KC86
4939
SA02
3Xeniid
aeAnthelia
sp.
Thu
wal,Shi’b
Nazar
22°19′N,
38°51′E
10m
1201
957
KC86
5001
KC86
4812
KC86
4875
KC86
4940
SA02
4Xeniid
aeOvabu
nda
biseriata
Thu
wal,Shi’b
Nazar
22°19′N,
38°51′E
8m
1201
958
KC86
5035
KC86
4813
KC86
4876
KC86
4941
SA02
6Xeniid
aeHeteroxenia
fuscescens
Thu
wal,PalaceSou
th22
°15′N,
38°57′E
5m
1201
960
KC86
5009
KC86
4814
KC86
4877
KC86
4942
SA02
7Xeniid
aeAnthelia
sp.
Thu
wal,Shi’b
Nazar
22°19′N,
38°51′E
11m
1201
961
KC86
5000
KC86
4815
KC86
4878
KC86
4943
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Tab
le1
(con
tinued)
Fam
ilyGenus
Species
Site
Coo
rdinates
Depth
USNM
ATPSα
17ITS
MtM
utS
COI71
SA02
8Nephtheidae
Lito
phyton
sp.
AlLith
,North
BrownReef
19°52′N,
40°06′E
9m
1201
962
KC86
5011*
KC86
4816
*KC86
4879
KC86
4944
SA02
9Tub
iporidae
Tubipo
ramusica
AlLith
,North
BrownReef
19°52′N,
40°06′E
7m
1201
963
KC86
4817
*KC86
4880
KC86
4945
SA03
0Tub
iporidae
Tubipo
ramusica
AlLith
,DoraReef
19°49′N,
39°53′E
6m
1201
964
SA03
1Tub
iporidae
Tubipo
ramusica
AlLith
,DoraReef
19°49′N,
39°53′E
12m
1201
965
KC86
4818
*KC86
4946
SA03
2Xeniid
aeAnthelia
sp.
AlLith
,DoraReef
19°49′N,
39°53′E
10m
1201
966
KC86
4998
KC86
4819
KC86
4881
KC86
4947
SA03
4Alcyo
niidae
Rhytisma
fulvum
fulvum
AlLith
,DoraReef
19°49′N,
39°53′E
9m
1201
968
KC86
5044
KC86
4820
KC86
4882
KC86
4948
SA03
5Xeniid
aeSympo
dium
caeruleum
AlLith
,DoraReef
19°49′N,
39°53′E
6m
1201
969
KC86
5027
KC86
4949
SA03
6Nephtheidae
Dendron
ephthya
sp.
AlLith
,DoraReef
19°49′N,
39°53′E
9m
1201
970
KC86
5007
KC86
4821
KC86
4883
KC86
4950
SA03
7Xeniid
aeXenia
sp.
Jedd
ah,Obh
urCreek
21°43′N,
39°06′E
1m
1201
971
KC86
5033
KC86
4822
KC86
4884
KC86
4951
SA03
8Nephtheidae
Dendron
ephthya
sp.
AlLith
,DoraReef
19°49′N,
39°53′E
10m
1201
972
KC86
5006
KC86
4823
KC86
4885
KC86
4952
SA03
9Tub
iporidae
Tubipo
ramusica
AlLith
,DoraReef
19°49′N,
39°53′E
12m
1201
973
KC86
4824
*KC86
4886
KC86
4953
SA04
0Xeniid
aeOvabu
nda
impu
lsatilla
AlLith
,DoraReef
19°49′N,
39°53′E
10m
1201
974
KC86
4825
KC86
4887
KC86
4954
SA04
1Tub
iporidae
Tubipo
ramusica
AlLith
,DoraReef
19°49′N,
39°53′E
7m
1201
975
KC86
5025
KC86
4888
KC86
4955
SA04
2Xeniid
aeAnthelia
sp.
