Sequencing of the rbcL Marker Reveals the Nonnative Red Alga Grateloupia taiwanensis (Halymeniaceae, Rhodophyta) in Alabama

Abstract

Mobile Bay, AL has been the site for the introduction of several terrestrial and
freshwater invasive species, including red imported fire ants (Solenopsis invicta) and
spike-topped apple snails (Pomacea bridgesii). The Gulf of Mexico has also been
invaded by several marine animal species, such as zebra mussels (Dreissena
polymorpha). To date, no invasive marine macroalga has been reported in the Mobile
Bay area. However, recent collections of an unusual species of Grateloupia
(Halymeniaceae, Rhodophyta) in Alabama indicate that an introduction has been
made. On the basis of phylogenetic analysis of the large subunit of ribulose-1,5-
bisphosphate carboxylase/oxygenase (rbcL) marker, the species has been identified as
Grateloupia taiwanensis S.M. Lin & H.Y. Liang. This is the first report of G. taiwanensis
outside its native range.

Full text

Sequencing of the rbcL Marker Reveals the Nonnative Red Alga Grateloupia
taiwanensis (Halymeniaceae, Rhodophyta) in Alabama
MICHAEL S. DEPRIEST AND JUAN M. LO
´PEZ-BAUTISTA
Mobile Bay, AL has been the site for the introduction of several terrestrial and
freshwater invasive species, including red imported fire ants (Solenopsis invicta) and
spike-topped apple snails (Pomacea bridgesii). The Gulf of Mexico has also been
invaded by several marine animal species, such as zebra mussels (Dreissena
polymorpha). To date, no invasive marine macroalga has been reported in the Mobile
Bay area. However, recent collections of an unusual species of Grateloupia
(Halymeniaceae, Rhodophyta) in Alabama indicate that an introduction has been
made. On the basis of phylogenetic analysis of the large subunit of ribulose-1,5-
bisphosphate carboxylase/oxygenase (rbcL) marker, the species has been identified as
Grateloupia taiwanensis S.M. Lin & H.Y. Liang. This is the first report of G. taiwanensis
outside its native range.
G
rateloupia C. Agardh is a genus of benthic
marine red algae (Rhodophyta), currently
containing around 90 species (Guiry and Guiry,
2012). It is the largest genus in the family
Halymeniaceae. Species of this genus occur
throughout the world in warm temperate to
tropical marine waters. Several Grateloupia spe-
cies are known in the Gulf of Mexico, specifically
Grateloupia gibbesii Harvey and Grateloupia ptero-
cladina (M.J. Wynne) S. Kawaguchi & H.W.
Wang, as well as many reports of unidentified
Grateloupia species (see Fredericq et al., 2009).
Wynne (2011) listed a total of 11 species in the
western Atlantic, including G. gibbesii,G. pterocla-
dina, and Grateloupia filicina (J.V. Lamouroux) C.
Agardh. Wynne, however, remarked that past
identifications of G. filicina in the western
Atlantic, including the Gulf of Mexico, are
doubtful due to the results of De Clerck et al.
(2005b), which indicated that true G. filicina may
be restricted to the Mediterranean Sea and
Macaronesia. This suggests that tropical collec-
tions of G. filicina actually belong to a different
species.
The genus Grateloupia is known for having
simple morphologies that make distinguishing
species difficult, and DNA sequencing has been
instrumental in generic and species-level circum-
scriptions (e.g., Wang et al., 2001; De Clerck et
al., 2005b; Lin et al., 2008). Specimens previously
identified as G. filicina, frequently reported
throughout the world, actually show an unex-
pectedly high amount of genetic diversity and
are therefore morphologically static. As a result
of G. filicina being demonstrated to be polyphy-
letic, several new species have been split from the
group (e.g., Kawaguchi et al., 2001; De Clerck et
al., 2005b). Even in the past few years, the genus
has gained many new species, including Grate-
loupia huangiae S.-M. Lin & H.-Y. Liang, Grate-
loupia dalianensis H.W. Wang & D. Zhao, and
Grateloupia yinggehaiensis H.W. Wang & R.X.
