Botanica Marina 47 (2004): 389–394 2004 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BOT.2004.053
Recent introduction of Polysiphonia morrowii (Ceramiales,
Rhodophyta) to Punta Arenas, Chile
Myung-Sook Kim1, Eun Chan Yang2, Andres
Mansilla3 and Sung Min Boo2,*
Research Institute for Basic Sciences, Pusan National
University, Busan 609-735, Korea
2
Department of Biology, Chungnam National University,
Daejon 305-764, Korea, e-mail: smboo@cnu.ac.kr
3
Department of Sciences and Natural Resources,
University of Magallanes, Casilla 113-D, Punta Arenas,
Chile
1
*Corresponding author
Abstract
Polysiphonia morrowii (Rhodomelaceae, Rhodophyta) is
abundant in intertidal zones of the northwestern Pacific
Ocean in spring, and has been introduced to the Mediterranean Sea. We collected specimens of this species
from intertidal rocks in February in Punta Arenas, in
southernmost Chile. Thalli were densely tufted, slender,
and elongate, with four pericentral cells, no cortication
and short tetrasporangial ultimate branchlets. The plastid
protein-coding rbcL was analyzed from specimens collected in Chile and Korea, as well as from putative relatives. The rbcL sequences of the Chilean specimens
were almost identical to those from Korea, and were
clearly separated from other related taxa from Chile and
other areas. These results suggest that P. morrowii has
been introduced recently from the northwestern Pacific
Ocean to Chile.
Keywords: Chile; invasive species; Polysiphonia
morrowii; rbcL, Rhodomelaceae, Rhodophyta.
Introduction
Due to the development of inter-oceanic transport,
coastal marine algal ecosystems worldwide are now
threatened by invasive species (Carlton and Geller 1992,
McIvor et al. 2001). Many reports have dealt with the
invasion of marine algae from the North Pacific Ocean to
the North Atlantic Ocean (Farnham 1980, Curiel et al.
1998, Maggs and Stegenga 1999, Verlaque 2001). However, only three invasive seaweed species from the
Northwest Pacific Ocean have been reported in the
Southern Atlantic and Pacific Oceans: Undaria pinnatifida
(Harvey) Suringar in Argentina (Casas and Piriz 1996),
Anotrichium yagii (Okamura) Baldock in Brazil (Horta and
Oliveira 2000), and Codium fragile ssp. tomentosoides
(van Goor) P.C. Silva in Chile (González and Santelices
2004).
Polysiphonia morrowii Harvey, a rhodomelacean red
alga, occurs commonly within intertidal zones along
Northwest Pacific coasts. It is an annual species and all
life history stages occur in the same period from February to May in Korea (Kim et al. 1994). Polysiphonia morrowii is characterized by prostrate and erect axes with
four pericentral cells, rhizoids in open connection with
the pericentral cells, axillary tetrasporangial branchlets,
and endogenous axillary branchlets produced from the
central axial cell (Harvey 1857, Segi 1951, Yoon 1986,
Kudo and Masuda 1992, Kim et al. 1994). It is distributed
in Korea (Kim et al. 1994), Japan (Kudo and Masuda
1992), China (Segi 1951), and far-eastern Russia (Perestenko 1980). It also occurs as an introduced species in
the Mediterranean Sea (Verlaque 2001, Curiel et al. 2002).
We collected specimens of Polysiphonia morrowii
recently from a littoral zone in Punta Arenas, Chile, and
compared thallus morphology with specimens from
Korea. To confirm the taxonomic identification of the
materials, we also analyzed the large subunit of the
RuBisCO gene (rbcL) from P. morrowii collected in Chile
and Korea, as well as from putative relatives. Analyses
of the rbcL sequences were selected because they are
more variable than the SSU rDNA gene sequences of
some ceramialean red algae (Cho et al. 2003a,b), and the
rbcL proved useful for tracking separate invasions of
Neosiphonia harveryi (Bailey) M.S. Kim et al. (as Polysiphonia harveyi Bailey, McIvor et al. 2001). Although many
Polysiphonia species are very variable in their morphology (Kim et al. 2000), and members of this cosmopolitan
genus have often been confused (McIvor et al. 2001),
molecular analyses are beginning to elucidate the taxonomic status of morphologically similar species (Choi et
al. 2001, McIvor et al. 2001). The main goal of this study
was to elucidate the biogeography of P. morrowii from
Punta Arenas, and to provide a description of its possible
route of introduction. This is the first report of P. morrowii
from the southeastern Pacific Ocean.
Materials and methods
Samplings and morphological observations
Eight samples of Polysiphonia morrowii were collected
from the intertidal zones in Korea and Chile. The species
inhabits a variety of substrata, such as rocks, wooden
piles, ropes, mussels, and other large algae. Seven other
putative relatives were sampled from Chile and the USA.
Specimens were fixed in 5% formalin/seawater, or were
air-dried, and deposited in the herbarium of the Department of Biology, Chungnam National University, Daejon,
Korea (CNUK). Photographs were taken through an
Olympus microscope (Vanox AHBT3, Tokyo, Japan).
390 M.-S. Kim et al.: Introduction of Polysiphonia morrowii to Chile
Table 1 Species studied.
Taxa
Locality
Voucher
GenBank accession no.