AlLith
,Marmar
Reef
19°50′N,
39°56′E
6m
1201
976
KC86
4826
KC86
4889
KC86
4956
SA04
3Alcyo
niidae
Rhytisma
fulvum
fulvum
AlLith
,Marmar
Reef
19°50′N,
39°56′E
5m
1201
977
KC86
4827
KC86
4890
KC86
4957
SA04
4Tub
iporidae
Tubipo
ramusica
AlLith
,DoraReef
19°49′N,
39°53′E
10m
1201
978
KC86
4996
KC86
4828
*KC86
4891
KC86
4958
SA04
5Xeniid
aeOvabu
nda
macrospiculata
AlLith
,Marmar
Reef
19°50′N,
39°56′E
5m
1201
979
KC86
5039
KC86
4829
KC86
4892
KC86
4959
SA04
6Tub
iporidae
Tubipo
ramusica
AlLith
,DoraReef
19°49′N,
39°53′E
10m
1201
980
KC86
4893
KC86
4960
SA04
7Xeniid
aeAnthelia
sp.
AlLith
,Marmar
Reef
19°50′N,
39°56′E
10m
1201
981
KC86
4999
KC86
4830
*KC86
4894
KC86
4961
SA04
8Xeniid
aeSympo
dium
caeruleum
AlLith
,Marmar
Reef
19°50′N,
39°56′E
5m
1201
982
KC86
5024
KC86
4962
SA04
9Alcyo
niidae
Paralem
nalia
eburnea
AlLith
,Marmar
Reef
19°50′N,
39°56′E
5m
1201
983
KC86
4831
KC86
4895
KC86
4963
SA05
0Xeniid
aeAnthelia
sp.
AlLith
,Marmar
Reef
19°50′N,
39°56′E
6m
1201
984
KC86
4997
KC86
4832
*KC86
4896
KC86
4964
SA05
1Alcyo
niidae
Sinu
laria
sp.
Farasan
Island
s,Abu
latt
16°47′N,
42°11′E
8m
1201
985
KC86
5022
KC86
4833
KC86
4965
SA05
2Alcyo
niidae
Rhytisma
fulvum
fulvum
Farasan
Island
s,Abu
latt
16°47′N,
42°11′E
8m
1201
986
KC86
5043
*KC86
4834
*KC86
4897
KC86
4966
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Tab
le1
(con
tinued)
Fam
ilyGenus
Species
Site
Coo
rdinates
Depth
USNM
ATPSα
17ITS
MtM
utS
COI71
SA05
3Alcyo
niidae
Sarcop
hyton
sp.
Farasan
Island
s,Abu
latt
16°47′N,
42°11′E
7m
1201
987
KC86
4835
SA05
4Alcyo
niidae
Sarcop
hyton
glau
cum
Farasan
Island
s,Abu
latt
16°47′N,
42°11′E
10m
1201
988
KC86
5018
*KC86
4836
*KC86
4898
KC86
4967
SA05
5Alcyo
niidae
Paralem
nalia
thyrsoides
Farasan
Island
s,Abu
latt
16°47′N,
42°11′E
10m
1201
989
KC86
4991
KC86
4837
KC86
4899
KC86
4968
SA05
6Alcyo
niidae
Sinu
laria
querciform
isFarasan
Island
s,Abu
latt
16°47′N,
42°11′E
11m
1201
990
KC86
5021
KC86
4838
KC86
4900
KC86
4969
SA05
7Alcyo
niidae
Rhytisma
fulvum
fulvum
Farasan
Island
s,Abu
latt
16°47′N,
42°11′E
9m
1201
991
KC86
4839
KC86
4901
KC86
4970
SA05
8Alcyo
niidae
Sinu
laria
leptoclado
sFarasan
Island
s,Abu
latt
16°47′N,
42°11′E
8m
1201
992
KC86
5020
KC86
4902
SA05
9Nephtheidae
Lito
phyton
sp.