Luan. The publications in which these species
are described (Lin and Liang, 2011; Zhao et al.,
2012) include molecular phylogenetic analyses
to more clearly delineate these taxa. Additional
taxonomic work is definitely necessary to contin-
ue to resolve systematic inconsistencies and to
account for unexpected, newly discovered diver-
sity in the genus Grateloupia.
In addition to these taxonomic concerns, it is
important to consider that Grateloupia contains
species that are known to be aggressively invasive,
most notably Grateloupia turuturu Yamada. Grate-
loupia turuturu, along with several other Grate-
loupia species, has been introduced in Italy
(Cecere et al., 2011), New Zealand (D’Archino
et al., 2007), Great Britain (Farnham and Irvine,
1973), France (Cabioch et al., 1997; Verlaque,
2001; Verlaque et al., 2005; Figueroa et al.,
2007), and the Atlantic coast of the United States
(Villalard-Bohnsack and Harlin, 1997; Gavio and
Fredericq, 2002; Marston and Villalard-Bohn-
sack, 2002). Due to the difficulty and cost of
stopping an invasive marine algal species—for
example, the 2000 accidental introduction of the
green alga Caulerpa taxifolia (M. Vahl) C. Agardh
in California (Anderson, 2005)—efforts to pre-
vent species introductions or to detect the
presence of a potential species are imperative
for conservation of native diversity. Recent
collections of Grateloupia made by the authors
on the Alabama coast have included specimens
that could not be morphologically identified
according to known taxa from the area. The
Gulf of Mexico Science, 2012(1–2), pp. 7–13
E2012 by the Marine Environmental Sciences Consortium of Alabama

current paper presents the identification of a
previously unknown nonnative species of Grate-
loupia from the Alabama Gulf Coast using large
subunit of ribulose-1,5-bisphosphate carboxyl-
ase/oxygenase (rbcL) sequence analysis, demon-
strates its position within Grateloupia using
phylogenetics, and suggests hypotheses regard-
ing the possible causes and circumstances of its
colonization.
MATERIALS AND METHODS
Twenty-one samples of Grateloupia of unknown
species and four samples of G. gibbesii were
collected from the locations listed in Table 1.
Individuals were found growing in the intertidal
or higher subtidal zone on rocks or cast ashore.
Upon collection, a small portion of thallus was
taken from each individual and desiccated in a
plastic bag with silica gel for later molecular
analysis. The remainder of each individual was
vouchered on a herbarium sheet; specimens
were deposited in The University of Alabama
Herbarium. DNA extraction of the desiccated
samples was performed using the DNEasy Plant
Mini Kit (Qiagen, Valencia, CA). The manufac-
turer’s recommendations were followed until the
final elutions, which were performed with
deionized water preheated to 65uC instead of
the elution buffer.
The rbcL marker, widely used for red algae in
both species identification (e.g., Saunders, 2009)
and phylogenetics (e.g., De Clerck et al., 2005a),
was amplified for all specimens. Polymerase
chain reaction (PCR) followed the methods of
Rindi et al. (2009). Primer sequences were
provided by G. W. Saunders (University of New
Brunswick, Fredericton, Canada, pers. comm.)
after standard primers failed to amplify. Proce-
dures for agarose gel electrophoresis, cleaning,
quantification of DNA, and capillary sequencing
were carried out according to Rindi et al. (2009).
Sequences were assembled using Geneious
Pro v5.1.7 (Drummond et al., 2010) and added
to a database of published rbcL sequences
from GenBank ,http://www.ncbi.nlm.nih.gov/
genbank/.for 18 Grateloupia samples selected
as an accurate representation of genetic diversity
in the genus (Table 1). The species Yonagunia
formosana (Okamura) Kawaguchi & Masuda was
selected as the outgroup after Lin and Liang
(2011). Sequences were aligned using MUSCLE
sequence alignment (Edgar, 2004) in Geneious.
After alignment, sequences were manually
checked for accuracy and truncated to uniform
length to avoid including ‘‘missing’’ data due to
incomplete and partial published sequences.
Other than trimming, no adjustments were made
to the alignment. Pair-wise distances between
sequences were calculated in Geneious when
applicable.
