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Algae 2012, 27(2): 83-94http://dx.doi.org/10.4490/algae.2012.27.2.083

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Research Article

Copyright © The Korean Society of Phycology 83 http://e-algae.kr pISSN: 1226-2617 eISSN: 2093-0860

Phylogenetic relationships and distribution of Gelidium crinale and G. pusillum (Gelidiales, Rhodophyta) using cox1 and rbcL sequences

Kyeong Mi Kim1 and Sung Min Boo1,*1Department of Biology, Chungnam National University, Daejeon 305-764, Korea

The taxonomic distinctiveness and cosmopolitan distributions of the red algae Gelidium crinale and G. pusillum re-

main unclear. Both species were first described in Devon in southwestern England; namely in Ilfracome for G. crinale

and Sidmouth for G. pusillum. We analyzed mitochondrial cox1 and plastid rbcL sequences from specimens collected in

East Asia, Australia, Europe and North America. In all phylogenetic analyses of cox1 and rbcL sequences, G. crinale was

distinct from congeners of the genus. The analyses also revealed a sister relationship with the G. coulteri and G. capense

clade. Nineteen cox1 haplotypes were identified for G. crinale, and they were likely geographically structured. Despite the

distinctiveness in both cox1 and rbcL datasets, the sister relationship of G. pusillum in the genus was not resolved. Our

cox1 and rbcL datasets indicate that G. crinale is a cosmopolitan species, found in East Asia, Australia, Europe and North

America, while the distribution of G. pusillum is restricted to Europe and Atlantic North America. Our results suggest that

infraspecific classification of G. pusillum may be abandoned.

Key Words: cox1; distribution; Gelidium crinale; Gelidium pusillum; phylogeny; rbcL

INTRODUCTION

Gelidium Lamour. is composed of approximately 127

described species distributed globally along tropical, sub-

tropical, and artic shorelines (Freshwater and Rueness

1994, Shimada et al. 2000, Millar and Freshwater 2005,

Kim et al. 2011, in press, Guiry and Guiry 2012). Members

of the genus can be the most abundant organisms within

intertidal algal assemblages. Gelidium is economically

important as food, and one of the most promising agar

sources in rhodophytes. It has recently been used for in-

dustrial paper pulp production in Korea (Seo et al. 2010).

However, identification of individual Gelidium speci-

mens is notoriously difficult because of the high degree of

morphological variation, particularly in the smaller and

medium-sized species (Dixon and Irvine 1977a).

Gelidium crinale (Hare ex Turner) Gallion and G. pusil-

lum (Stackhouse) Le Jolis, which are small and morpho-

logically diverse, are among the most difficult species to

identify in red algae, and their distributions are unclear.

These species are traditionally recognized as two distinct

species (Feldmann and Hamel 1936, Silva et al. 1996), and

Womersley and Guiry (1994) found a difference between

the types of G. crinale and G. pusillum. On the contrary, G.

crinale and G. pusillum were merged by Dixon and Irvine

(1977a, 1977b). Seven to nine varieties or formas have

been described in each of G. crinale and G. pusillum (Silva

et al. 1996, Guiry and Guiry 2012). The name G. pusillum

is commonly used for any small tuft-forming Gelidium

(Silva et al. 1996). Although G. crinale and G. pusillum are

considered as the most widely distributed species in the

genus AlgaeBase (Guiry and Guiry 2012), the occurrence

Received February 27, 2012, Accepted May 28, 2012

*Corresponding Author

E-mail: [email protected]: +82-42-821-6555, Fax: +82-42-822-9690

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://cre-ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Algae 2012, 27(2): 83-94

http://dx.doi.org/10.4490/algae.2012.27.2.083 84

and Fredericq 2002); and for cox1, cox143F-cox11549R

(Geraldino et al. 2006) and C622F-C880R (Yang et al.

2008).

Ninety-three rbcL sequences including 13 new se-

quences and 65 cox1 sequences including 24 new Gelid-

ium sequences were collated using the Se-Al version

2.0a11 software (Rambaut 1996) and aligned visually.