AlLith
,Mulathu
19°44′N,
39°54′E
10m
1201
993
KC86
5012
KC86
4903
KC86
4971
SA06
0Alcyo
niidae
Sarcop
hyton
auritum
AlLith
,Mulathu
19°44′N,
39°54′E
7m
1201
994
KC86
5014
KC86
4840
KC86
4904
KC86
4972
SA06
1Nephtheidae
Dendron
ephthya
sp.
Farasan
Island
s,Dahik
16°54′N,
52°07′E
9m
1201
995
KC86
4841
KC86
4973
SA06
2Tub
iporidae
Tubipo
ramusica
Farasan
Island
s,Dahik
16°54′N,
52°07′E
7m
1201
996
KC86
4905
KC86
4974
SA06
3Nephtheidae
Dendron
ephthya
sp.
Farasan
Island
s,Dahik
16°54′N,
52°07′E
8m
1201
997
KC86
5002
KC86
4842
KC86
4906
KC86
4975
SA06
4Nephtheidae
Dendron
ephthya
sp.
Farasan
Island
s,Dahik
16°54′N,
52°07′E
7m
1201
998
KC86
5003
KC86
4843
KC86
4907
KC86
4976
SA06
5Nephtheidae
Dendron
ephthya
sp.
Farasan
Island
s,Dahik
16°54′N,
52°07′E
5m
1201
999
KC86
5005
KC86
4844
KC86
4908
KC86
4977
SA06
6Alcyo
niidae
Sarcop
hyton
glau
cum
Farasan
Island
s,Dahik
16°54′N,
52°07′E
6m
1202
000
KC86
5017
*KC86
4845
*KC86
4978
SA06
7Alcyo
niidae
Sarcop
hyton
auritum
Farasan
Island
s,Dahik
16°54′N,
52°07′E
7m
1202
001
KC86
5015
KC86
4846
KC86
4909
KC86
4979
SA06
8Nephtheidae
Dendron
ephthya
sp.
Farasan
Island
s,Abu
Shariah
16°43′N,
42°15′E
6m
1202
002
KC86
5026
KC86
4910
SA06
9Nephtheidae
Dendron
ephthya
sp.
Farasan
Island
s,Abu
Shariah
16°43′N,
42°15′E
6m
1202
003
KC86
5004
KC86
4847
KC86
4911
KC86
4980
SA07
1Xeniid
aeXenia
umbella
taJedd
ah,Obh
urCreek
21°43′N,
39°06′E
8m
1202
005
KC86
5030
KC86
4848
KC86
4912
KC86
4981
SA07
2Nephtheidae
Paralem
nalia
thyrsoides
Jedd
ah,Obh
urCreek
21°43′N,
39°06′E
8m
1202
006
KC86
4992
KC86
4849
KC86
4913
KC86
4982
SA07
4Xeniid
aeOvabu
nda
verseveldti
Jedd
ah,Obh
urCreek
21°43′N,
39°06′E
13m
1202
008
KC86
4850
KC86
4914
KC86
4983
SA07
5Xeniid
aeOvabu
nda
goha
riJedd
ah,Obh
urCreek
21°43′N,
39°06′E
14m
1202
009
KC86
5034
*KC86
4851
KC86
4915
KC86
4984
SA07
6Xeniid
aeXenia
sp.
Jedd
ah,Obh
urCreek
21°43′N,
39°06′E
11m
1202
010
KC86
5032
KC86
4852
KC86
4916
KC86
4985
SA07
7Alcyo
niidae
Sarcop
hyton
gemmatum
Jedd
ah,Obh
urCreek
21°43′N,
39°06′E
8m
1202
011
KC86
5016
KC86
4853
KC86
4917
KC86
4986
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(Seattle, WA, USA) for sequencing following the methodsin McFadden et al. (2011).