Parameters for maximum likelihood (ML) and
Bayesian inference (BI) were determined using
jModelTest 2.1 (Guindon and Gascuel, 2003;
Posada, 2008). ML analysis was executed in
GARLI v2.0 (Zwickl, 2006) with 500 bootstrap
replicates, starting from a random tree. Boot-
strap confidence values were obtained via Con-
sense (Felsenstein, 2005) on the CIPRES Science
Gateway (Miller et al., 2010). Values obtained
from Consense were converted to a percentage
value and rounded down. BI was executed in
MrBayes v3.1.2 (Huelsenbeck and Ronquist,
2001; Ronquist and Huelsenbeck, 2003).
The final tree was obtained in the NEXUS file
format, rooted with Y. formosana, and processed
in FigTree v1.3.1 ,http://tree.bio.ed.ac.uk/
software/figtree/.and Adobe Illustrator CS3
(Adobe Systems Incorporated, San Jose, CA) for
publication.
RESULTS
After alignment, the rbcL data set consisted of
20 taxa with 1,195 base pairs each. All 21
sequences of Grateloupia from Alabama were
identical, and all four sequences of G. gibbesii
were identical; therefore, only one sequence was
included in the alignment for each taxon. The
alignment contained no gaps, reflecting an
accurate alignment and the absence of insertions
and deletions in rbcL of red algae. The TrN +G
model was selected by ModelTest. Nucleotide
frequencies, substitution rates, and gamma
shape parameter were estimated by GARLI.
The resulting phylogram, with bootstrap confi-
dence values, is given in Figure 1. The alignment
and tree produced in this analysis are available in
TreeBASE: ,http://purl.org/phylo/treebase/
phylows/study/TB2:S13391..
DISCUSSION
The current study presents samples from an
unidentified Grateloupia population in Alabama.
The rbcL sequences for the Alabama Grateloupia
samples are identical to each other and nearly
identical (,0.1% divergent) to G. taiwanensis S.-
M. Lin & H.Y. Liang in Lin et al. (2008). Near-
complete similarity indicates that these speci-
mens are conspecific. The unidentified Grate-
loupia in Alabama is therefore determined to be
G. taiwanensis. This assertion is supported by the
sequence divergences among G. taiwanensis and
some of its most closely related taxa (.3.1%
divergent from G. huangiae and relatives) in this
8 GULF OF MEXICO SCIENCE, 2012, VOL. 30(1–2)

TABLE 1. List of species used in this study, with collection information and references.
Species Locality Collection data GenBank accession Reference
Grateloupia americana S. Kawaguchi
& H.W. Wang
Whale Park, near Sitka
(Baranof Island), AK
S. Lindstrom, 21 April 2000 AF488814 Gavio and Fredericq (2002)
Grateloupia belangeri (Bory) De Clerck,
Gavio, Fredericq, Cocquyt & Coppejans
Yzerfontein, Western Cape Province,
South Africa
O. De Clerck, 24 Nov. 1999 AY772035 De Clerck et al. (2005a)
Grateloupia capensis O. De Clerck Kommetjie, Cape Peninsula, South Africa O. De Clerck, 1 June 2003 AJ868466 De Clerck et al. (2005b)
Grateloupia doryphora (Montagne)
M.A. Howe
Playa de San Francisco, Bahı´a de Anco´n,
Anco´ n, Lima, Peru
P. Carba´jal, 15 Sep. 2001 AF488817 Gavio and Fredericq (2002)
Grateloupia elliptica Holmes Oryu
¯zako, Miyazaki Prefecture, Japan
(cultured material)
21 Dec. 1997 AB038605 Wang et al. (2000)
Grateloupia filicina (Lamouroux)
C. Agardh
Cala Aiguafreda, Begur, Province of
Girona, Spain
L. Lavelli, 1 May 2002 AJ868474 De Clerck et al. (2005b)
Grateloupia gibbesii Harvey Charleston Harbor, Charleston, SC M.S. DePriest, 22 June 2012 JX645160 This study
Grateloupia huangiae S.-M. Lin
& H.-Y. Liang
Makang, Taipei County, N.E. Taiwan S.-M. Lin, 30 April 2002 HM590410 Lin & Liang (2011)
Grateloupia longifolia Kylin Yzerfontein, Western Cape Province,
South Africa
O. De Clerck, 2 June 2003 AY772023 De Clerck et al. (2005a)
Grateloupia minima P.L. Crouan
& H.M. Crouan
Yzerfontein, Western Cape Province,
South Africa
O. De Clerck, 2 June 2003 AJ868487 De Clerck et al. (2005b)
Grateloupia phuquocensis Tanaka
& Pham-Hoa`ng Hoˆ
Kaalawai, Oahu, HI O. De Clerck, 26 April 2003 AY772022 De Clerck et al. (2005a)
Grateloupia somalensis Hauck Plage de Monseigneur, Fort Dauphin,
Madagascar
E. Coppejans, 31 Aug. 2002 AY772021 De Clerck et al. (2005a)
Grateloupia sparsa (Okamura) Chiang O
–hara, Chiba Prefecture, Japan 9 April 2000 AB055473 Kawaguchi et al. (2001)
Grateloupia subpectinata Holmes Irago-misaki, Atsumi Peninsula, Aichi
Prefecture, Japan
3 July 2000 AB114213 Faye et al. (2004)
Grateloupia taiwanensis S.-M.