Outgroup taxa used were representatives from Gelidiella

Feldmann et G. Hamel, Pterocladia J. Agardh, Pterocla-

diella Santelices et Hommersand, and Ptilophora (Suhr)

Kützing (Freshwater et al. 1995, Kim et al. 2011).

Maximum likelihood (ML) phylogenetic analysis of

rbcL was performed using the GTR + Γ + I model imple-

mented in RAxML software (Stamatakis 2006). We used

200 independent tree inferences with the “number of

run” option, with default optimized subtree pruning and

regrafting (SPR) rearrangement and 25 distinct rate cate-

gories to identify the best tree. Statistical support for each

branch was obtained from 1,000 bootstrap replications

using the same substitution model and RAxML program

settings.

Bayesian analyses (BA) were performed for combined

and individual datasets with MrBayes v.3.1.1 (Ronquist

and Huelsenbeck 2003) using the Metropolis-coupled

Markov chain Monte Carlo (MC3) with the GTR + Γ + I

model. For each matrix, one million generations of two

independent runs were performed with four chains and

sampling trees every 100 generations. The burn-in peri-

od was identified graphically by tracking the likelihoods

at each generation to determine whether they reached

a plateau. The 15,001 trees for rbcL and 22,501 trees for

cox1 sampled at the stationary state were used to infer the

Bayesian posterior probability.

A statistical parsimony network of cox1 haplotypes was

created using TCS version 1.21 software (Clement et al.

2000). Haplotype and nucleotide diversity measurements

were performed using DnaSP software (Rozas and Rozas

1999).

RESULTS

Molecular analyses

A total of 93 sequences from Gelidium and outgroups

were aligned using a 1,266-nucleotide (nt) portion of

rbcL. Variable sites were found at 492 portions (38.9%),

and 393 portions (31%) were parsimoniously informa-

tive. All GenBank accessions of G. crinale from nine coun-

tries formed a single monophyletic group with maximum

of both species in many countries should be reassessed.

In this study we characterized two species, namely G.

crinale and G. pusillum, using two molecular markers.

To evaluate the relationship and distribution of G. crinale

and G. pusillum, we analyzed plastid rbcL, and mitochon-

drial cox1 including type locality materials. Plastid rbcL is

commonly used for Gelidium phylogeny (Freshwater and

Rueness 1994, Freshwater et al. 1995, Shimada et al. 2000,

Millar and Freshwater 2005, Nelson et al. 2006, Kim et al.

2011). Recent studies have revealed that mitochondrial

cox1 is useful for both DNA bar-coding of gelidioid red

algae and to examine their distribution patterns (Fresh-

water et al. 2010, Wiriyadamrikul et al. 2010, Kim et al.

2012). In this study, we included published rbcL sequenc-

es analyzed from G. crinale type material (Freshwater et

al. 2010) and samples collected in the G. pusillum type

locality.

MATERIALS AND METHODS

Taxon sampling and morphological observation

A total of 18 specimens of G. crinale were obtained for

this study: 17 collected from 10 locations including Chi-

na, Hong Kong, Korea, Spain and the UK, and one strain

from the culture collection of the University of Texas,

UTEX (Appendix A). Ten G. pusillum field collections

were made at five locations in France, Spain and the UK.

Materials for morphological observations were mounted

on herbarium sheets, while clean apical parts of the spec-

imens were desiccated in silica gel for DNA extraction.

Tissues were sectioned using a freezing microtome (FX-

802A; Coper Electronics Co., Ltd., Kanagawa, Japan), and

sectioned preparations were stained with 1% aqueous

aniline blue. Photographs were taken with an FX-35DX

camera (Nikon, Tokyo, Japan) attached to a Vanox AHBT3

microscope (Olympus, Tokyo, Japan). Voucher specimens

were deposited at the herbarium of Chungnam National

University (CNUK), Daejeon, Korea.

DNA extraction, sequencing and phylogenetic analyses

Twenty-eight specimens were available for DNA extrac-

tion (Appendix A). DNA extraction, PCR amplification,

and sequencing are described in Geraldino et al. (2010).