DNA for nuclear locus amplification was extracted fol-lowing the coral DNA extraction protocol outlined inConcepcion et al. (2006, 2008). The nuclear marker ATPSα( ∼280 bp, n=53) was amplified using the published primersATPSαf1 and ATPSαr1 (Jarman et al. 2002), and with thefollowing PCR protocol: 94 °C for 2 min; 94 °C for 20 s,57 °C for 60 s, 72 °C for 60 s (35 cycles); followed by afinal extension of 72 °C for 6 min. The ITS region(∼1,100 bp, n=61) was amplified using the publishedprimers 1S-f and 2SS-r (annealing temperature: 50 °C; Weiet al. 2006), in addition to the newly designed primersITSRHF 5 ′-TTGGCACCTGTCAGATGRKY-3 ′ andITSRHR 5′-CACCCRTTTTRGGCTGCATT-3′ (45 °C).Primers were designed with Primer3 (Rozen and Skaletsky2000), and PCR amplifications were performed on a Bio-Rad MyCycler™ with the following PCR protocol: 96 °Cfor 9 min; 96 °C for 10 s, 45 or 50 °C for 30 s, 70 °C for4 min (33 cycles); followed by a final extension of 70 °C for5 min (1 cycle). Each 25 μl PCR reaction contained 1.0 μlof template DNA (∼5 ng μL), 12.5 μl BioMix Red 2× PCRreaction mix (Bioline), 0.325 μl of each primer (10 μM),0.75 μl of BSA (10 mg ml), and 11.1 μl deionized sterilewater. For some reactions, an additional 1 μl of DNAtemplate was added to improve PCR product (final volume26 μl). PCR products were visualized using 1.0 % agarosegels in 1× SB (sodium borate; Brody and Scott 2004) bufferstained with Gelstar® (Lonza). Amplification reactions forsequencing of nuclear genes were first treated with Exonu-clease I and FastAP™ thermo sensitive alkaline phosphatase(Fermentas) using the following thermocycler profile: 37 °Cfor 60 min, 85 °C for 15 min. Treated PCR products werethen cycle-sequenced using BigDye Terminators (AppliedBiosystems) run on an ABI-3730XL DNA Analyzer at theAdvanced Studies of Genomics, Proteomics and Bioinformat-ics (ASGPB) facility at UH Mānoa. Sequences are availablefromGenBank (accession numbers: KC864797 - KC865044).
Sequencing and phasing
For nuclear loci, Phase (Stephens et al. 2001) as implementedin DNAsp 5 (Librado and Rozas 2009) was used to resolve thealleles for each heterozygous colony, when alignment ofdirect-sequenced forward and reverse reads was possible.The PCR products of heterozygous colonies that could notbe phased reliably from direct sequences were ligated into thepGEM®-T Easy cloning vector (Promega) and transformedinto JM109 competent cells following the manufacturer’sprotocol. After blue/white colony selection, up to 12 colonieswere screened by PCR to verify an insert of the correct sizeusing the M13 vector primers. Initially, four colonies weresequenced with the original primers. Heterozygote allelesT
able
1(con
tinued)
Fam
ilyGenus
Species
Site
Coo
rdinates
Depth
USNM
ATPSα
17ITS
MtM
utS
COI71
SA08
1Nephtheidae
Stereoneph
thya
sp.
Farasan
Island
s,EastFarasan
Island
16°44′N,
42°13′E
10m
1202
012
KC86
4854
KC86
4918
KC86
4987
SA08
3Nephtheidae
Stereoneph
thya
sp.