Lin & H.-Y. Liang
Sail Rock, Kenting National Park,
S. Taiwan
S.-M. Lin, 2 Oct. 2002 EU292742 Lin et al. (2008)
Grateloupia turuturu Yamada Onahama, Iwaki, Fukushima Prefecture,
Japan
16 Sep. 1999 AB055475 Kawaguchi et al. (2001)
Grateloupia turuturu The Kench, Hayling Island, Langstone
Harbour, Hampshire, UK
R.L. Fletcher, 28 March 2002 AY100002 Gavio and Fredericq (2002)
Grateloupia turuturu Lagoon of Venice, Venice, Italy A. Sfriso FN821950 Cecere et al. (2011)
Grateloupia sp. Fort Morgan, AL J.M. Lo´ pez-Bautista, 5 May 2004 JX645159 This study
DEPRIEST AND LO
´PEZ-BAUTISTA—GRATELOUPIA TAIWANENSIS IN ALABAMA 9

analysis and previously published phylogenies.
Grateloupia taiwanensis has not been previously
reported from the Gulf of Mexico (Fredericq et
al., 2009) or the western Atlantic (Wynne, 2011).
Before the current study, the distribution of G.
taiwanensis was known to include only Taiwan
(Lin et al., 2008). Therefore, we consider G.
taiwanensis to be a nonnative species in the Gulf
of Mexico. We also consider the introduction of
G. taiwanensis to be recent; this is supported by
the lack of previous reports of Grateloupia
specimens from the Gulf of Mexico with the
morphological features typical of G. taiwanensis
(large size and proliferous blades, see Figure 2,
which make it very conspicuous in intertidal
habitats) and the previous experience of the
authors collecting in the Gulf of Mexico.
On the basis of its rbcL sequence, G. gibbesii
does not appear to be conspecific with any
species in this analysis or with any species with a
published rbcL sequence in GenBank. The
samples collected of this species are very close
(,5 km) to the type locality of this alga,
Sullivan’s Island, SC (Harvey, 1853). Therefore,
we conclude that our identification of this alga is
correct and that G. gibbesii represents a unique
evolutionary lineage. Before the current study’s
publication, the authors became aware of the
possibility that the unknown Grateloupia found in
Alabama might not be G. taiwanensis but G.
gibbesii because this species had already been
known in the Gulf of Mexico and sequence data
had not been generated for it. However, because
these two species show a sequence divergence of
6.8% in rbcL, this is not the case. Future
collections and sequencing of G. gibbesii from
the Gulf of Mexico are needed to confirm its
presence.
