· 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,

Page 1

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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

Page 2

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: [email protected]

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.

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