Farasan
Island
s,Abu
latt
16°47′N,
42°11′E
8m
1202
014
KC86
5023
KC86
4855
KC86
4919
KC86
4988
SA08
4Alcyo
niidae
Sinu
laria
compressa
Farasan
Island
s,Abu
latt
16°47′N,
42°11′E
11m
1202
015
KC86
5019
KC86
4856
KC86
4920
KC86
4989
SA08
5Xeniid
aeXenia
umbella
taJedd
ah,Obh
urCreek
21°43′N,
39°06′E
8m
1202
016
KC86
5031
KC86
4857
KC86
4921
KC86
4990
Total
5361
6469
aHeterozyg
otecolonies
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Fig. 2 Phylogeneticrelationships among 13 generain the anthozoan orderAlcyonacea (subclassOctocorallia). Left combinedphylogenetic tree of nuclear lociATPSα and ITS. Rightcombined phylogenetic tree ofmitochondrial loci COI andmtMutS. Values at nodesrepresent: top maximumlikelihood bootstrap values, andbottom Bayesian Inferenceposterior probabilities. Xeniagroup A and B are marked
Fig. 3 All non-gorgonianoctocoral genera, andmorphotypes within genera,that were surveyed along thewest coast of the Kingdom ofSaudi Arabia in Jeddah,Thuwal, Al Lith and theFarasan Islands. Scale bars1 cm
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were accepted only if two or more copies of the same allelewere found and could be matched with the direct sequenceread from the original mixed template. If the first four coloniesdid not recover two copies of each allele, four additionalcolonies were sequenced until two copies of each allele wererecovered.
Phylogenetic analyses
All mitochondrial sequences were inspected using Lasergene(DNAStar) and aligned using ClustalW 2.0.11 (Larkin et al.2007) and MUSCLE 3.6 (Edgar 2004). All nuclear sequenceswere visually inspected using Geneious Pro 4.7.6 (Drummondet al. 2010) and aligned using both ClustalW and MUSCLEplugins in Geneious. Subsequently, phylogenetic relationshipswere reconstructed using Bayesian Inference (BI) (chainlength=1.1×10^6, burn-in length=100,000) and maximumlikelihood (ML) (10,000 bootstrap replicates) methods inGeneious Pro. BI trees were generated with MrBayes 2.0.2(Huelsenbeck and Ronquist 2001), and ML trees were gener-ated from PHYML 1.0 (Guindon and Gascuel 2003). Bestsubstitution models were found with MrModeltest plugin inGeneious Pro. Because both nuclear and both mitochondrialloci fit the same best substitution model, no gene-specificmodels were selected in BI and ML analyses. In all analyses,the family Tubiporidae was used as outgroup based on earlieranalyses (McFadden et al. 2006a).
Results
Classical taxonomy
Based on morphological taxonomic analyses, the surveyyielded 28 species from the four families Alcyoniidae (n=16), Nephtheidae (n=16), Tubiporidae (n=9), and Xeniidae(n=33). Based on our field observations, the outermost reefsin Al Lith had the highest number of species, and thesouthernmost sites in the Farasan Islands had the lowestnumber of species.
Several morphotypes were recognized during collections(Fig. 3). For example, we recognized three distinctmorphotypes of Tubipora musica in the field based onvarying polyp structure: (1) polyps with long curving tenta-cles displaying uniform width and short pinnules, (2) polypswith feathery shaped tentacles, no pinnules proximal to themouth, and a decrease in pinnule length in the distal part ofthe tentacles, and (3) polyps with tentacles bearing shortpinnules and a soft tissue appearance,waving with the cur-rent. We recognized two distinct morphotypes of Sinulariain the field based on varying colony structure: (1) shortbulky bumpy stalks, and (2) longer finger-like stalks. Fur-thermore, two color morphs were recognized within thegenus Dendronephthya: (1) dark red and white, and (2)orange and pink. Two morphotypes were recognized withinParalemnalia: (1) branching, and (2) encrusting with shortextensions. Finally, we recognized two color morphs withinRhytisma: (1) green, and (2) gray.
Phylogenetic analyses
Among the four families collected, the mitochondrial markermtMutS was successfully amplified from 67 colonies and themitochondrial marker COI from 73 colonies (Table 1). Thenuclear marker ATPSα was successfully amplified from 53colonies, and the nuclear marker ITS from 51 colonies(Table 1).