Primer pairs for amplification and sequencing of each

gene were as follows: for rbcL, F7-R753 and F645-RrbcS

start (Freshwater and Rueness 1994, Lin et al. 2001, Gavio

Kim & Boo Gelidium crinale and G. pusillum

85 http://e-algae.kr

Fig. 1. Maximum likelihood tree of Gelidium using 93 rbcL sequences calculated using the GTR + Γ + I evolution model (-lnL = 10511.506124; substitution rate matrix RAC = 0.951940, RAG = 5.742494, RAT = 1.371355, RCG = 1.258690, RCT = 10.348932, RGT = 1; shape parameter [α] = 1.575834). Maximum likelihood bootstrap values and Bayesian posterior probabilities are shown for each clade. Only bootstrap values ≥50% and ≥0.95 Bayesian posterior probabilities are shown.

Algae 2012, 27(2): 83-94


Fig. 2. Maximum likelihood tree of Gelidium using 65 cox1 sequences calculated using the GTR + Γ + I evolution model (-lnL = 9341.027999; substitution rate matrix RAC = 1.153854, RAG = 10.881554, RAT = 1.077417, RCG = 0.000017, RCT = 20.342285, RGT = 1; shape parameter [α] = 1.101521). Maximum likelihood bootstrap values and Bayesian posterior probabilities are shown for each clade. Only bootstrap values ≥50% and ≥0.95 Bayesian posterior probabilities are shown.



terete to flattened distally (up to 550 µm wide). Spanish

specimen appears to have cylindrical branches (Fig. 4D).

Large dome-shaped apical cells are evident at the apices

and project over the cortical margin (Fig. 4E). Axes and

branches consist of cortex and medulla (Fig. 4F). Three

to five layered cortex consist of small, pigmented, rect-

angular surface, and oval inner cortical cells. Medulla is

composed of large, colourless medullary cells that were

intermixed with internal rhizoidal filaments.

G. pusillum thalli are cartilaginous and prostrate axes

are terete, quite regular in diameter, and erect branches

(Fig. 5A-C). Erect axes arise from the dorsal sides of inde-

terminate, prostrate axes, are compressed and up to 1.5

cm in height. Branches develop irregularly branching of

up to three orders. Dome-shaped apical cells are present

at the apices (Fig. 5D). Axes and branches consist of cor-

tex and medulla (Fig. 5E). The cortex consists of three to

four layers of small pigmented cells, whereas the medulla

is composed of large colorless cells.

DISCUSSION

Taxonomy of Gelidium crinale and G. pusillum

Based on both mitochondrial cox1 and plastid rbcL

datasets, Gelidium crinale was distinct from G. pusil-

lum and other congeners of the genus. All G. crinale rbcL

sequences from East Asia, Europe, Australia and North

America formed a monophyletic clade with a published

sequence (AF308786) from the type material (Freshwater

et al. 2010). G. crinale specimens from East Asia, Australia

and North America corresponded to the description by

Feldmann and Hamel (1936), and were identified based

on the thalli being caespitose and having cylindrical to

compressed prostrate and cylindrical erect axes with fili-

form branches. G. crinale formed a clade with G. capense

from South Africa and G. coulteri from USA. It was diffi-

cult to identify a synapomorphic characteristic for these

three species.

The intraspecific divergence (0.00-2.74%) of G. crinale

from Asia, Australasia, Europe and North America is simi-

lar to that (up to 2.65%) of the species in previous studies

(Freshwater et al. 2010) and that (0.00-2.45%) of Hypnea

flexicaulis Yamaighi et Masuda (Geraldino et al. 2006).

Seven varieties or formas have been described in G.

crinale; f. luxurians Collins (type locality, Pacific Beach,

San Diego Co. California) (Collins et al. 1906), var. lu-

bricum (Kützing) Hauck, var. spathulatum (Kützing)

Hauck (Adriatic Sea) (Womersley and Guiry 1994), var.

support (Fig. 1). The G. crinale clade consisted of three

subgroup; Asian, Australian, and European / American

group. Piarwise divergence of G. crinale was up to 2.74%.