The phylogeny reconstructed in the current
study shows that G. taiwanensis is closely related
to other taxa known primarily from the Pacific
Ocean. Of the species included in the analysis, G.
huangiae was described most recently (Lin and
Liang, 2011) and is found in Taiwan. Grateloupia
sparsa is widely distributed in the Asian Pacific,
along with G. turuturu. However, G. turuturu is
found throughout the world as an invasive
species. None of these species has been found
Fig. 1. Maximum likelihood (ML) phylogram of the rbcL marker of selected species of Grateloupia, using the TrN
+G model. Numbers above branches indicate ML bootstrap confidence values (500 replicates). Numbers below
branches indicate Bayesian posterior probabilities. ‘‘X’’ indicates support ,50%. Scale bar 5substitutions per site.
10 GULF OF MEXICO SCIENCE, 2012, VOL. 30(1–2)

Fig. 2. Herbarium sample of Grateloupia taiwanensis from Alabama.
DEPRIEST AND LO
´PEZ-BAUTISTA—GRATELOUPIA TAIWANENSIS IN ALABAMA 11

in the Gulf of Mexico, and of these, only G.
turuturu is known outside the Pacific Ocean. On
the basis of these distributions, it appears likely
that the most recent common ancestor of this
group occurred in Asia and that G. taiwanensis
was introduced to Alabama from Taiwan, rather
than vice versa. This is concordant with the
pattern of introductions of species of Grateloupia
from Asia, most notably G. turuturu but also
including several other species (Verlaque et al.,
2005).
Apart from the current study, which reports G.
taiwanensis for the first time from Alabama, the
extent of the occurrence of G. taiwanensis in the
Gulf of Mexico is currently unknown. Additional
collections are being made to Grateloupia taiwa-
nensis to determine their current expansion and
any possible detrimental effects this introduction
might have on marine communities.
ACKNOWLEDGMENTS
This study was funded by the U.S. National
Science Foundation (NSF) Assembling the Tree
of Life Program (DEB 0937978 to JLB) and the
NSF Research Experiences for Undergraduates
(DEB 1027012 to JLB). The authors express
sincere thanks to Dr. Showe-Mei Lin (National
Taiwan Ocean University, Keelung, Taiwan) for
providing samples and sequences of Grateloupia
taiwanensis; to Dr. Michael J. Wynne (University
of Michigan Herbarium, Ann Arbor, MI) for his
insightful comments on this manuscript; and to
Dr. D. Reid Wiseman (College of Charleston,
Charleston, SC) for providing samples of Grate-
loupia gibbesii and for collection assistance in
Charleston.
LITERATURE CITED
ANDERSON, L. W. J. 2005. California’s reaction to
Caulerpa taxifolia: a model for invasive species rapid
response. Biol. Invasions 7:1003–1016.
CABIOCH, J., A. CASTRIC-FEY, M.-T. L’HARDY-HALOS,AND A.
RIO. 1997. Grateloupia doryphora et Grateloupia filicina
var. luxurians (Rhodophyta, Halymeniaceae) sur les
coˆtes de Bretagne (France). Cryptogamie Algol.
18:117–137.
CECERE, E., I. MORO,M.A.WOLF,A.PETROCELLI,M.
VERLAQUE,AND A. SFRISO. 2011. The introduced
seaweed Grateloupia turuturu (Rhodophyta, Halyme-
niales) in two Mediterranean transitional water
systems. Bot. Mar. 54:23–33.
D’ARCHINO, R., W. A. NELSON,AND G. C. ZUCCARELLO.
2007. Invasive marine red alga introduced to New
Zealand waters: first record of Grateloupia turuturu
(Halymeniaceae, Rhodophyta). N. Z. J. Mar. Freshw.
Res. 41:35–42.
DECLERCK, O., B. GAVIO,S.FREDERICQ,I.BA
´RBARA,AND E.
COPPEJANS. 2005b. Systematics of Grateloupia filicina
(Halymeniaceae, Rhodophyta) based on rbcL se-
quence analyses and morphological evidence, in-
cluding the reinstatement of G. minima and the
description of G. capensis sp. nov. J. Phycol.
41:391–410.
———, ———, ———, E. COCQUYT,AND E. COPPEJANS.
2005a. Systematic reassessment of the red algal genus
Phyllymenia (Halymeniaceae, Rhodophyta). Eur. J.
Phycol. 40:169–178.
DRUMMOND, A. J., B. ASHTON,S.BUXTON,M.CHEUNG,A.