COI and mtMutS provided very similar resolution, andmembers of different genera were clearly resolved, with theexception of Ovabunda and Xenia (Fig. 2). COI clearlyseparated 11 out of 13 genera, and mtMutS clearly separated11 out of 12 genera. In both the COI and mtMutS phylogeny,Xenia had two lineages: one within the Ovabunda clade, andone as a sister group to Heteroxenia. ATPSα provided theleast resolution among all markers tested, and 3 out of 13genera were clearly resolved: Paralemnalia, Rhytisma, andOvabunda (Fig. 2). Xenia colonies were divided into twolineages, one grouped close to Ovabunda, and the secondpart of a large clade containing all remaining genera, includ-ing Heteroxenia. ITS clearly separated 9 out of 12 genera,
Fig. 4 Colony morphologydifferences between Xeniaclade A (top) and Xenia clade B(bottom). Scale bars 1 cm
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the exceptions being Sarcophyton, Ovabunda, and Xenia(Fig. 2).
Mitochondrial (COI + mtMutS) and nuclear (ITS +ATPSα) phylogenetic analyses were generally congruent(Fig. 2). In the nDNA phylogeny, but not in the mtDNAphylogeny, two Sarcophyton colonies grouped as sister taxato Sinularia with 100 % ML bootstrap support. Further-more, among all loci, a group of four Xenia colonies con-sistently grouped as a sister group to Heteroxenia, whereasall other Xenia species sampled grouped within Ovabunda.In all analyses, the family Tubiporidae was designated as theoutgroup.
Within the genus Sinularia, S. querciformis revealeda different colony stucture than the other species (S.leptoclados and S. compressa) (Fig. 3) and all specieswere genetically different (Fig. 2). The two color morphswithin Dendronephthya revealed no corresponding geneticdifferentiation. No Dendronephthya colonies were identifiedto species level, and no intrageneric phylogenetic groups wererevealed to suggest cryptic diversity. Likewise, the differentmorphotypes within Paralemnalia, Rhytisma f. fulvum, andTubipora musica did not correspond to different haplotypes inthe preliminary phylogenetic analysis (Fig. 2).
Among the five Xeniidae genera, Anthelia, Heteroxenia,and Sympodium were consistently and clearly distinguishedwith both mitochondrial and nuclear loci, whereasOvabunda and Xenia were not. Members of the Alcyoniidaeand Nephtheidae consistently grouped in three clades inboth mtDNA and nDNA phylogenies. One of thesecontained the Nephtheidae genera Dendronephthya,Litophyton, and Stereonephthya, the second contained theAlcyoniidae genera Sarcophyton and Sinularia, and the lastwas a mixed clade containing Rhytisma from Alcyoniidaeand Paralemnalia from Nephtheidae (Fig. 2).
As an example of how our Saudi Arabian data canfunction to compare geographic biodiversity with other lo-cations, we combined our mtMutS and COI dataset with aprevious soft coral mtMutS and COI dataset from Eilat,Israel (McFadden et al. 2011). The majority of the mito-chondrial gene haplotypes that were found among SaudiArabian specimens were identical, or nearly so, to those fromEilat. The few exceptions with different haplotypes in SaudiArabia were: one of the two distinct haplotypes of Tubipora;one of the two specimens identified as Sarcophyton auritum;and, among the Xeniidae, Xenia group ‘A’ (Figs. 2, 3, 4;discussed further below), Anthelia spp., and the Ovabundagroup of SA007 and SA009.
Discussion
Here, we present our preliminary survey of Saudi ArabianRed Sea alcyonacean octocorals that combines classical
taxonomy, molecular studies, and in situ photographs todocument a total of 28 species in four families. Concordantwith surveys of scleractinian corals (Sheppard 1985;Sheppard and Sheppard 1985, 1991), the lowest cover anddiversity was observed at our southernmost site. However,we examined only a portion of the coast covered in theSheppard’s survey.