G. crinale was sister to the clade of G. coulteri Harvey and

G. capense (S. G. Gmelin) P. C. Silva (93% for ML and 1.0

for BA). Eleven of G. pusillum sequences from France,

Norway, Spain, and UK formed a monophyletic group

(100% for ML and 1.0 for BA).

A total of 65 sequences from Gelidium and three out-

groups were aligned using a 1,200 nt region of the cox1

gene. Among the 447 (37.3%) variable sites, 400 portions

(33.3%) were parsimoniously informative. The topology

based on cox1 sequences was congruent with the rbcL

phylogeny (Fig. 2). The cox1 ML tree showed that all G.

crinale from eight countries were monophyletic (99% for

ML and 1.0 for BA). Twelve G. pusillum from France, Nor-

way, Spain, UK, and USA were monophyletic with maxi-

mum support.

Because of short sequences of cox1 in GenBank, in

haplotype analyses, we used 618 nt cox1 fragment of G.

crinale from 28 individuals collected in Australia, China,

Hong Kong, Korea, Puerto Rico, Spain, UK, and USA. A to-

tal of 19 haplotypes among 33 polymorphic sites (5.3%)

were found. Haplotype and nucleotide diversities of cox1

within G. crinale were 0.912 ± 0.049 (H) and 0.014 ± 0.005

(π), respectively. The 19 haplotypes were placed in three

geographically distinct groups; Asian, Australian, and Eu-

ropean / American groups (Fig. 3). All haplotypes in Asia

were closely related. However, H7 from Hainan, China

was linked to H5 (with six missing haplotypes) and to H3

and H4 (with seven missing haplotypes). Haplotypes H9-

H11 were found in Australia, and haplotypes H12-H19

were found in Puerto Rico, Spain, UK, and USA.

Eleven G. pusillum sequences from France, Norway,

Spain, and UK formed a monophyletic group with maxi-

mum support. Pairwise divergence of G. pusillum was

0.24%. Four haplotypes were found from nine individu-

als of G. pusillum from France, Spain and UK (data not

shown). Four specimens from France and UK shared

same haplotype. Three short cox1 sequences (450 nt long)

from NCBI (HQ412445-7) from France, Norway and USA

were also dentical.

Morphology of Gelidium crinale and G. pusillum

Specimens of G. crinale confirmed by cox1 and rbcL are

shown in Fig. 4. Thalli (Fig. 4A & B) from Jeoncheon, Ko-

rea and Qingdao, China are purple-red, cartilaginous and

composed of terete prostrate axes and erect axes (up to 2

cm high). Specimen from Seoko, Hong Kong (Fig. 4C) are

Algae 2012, 27(2): 83-94


lum, three are considered as synonyms of the species; var.

conchicolum Piccone et Grunow (type locality, Massawa,

Eritrea, Ethiopia) (Piccone 1884), f. foliaceum Okamura

(Type locality, Shiso-dima, Seto, Kii Province, Japan)

(Okamura 1934), and var. minusculum Weber-van Bosse

(type locality, Daram Inlet, East coast of Misoöl Island,

Indonesia) (Weber-van Bosse 1921) (see AlgaeBase, Guiry

and Guiry 2012). However, six have still been flagged in

the AlgaeBase; var. cylindricum W. R. Taylor (type locality,

Bahia San Francisco, Ecuador) (Taylor 1945), var. mucro-

natum P. J. L. Dangeard, var. pacificum W. R. Taylor (Isla

Santa Maria, Galapagos Islands, Ecuador) (Taylor 1945),

f. pakistancium Afaq-Husain et Shameel (type locality,

Gadani, Karachi, Pakistan) (Afaq-Husain and Shameel

1999), var. pulvinatum (C. Agardh) Feldmann (type local-

ity, Cadíz, Spain) (Feldmann and Hamel 1936), and var.

simplex P. J. L. Dangeard (type locality, Marocco) (Dan-

geard 1949).