COOPER,C.DURAN,M.FIELD,J.HELED,M.KEARSE,S.
MARKOWITZ,R.MOIR,S.STONES-HAVAS,S.STURROCK,T.
THIERER,AND A. WILSON. 2010. Geneious v5.0,
available from http://www.geneious.com, accessed
12 September 2012.
EDGAR, R. C. 2004. MUSCLE: multiple sequence
alignment with high accuracy and high throughput.
Nucl. Acids Res. 32:1792–1797.
FARNHAM, W. F., AND L. M. IRVINE. 1973. The addition of
a foliose species of Grateloupia in the British marine
flora. Br. Phycol. J. 14:208–209.
FAYE, E. J., H. W. WANG,S.KAWAGUCHI ,S.SHIMADA,AND M.
MASUDA. 2004. Reinstatement of Grateloupia subpecti-
nata (Rhodophyta, Halymeniaceae) based on mor-
phology and rbcL sequences. Phycol. Res. 52:59–67.
FELSENSTEIN, J. 2005. PHYLIP (phylogeny inference
package). Department of Genome Sciences, Univer-
sity of Washington, Seattle, WA.
FIGUEROA, F. L., N. KORBEE,O.DECLERCK,I.BA
´RBARA,AND
E. A. R. GALL. 2007. Characterization of Grateloupia
lanceola (Halymeniaceae, Rhodophyta), an obscure
foliose Grateloupia from the Iberian Peninsula, based
on morphology, comparative sequence analysis and
mycosporine-like amino acid composition. Eur. J.
Phycol. 42:231–242.
FREDERICQ, S., T. O. CHO,S.A.EARLE,C.F.GURGEL,D.M.
KRAYESKY,L.E.MATEO-CID,A.M.MENDOZA-GONZA
´LES,J.
N. NORRIS,AND A. M. SUA
´REZ. 2009. Seaweeds of the
Gulf of Mexico, p. 187–259. In: Gulf of Mexico: its
origins, waters and biota. I. Biodiversity, D. L. Felder,
and D. K. Camp (eds.). Texas A&M University Press,
College Station, TX.
GAVIO, B., AND S. FREDERICQ S. 2002. Grateloupia turuturu
(Halymeniaceae, Rhodophyta) is the correct name
of the non-native species in the Atlantic known as
Grateloupia doryphora. Eur. J. Phycol. 37:349–359.
GUINDON, S., AND O. GASCUEL. 2003. A simple, fast, and
accurate algorithm to estimate large phylogenies by
maximum likelihood. Syst. Biol. 52:696–704.
GUIRY, M. D., AND G. M. GUIRY. 2012. AlgaeBase. World-
wide electronic publication, National University of
Ireland, Galway. http://www.algaebase.org; searched
on 6 September 2012.
HARVEY, W. H. 1853. Nereis Boreali-Americana: or,
Contributions to a History of the Marine Algae of
North America. Part II, Rhodospermeae. V. Crypto-
nemiaceae, XXII. Grateloupia. Smithsonian Contri-
butions to Knowledge 5(5). Reprint in Bibliotheca
Phycologica Band 20, p. 199–200. J. Cramer Publish-
ing, Lubrecht & Cramer LTD, Port Jervis, NY.
HUELSENBECK, J. P., AND F. RONQUIST. 2001. MRBAYES:
Bayesian inference of phylogeny. Bioinformatics
17:754–755.
12 GULF OF MEXICO SCIENCE, 2012, VOL. 30(1–2)

KAWAGUCHI, S., H. W. WANG,T.HORIGUCHI,G.SARTONI,
AND M. MASUDA. 2001. A comparative study of the red
alga Grateloupia filicina (Halymeniaceae) from the
northwestern Pacific and Mediterranean with the
description of Grateloupia asiatica sp. nov. J. Phycol.
37:433–442.
LIN, S.-M., AND H.-Y. LIANG. 2011. Grateloupia huangiae
(Halymeniaceae, Rhodophyta), a new species from
Taiwan previously confused with Polyopes lancifolius,
with emphasis on the development of the auxiliary-
cell ampullae. Phycologia 50:232–240.