In our study we found four families, 13 genera, and 28species of non-gorgonian octocorals. By comparison, a recentoctocoral survey of the Red Sea at Eilat, Israel (Gulf of Aqaba,northern Red Sea) included six families: Alcyoniidae,Briareidae, Nephtheidae, Nidaliidae, Tubiporidae, andXeniidae, comprising 19 genera and 43 species (McFaddenet al. 2011). The latter study included only part of theoctocoral diversity known from that region. We attribute thegreater diversity on Eilat reefs to the intense studies conductedhere (Benayahu 1985; van Ofwegen 2002). A study in theDahlak Archipelago (southern Red Sea) reported five fami-lies: Alcyoniidae, Nephtheidae, Nidaliidae, Tubiporidae, andXeniidae, comprising 14 genera and 28 species (Benayahu etal. 2002), which is very similar to our finding along the SaudiArabian coastline. In comparison to octocoral biodiversitysurveys in the Red Sea, a survey in the southern RyukyoArchipelago reported four octocoral families: Alcyoniidae,Briareidae, Clavulariidae and Tubiporidae, comprising 11genera and 56 species (Benayahu 2002). Furthermore, incomparison to our study with specimens from 14 sites,Benayahu (2002) collected from 18 sites and Benayahu etal. 2002 collected from 17 sites. In a combined analysis of ourdataset and a previously published octocoral dataset from Eilat(McFadden et al. 2011), we found that the majority of themitochondrial gene haplotypes (mtMutS + COI) that werefound among Saudi Arabian specimens were identical, ornearly so, to those from Eilat.
Phylogenetic analyses were generally consistent amongnuclear and mitochondrial loci. Nine out of 13 genera thatwere included in this study were clearly and consistentlydistinct by both classical taxonomic and molecular systematicdata, the exceptions being Sarcophyton, Ovabunda, and Xe-nia. It is important to note that sister group relationshipsreported here are tentative because of limited taxon samplingin this preliminary biodiversity survey of octocoral diversity.Regardless of the exact relationships, however, these studiesare useful to highlight a number of taxa for additional study,such as the genus Sarcophytonwhich was recovered as mono-phyletic inmitochondrial analyses, but in the nuclear analyses,colonies of Sarcophyton auritum grouped as a sister group tothe genus Sinularia (Fig. 2). A cryptic clade of Lobophytumand Sarcophyton species has previously been recognized(McFadden et al. 2006b), and the Sarcophyton auritum inthe current study falls within that cryptic clade. However,the apparent sister relationship between Sinularia and themixed clade of Sarcophyton/Lobophytum (Fig. 2) is not
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consistent with phylogenies reconstructed previously fromboth mtDNA and nDNA (McFadden et al. 2009; Brockmanand McFadden 2012). We attribute this incongruence to lim-ited taxon sampling in this Saudi Arabian octocoral survey.
Even with limited taxon sampling in this preliminarysurvey, we discovered clear polyphyly within the nominalgenus Xenia. Four Xenia colonies group as a sister taxon toHeteroxenia, hereafter called Xenia group ‘A’, whereas theother Xenia colonies group as a sister taxon to Ovabunda,hereafter called Xenia group ‘B’ (Fig. 2; Stemmer et al.2012). After discovering this cryptic diversity, reverse tax-onomy (sensu Markmann and Tautz 2005) was applied; thatis, we searched for supporting morphological characters thatwe may have previously overlooked, using phylogeneticpatterns as a guide. Interestingly, we were able to detectdifferences in colony growth forms (Fig. 4), as well asskeletal features between the two Xenia groups. Coloniesof Xenia clade ‘A’ are always small, the stem and tentaclesare not very long, and the color pattern is white tentacles andpolyps, and brown pinnules. The specimens of Xenia clade‘B’ vary in appearance: some have the same colony mor-phological features as Xenia group ‘A’, but are much lighterin color (SA037, SA071), whereas others have differentmorphological features than Xenia group ‘A’: the stemsand polyp tentacles are much longer, but the color patternis the same as seen in colonies from Xenia group ‘A’(SA076, SA085). Furthermore, Xenia colonies from group‘B’ contain varying microscopic features: SA037, SA076both contain two rows of pinnules, with 18–19 and 20–24pinnules, respectively, at the outmost row, whereas SA71and SA85 both contain three rows of pinnules and 19–26and 21–27 pinnules, respectively, at the outermost row. OfXenia group ‘A’, only specimen SA11 could be identifieddue to poor preservation. The colonies’ tentacles have threeto four pinnule rows and 22–26 pinnules at the outmost row.Despite the limited identification due to poor preservation,however, we found that all Xenia group ‘B’ colonies containtypical Xenia sclerites, whereas none of the Xenia coloniesexamined from group ‘A’ contain sclerites, thus indicatingthe importance of sclerites in taxonomic studies of thisgroup. There are no morphological features that support aclose relationship of Xenia group ‘A’ to Heteroxenia. More-over, in a more extensively sampled analysis of Xenia in-cluding both samples from group ‘A’ and Xeniidae coloniesfrom Indonesia, the colonies from group ‘A’ do not group asa sister taxon to Heteroxenia, but appear more related to anIndonesian species, X. lillieae (C.S. McFadden, unpublisheddata). Thus, the sister relationship observed in our prelimi-nary analyses is tentative because of limited taxon sampling,but there is evidence of two polyphyletic groups in thenominal genus Xenia that merits further attention.