The monophyly and low genetic variation (up to 0.24%)

of G. pusillum reveal that G. pusillum is a species with

less genetic diversities and indicate the above six varieties

or formas may be synonyms of the species or belong to

other species. For example, four varieties of G. pusillum

(e.g., var. conchicola, var. cylindricum, var. pacificum, and

var. pulvinatum) reported in Korea (Lee 1994, Lee and

Kim 1995) have not been found in our recent studies de-

spite many trips in their collection sites (Kim et al. 2011,

corymbosum (Kützing) Feldmann et G. Hamel (type lo-

cality, Mediterranean Sea, Italy) (Feldmann and Hamel

1936), var. perpusillum Piccone et Grunow (Eritrea, Mas-

sawa, Italy) (Piccone 1884), var. platycladum W. R. Taylor

(type locality, Port Aransas, Texas, USA) (Taylor 1943) and

var. polycladum (Kützing) Hauck. Of these, var. corymbo-

sum, var. platycladum, and var. perpusillum have been

accepted taxonomically in AlgaeBase (Guiry and Guiry

2012).

Our cox1 haplotype network revealed geological struc-

ture of the G. crinale populations, but pairwise diver-

gence (up to 2.74% in cox1) is in a range of other species,

as mentioned in above. It is therefore difficult to conclude

whether our molecular data support infraspecific classifi-

cation or not. Further sampling is necessary for confirm-

ing the infraspecific categories.

G. pusillum specimens from France, Norway, Spain,

and USA formed a monophyletic clade with those collect-

ed from the type locality (Sidmouth, Devon, England) in

both the cox1 and rbcL trees. G. pusillum is characterized

by tufted thalli having compressed, oval, or lanceolate

branches (Feldmann and Hamel 1936, Silva et al. 1996).

All specimens from UK, France and Spain are similar in

having compressed thalli and oval or lanceolate branch-

es. The sister relationship of G. pusillum in the genus was

not resolved in the cox1 and rbcL trees.

Of nine varieties or formas described within G. pusil-

Fig. 3. Nineteen cox1 haplotypes (H1-H19) network of Gelidium crinale from 22 localities in Australia, China, Hong Kong, Korea, Puerto Rico, Spain, UK, and USA. Small grey circles correspond to missing haplotypes and the size of each circle is proportional to the number of individuals analyzed. Numerals in parentheses refer to the number of specimens with identical sequences. AU, Australia; CH, China; ES, Spain; HK, Hong Kong; KR, Korea; PR, Puerto Rico; UK, United Kingdom; USA, United States.



A C

D

B

E F

Jeoncheon, KoreaSep 25, 2010

Cadíz, SpainApr 12, 2010

Qingdao, ChinaOct 11, 2010

Seoko, Hong KongApr 23, 2008

Fig. 4. Morphology of Gelidium crinale. (A) Specimen collected in Jeoncheon, Korea. (B) Specimen collected in Qingdao, China. (C) Specimen collected in Seoko, Hong Kong. (D) Specimen collected in Cadíz, Spain. (E) A dome-shaped apical cell at the tip of a branchlet. (F) Transverse section of an axis. Scale bars represent: A-D, 5 mm; E, 10 μm; F, 50 μm.

Fig. 5. Morphology of Gelidium pusillum. (A) Specimen collected in type locality (Sidmouth, Devon, UK). (B) Specimen collected in Cadíz, Spain. (C) Specimen collected in Caen, France. (D) A dome-shaped apical cell at the tip of a branchlet. (E) Transverse section of an axis. Scale bars represent: A-C, 5 mm; D, 10 μm; E, 50 μm.

A C

D

B

E

Sidmouth, UKApr 4, 2010

Cadíz, SpainApr 11, 2010

Caen, FranceNov 17, 2009

Algae 2012, 27(2): 83-94


Freshwater 2005, Freshwater et al. 2010)

Contrary to previous studies on its global distribution

(e.g., Silva et al. 1996, Guiry and Guiry 2012), our cox1 and

rbcL datasets revealed that G. pusillum is likely restricted

to Europe and Atlantic North America. Despite basic lo-

cal alignment search tool (BLAST) search of all sequences

registered in GenBank as well as sequences generated in

the present study, no accession of G. pusillum from Asia,

Australia and Pacific North America matched those from

Europe. However, G. pusillum specimens from Spain and

France formed a clade with those from Sidmouth, UK.