———, ———, AND M. H. HOMMERSAND. 2008. Two
types of auxiliary cell ampullae in Grateloupia
(Halymeniaceae, Rhodophyta), including G. taiwa-
nensis sp. nov. and G. orientalis sp. nov. from Taiwan
based on rbcL gene sequence analysis and cystocarp
development. J. Phycol. 44:196–214.
MARSTON, M., AND M. VILLALARD-BOHNSACK. 2002. Genetic
variability and potential sources of Grateloupia
doryphora (Halymeniaceae, Rhodophyta), an invasive
species in Rhode Island waters (USA). J. Phycol.
38:649–658.
MILLER, M. A., W. PFEIFFER,AND T. SCHWARTZ. 2010.
Creating the CIPRES Science Gateway for inference
of large phylogenetic trees, p. 1–8. In: Proceedings of
the Gateway Computing Environments Workshop
(GCE), 14 Nov. 2010, New Orleans, LA.
POSADA, D. 2008. jModelTest: phylogenetic model
averaging. Mol. Biol. Evol. 25:1253–1256.
RINDI, F., D. W. LAM,AND J. M. LO
´PEZ-BAUTISTA. 2009.
Phylogenetic relationships and species circumscrip-
tion in Trentepohlia and Printzina (Trentepohliales,
Chlorophyta). Mol. Phylogen. Evol. 52:329–339.
RONQUIST, F., AND J. P. HUELSENBECK. 2003. MRBAYES 3:
Bayesian phylogenetic inference under mixed mod-
els. Bioinformatics 19:1572–1574.
SAUNDERS, G. W. 2009. Routine barcoding of Canadian
Gracilariales (Rhodophyta) reveals the invasive spe-
cies Gracilaria vermiculophylla in British Columbia.
Mol. Ecol. Resourc. 9(s1):140–150.
VERLAQUE, M. 2001. Checklist of the macroalgae of
Thau Lagoon (He´ rault, France), a hot spot of
marine species introduction in Europe. Oceanol.
Acta 24:29–49.
———, P. M. BRANNOCK,T.KOMATSU,M.VILLALARD-
BOHNSACK,AND M. MARSTON. 2005. The genus Grate-
loupia C. Agardh (Halymeniaceae, Rhodophyta) in
the Thau Lagoon (France, Mediterranean): a case
study of marine plurispecific introductions. Phycolo-
gia 44:477–496.
VILLALARD-BOHNSACK, M., AND M. M. HARLIN. 1997. The
appearance of Grateloupia doryphora (Halymeniaceae,
Rhodophyta) on the northeast coast of North
America. Phycologia 36:324–328.
WANG, H. W., S. KAWAGUCHI,T.HORIGUCHI,AND M.
MASUDA. 2000. Reinstatement of Grateloupia catenata
(Rhodophyta, Halymeniaceae) on the basis of
morphology and rbcL sequences. Phycologia
39:228–237.
———, ——— , ———, AND ———. 2001. A morpho-
logical and molecular assessment of the genus Prionitis
J. Agardh (Halymeniaceae, Rhodophyta). Phycol. Res.
49:251–261.
WYNNE, M. 2011. A checklist of benthic marine algae of
the tropical and subtropical western Atlantic: third
revision. Nova Hewigia Beih. 140:166.
ZHAO, D., H. WANG,Y.SHENG,J.LU
¨,AND R. LUAN. 2012.
Morphological observation and rbcL gene sequences
studies of two new species, Grateloupia dalianensis
H.W. Wang et D. Zhao, sp. nov. and G. yinggehaiensis
H.W. Wang et R.X. Luan, sp. nov. (Halymeniaceae,
Rhodophyta) from China. Acta Oceanol. Sin.
31:109–120.
ZWICKL, D. J. 2006. Genetic algorithm approaches for
the phylogenetic analysis of large biological se-
quence data sets under the maximum likelihood
criterion. Ph.D. diss., The University of Texas at
Austin.
THE UNIVERSITY OF ALABAMA,TUSCALOOSA,ALABAMA
35487. Date accepted: October 16, 2012
DEPRIEST AND LO
´PEZ-BAUTISTA—GRATELOUPIA TAIWANENSIS IN ALABAMA 13