In many cases, variation in colony morphologies compli-cated the classification process. For example, within both
species, Rhytisma fulvum fulvum and Tubipora musica, andin both genera, Dendronephthya and Paralemnalia, therewas no correspondence between morphological differencesamong colonies and molecular haplotypes, indicating mor-phological polymorphism in these groups.
This survey of Saudi Arabian octocorals yields a number ofquestions for future study, and a wealth of specimens that canbe used for future molecular and zoogeographic comparativeanalyses. Ten out of the 13 octocoral genera were clearlyresolved with ITS sequence data, 11 out of 13 were clearlyresolved using mtMutS and COI, but only 2 of the 13 wereresolved using ATPSα. This finding confirms a relativelyaccurate understanding of the evolutionary relationshipsamong morphological characters used to distinguish theseoctocoral taxa on generic level, with the exception of ourdiscovery of polyphyly within the genus Xenia, which in-dicates cryptic diversity and merits additional taxonomic at-tention. Additionally, our sampling of four loci (two nDNA:ITS, ATPSα; and two mtDNA:mtMutS,COI) appears to strikethe desirable balance between resolution, cost, and labor forfuture phylogenetic studies of alcyonacean corals, althoughATPSα may be more useful at higher taxonomic levels. Fur-ther research is needed to determine whether Octocoralliamimic scleractinians in the diversity gradients across SaudiArabia as reported by Sheppard and Sheppard (1985, 1991)and Sheppard (1985), but the trend is suggested by our obser-vations. Overall, our study provides both a preliminary taxo-nomic survey of the region that includes classical taxonomy,multilocus molecular barcodes, and in situ photographs, andwe provide an initial phylogenetic survey to guide futureresearch efforts in the region and on octocoral relationshipswithin the Alcyonacea.
Acknowledgments Support for this project came from the BinationalScience Foundation #2008186 to Y.B., C.S.M. and R.J.T. and the Na-tional Science Foundation (NSF) OCE-0623699 to R.J.T. Fieldwork wasfunded in part by NSF grant OCE-0929031 to B.W. Bowen, NSF OCE-0623699 (R.J.T.), and the King Abdullah University of Science andTechnology (KAUST) Red Sea Research Center (M.L.B.) We thank J.DiBattista, the KAUST Reef Ecology Lab, and Coastal and MarineResources Core Lab for logistic assistance. Also, we thank members ofthe ToBo laboratory at the Hawai’i Institute ofMarine Biology, especiallyZac Forsman, for guidance and advice with laboratory work and analyses,S. Hou and A. G. Young for assistance with sequencing nuclear genes,and A. Lee for assistance with sequencing the mitochondrial genes. Thiswork was completed in partial fulfillment of the requirements for aMasters of Science degree at the University of Hawai’i at Manoa, andbenefited from support and guidance of B.W. Bowen and D. Rubinoff.
References
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