We suggest that records of G. pusillum from Europe and

Atlantic North America may be the result of misiden-

tifications, and the specimens filed under G. pusillum

should be treated with caution. We agree with the views

of Millar and Freshwater (2005) that G. pusillum, reputed

to be cosmopolitan (see Silva et al. 1996, Guiry and Guiry

2012), may be limited to areas around Europe and Atlan-

tic North America.

Conclusions

Identifications of G. crinale and C. pusillum based on

morphology alone is problematic and usually requires

molecular analyses for confirmation. Thus, reports of

G. pusillium outside from of Europe and Atlantic North

America should be treated with caution, and herbarium

specimens identified as G. pusillum in East Asia, Austra-

in press). Instead, at least one new species in Korea are

likely previously misidentified as variant of G. pusillum

(Kim et al. in press). Specimen (Fig. 5B) from the type

locality of G. pusillum var. pulvinatum, Cadíz, Spain did

not reveal variation in molecular data and morphology.

Therefore, we suggest that infraspecific classification of

G. pusillum may be abandoned.

Distribution of Gelidium crinale and G. pusillum

A distribution map of G. crinale and G. pusillum is

shown in Fig. 6. Two interesting biogeographic patterns

emerge when considering the overall distribution of both

species and their distinct lineages revealed by phyloge-

netic analyses. The parsimony network of cox1 haplo-

types revealed a geographic structure (Fig. 3); Asian, Aus-

tralian, and European / American groups. In the Asian

group, seven haplotypes were found and closely related.

Australian group is quite distinct from Asian, and Europe-

an / American groups. Specimens from North Carolina,

USA and Europe made one group with a single missing

haplotype. The genetic connectivity between Europe and

north America may be caused by similar oceanographic

conditions. Our results indicate that G. crinale is a cosmo-

politan species, although there are high morphological

variations and intraspecific divergences. This result is in

agreement with previous studies showing that G. crinale

is distributed globally (Shimada et al. 1999, Millar and

Fig. 6. Map showing current geographic distribution of Gelidium crinale and G. pusillum based on the present study and previous publications (Freshwater and Rueness 1994, Freshwater et al. 1995, 2010, Shimada et al. 1999, Millar and Freshwater 2005, Kim et al. 2011).



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ACKNOWLEDGEMENTS

We thank Jeong Kwang Park for preparing plates of

morphology, Juliet Brodie and Keith Hiscock for help in

collection trip in Devon, UK and Dr. José Lucas Perez in

Cadíz, Spain, respectively. This work was supported by

Marine Biotechnology Grants from the Ministry of Land,

Transportation Maritime Affairs and Basic Science Grant

(2012-0704) of Korean Research Foundation to SMB.

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Appendix A. Gelidium crinale and G. pusillum samples used in the present study

Collection sites (date)GenBank accession no.

SourcerbcL cox1

Gelidium crinale (Hare ex Turner) Gaillon

Australia; Fish Hook Bay, Rottnest Is. AF308788 - Freshwater et al. 2010

Australia; Fish Hook Bay, Rottnest Is. AY350780 - Freshwater et al. 2010

Australia; Green Is., Rottnest Is. (Aug 14, 2002) HQ412492 HQ412464 Freshwater et al. 2010

Australia; Green Is., Rottnest Is. (Aug 14, 2002) HQ412493 HQ412465 Freshwater et al. 2010

Australia; Old Gulch, Lord Howe Is. - HQ412466 Freshwater et al. 2010

Australia; Summer Cloud Bay, Jervis Bay AY350781 HQ412467 Freshwater et al. 2010

China; Donghai road, Qingdao (Oct 11, 2010) JX096509 JX096528 In this study

China; Donghai road, Qingdao (Oct 11, 2010) JX096510 - In this study

China; Hainan (May 11, 2009) JX096511 JX096529 In this study

China; Hainan (May 11, 2009) JX096512 JX096530 In this study

China; Oriental Hotel, Yantai (Sep 12, 2011) JX096513 JX096531 In this study

Hong Kong; Seoko (Apr 23, 2008) - JX096532 In this study

Hong Kong; Seoko (Apr 23, 2008) JX096514 JX096533 In this study

Hong Kong; Seoko (Apr 23, 2008) JX096515 JX096534 In this study

Japan; Awaji Is., Hyogo Pref. (Oct 1, 1996) AB017679 - Shimada et al. 1999

Korea; Bumseom, Jeju (Aug 23, 2009) JX096516 - In this study

Korea; Bumseom, Jeju (Jul 5, 2009) JX096517 JX096535 In this study

Korea; Dugok, Namhaegun (2010) - JX096536 In this study



Korea; Jeoncheon (Nov 25, 2010) JX096518 JX096539 In this study

Korea; Muchangpo, Boryeong (Jul 29, 2007) HM629823 HM629863 Kim et al. 2011

Korea; Pyeongdae, Jeju (May 29, 2010) - JX096540 In this study

Puerto Rico; Playa Esperanza, Manati U00983 HQ412463 Freshwater et al. 1995, 2010

Spain; Aramar, Asturias AF308791 - Freshwater et al. 2010

Spain; Aramar, Asturias AF308792 - Freshwater et al. 2010

Spain; La Arana, Malaga AF308789 - Freshwater et al. 2010

Spain; La Garita, Canary Is. AF308793 - Freshwater et al. 2010

Spain; Tarifa, Cadíz (Apr 12, 2010) JX096519 JX096541 In this study

UK; Ilfracombe, Devon AF308786 - Freshwater et al. 2010

UK; Sidmouth, Devon (Apr 4, 2010) JX096520 JX096542 In this study

USA; Beaufort Inlet, North Carolina AF308795 HQ412458 Freshwater et al. 2010

USA; Bogue Sound, North Carolina (Feb 16, 1991) HQ412489 HQ412460 Freshwater et al. 2010

USA; Fort Fisher, North Carolina (May 14, 1991) - HQ412461 Freshwater et al. 2010

USA; Marineland, Florida AF308794 - Freshwater et al. 2010

USA; Masonboro Inlet, North Carolina U00981 HQ412457 Freshwater et al. 1995

USA; North Carolina AF308790 - Freshwater et al. 2010

USA; Port Aransas, Texas U00982 - Freshwater et al. 1995

USA; Port Aransas, Texas (UTEX culture collection) JX096521 JX096543 In this study

USA; Radio Is., North Carolina (Feb 16, 1991) HQ412488 HQ412459 Freshwater et al. 2010

USA; Stump Sound, North Carolina (Jul 11, 2003) HQ412491 HQ412462 Freshwater et al. 2010

Algae 2012, 27(2): 83-94


Appendix A. Continued

Collection sites (date)GenBank accession no.

SourcerbcL cox1

Gelidium pusillum (Stackhouse) Le Jolis

France; Cancale, Brittany U01000 HQ412446 Freshwater and Rueness 1994, Freshwater et al. 2010

France; Lion sur-mer, Caen (Nov 17, 2009) HM629832 HM629872 Kim et al. 2011

France; Lion sur-mer, Caen (Nov 17, 2009) JX096522 - In this study

France; Wimereux U01001 - Freshwater and Rueness 1994

Norway; Fedje, Hordaland U00999 HQ412445 Freshwater et al. 1995, 2010

Spain; El Chato, Cadíz (Apr 11, 2010) JX096523 JX096544 In this study

Spain; El Chato, Cadíz (Apr 11, 2010) JX096524 JX096545 In this study

UK; Ilfracombe, Devon (Apr 3, 2010) - JX096546 In this study




UK; Penmon, Anglesey U01002 - Freshwater and Rueness 1994


UK; Sidmouth, Devon (Apr 4, 2010) JX096526 - In this study


USA; Masonboro Inlet, North Carolina (Dec 30, 2006) - HQ412447 Freshwater et al. 2010

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