Polysiphonia brodiaei
P. elongata (Hudson) Sprengel
P. morrowii
Ireland: N. Ireland: Portaferry
Ireland: Fanad
Korea: Yeosu: Dolsando
Korea: Gangreung:Anin
Korea: Jeju: Hansuri
Korea: Jindo: Hoidong
Korea: Namhaedo: Namhaegyo
Korea: Tongyong: Mireukdo
Chile: Punta Arenas: Fuerte Bulnes
Chile: Punta Arenas: Seno Otway
USA: Oregon: Seal Rock
USA: Washington: Orcas Island
Chile: Punta Arenas: Fuerte Bulnes
Chile: Punta Arenas: Seno Otway
USA: Oregon: Devil’s Punchbowl
New Zealand: Wellington: Titahi Bay
Chile: Punta Arenas: Fuerte Bulnes
Chile: Central Chile: Las Cruses
England: Hampshire: Hayling
USA: California: Beach Club Reef
South Africa: S. Kwa Zulu-Natal: Palm Beach
Germany: Kiel Bight
–
–
P148
P47
P174
P65
P173
P175
P176
P177
P193
P191
P179
P178
P192
–
P180
P183
–
–
–
–
AF3429161
AF3429111
AY396034
AY396030
AY396032
AY396033
AY396031
AY396027
AY396029
AY396028
AY396036
AY396035
AY396040
AY396041
AY396039
AF3429081
AY396037
AY396038
AF3429001
AY1725782
AF4658163
AF0833814
P. pacifica
P. paniculata Montagne
P. scopulorum
P. strictissima J.D. Hooker et Harvey
Polysiphonia sp.
Neosiphonia harveyi
Chondria californica
Laurencia natalensis
Rhodomela confervoides
1
McIvor et al. (2001);
2
McIvor et al. (2002);
3
Fujii et al. (unpublished); 4 de Jong et al. (1998).
Analysis of the rbcL sequence
The source of algal material used in this study and the
GenBank (http://www.ncbi.nlm.nih.gov) accession numbers of rbcL sequence data are listed in Table 1. Samples
from the field were transported live back to the laboratory
in sterilized seawater, then cleaned, and sorted carefully
under a dissecting microscope.
Genomic DNA was extracted from approximately
0.005 g of dry thalli ground in liquid nitrogen using the
Qiagen DNeasy Plant Mini Kit (Qiagen GmbH, Hilden,
Germany) or the Invisorb Spin Plant Mini Kit (Invitek, Berlin-Buch, Germany), according to the manufacturers’
instructions. The rbcL region was amplified using primers
F7-R753 and F645-RrbcS start and sequenced, using
primers F7, F645, R753, and RrbcS start (Freshwater and
Rueness 1994, Lin et al. 2001, Gavio and Fredericq
2002). The PCR products were purified using a High
PureTM PCR product purification kit (Roche Diagnostics
GmbH, Mannheim, Germany), in accordance with the
user’s guide. The sequences of the forward and reverse
strands were determined for all taxa using an ABI
PRISMTM 377 DNA Sequencer (Applied Biosystems, Foster City, CA, USA) at Research Center, Chungnam
National University, Daejon, Korea. The electropherogram
output for each specimen was edited using the program
Sequence Navigator v. 1.0.1 (Applied Biosystems, Foster
City, CA, USA). The alignment of each gene sequence
was based on the alignment of the inferred amino acid
sequence and was refined by eye. There were no gaps
in our alignments of rbcL. A total of 22 taxa including the
rbcL data from GenBank were used for phylogenetic
analysis. Chondria californica (Collins) Kylin, Laurencia
natalensis Kylin, and Rhodomela confervoides (Hudson)
Silva were used as outgroups.
The sequence data were analyzed evaluating maximum likelihood (ML), minimum evolution (ME), and maximum parsimony (MP), using PAUP* (Swofford 2002).
Modeltest (Posada and Crandall 1998), used to determine the correct parameters for the ML analyses, specified a general time reversible (GTR) model, with the
proportion of invariable sites set at 0.5367, and a gamma
distribution of 2.4477. The rate matrix specified was ACs1.1893, A-Gs4.7846, A-Ts6.4000, C-Gs1.3143, CTs20.5815, and G-Ts1, and the base frequencies
specified were As0.3275, Cs0.1626, Gs0.2030, and
Ts0.3068. Tree likelihoods were estimated using a heuristic search with 100 random addition sequence replicates, and tree bisection reconnection (TBR) branch
swapping. For all ME analyses, a ML distance matrix was
used as input, with parameters specified by Modeltest.
The ME method (Rzhetsky and Nei 1992) was used to
construct a tree using a heuristic search with 1,000 random addition sequence replicates, and TBR branch
swapping. Maximum parsimony analysis was performed
using a heuristic search algorithm with the following settings: 1,000 random addition sequences, TBR branch
swapping, MulTrees, all characters unordered and
unweighted, and branches with a maximum length of
zero collapsed to yield polytomies. Because the ME, ML,
and MP analyses formed congruent trees, only the ML
tree is presented here. The robustness for individual
nodes was determined by bootstrapping (BS) the rbcL
data set, 100 times for ML and 1,000 times for ME and
MP.
Results
Morphology
Polysiphonia morrowii from Chile is composed of a primary upright axis and prostrate filaments with four pericentral cells without cortication (Figure 1). The thallus is
densely tufted, slender and elongate, and tightly entangled, and is attached to the substratum with adventitious
M.-S. Kim et al. Introduction of Polysiphonia morrowii to Chile
391
Figures 1–3 Polysiphonia morrowii: vegetative and reproductive morphology of specimens collected in Chile.
(1) Ramification of branches, showing densely tufted, slender, and irregularly alternate branches with a sharply pointed apex. (2–3)
Branchlets with tetrasporangia (scale bars Figures 1–3s150 mm).
unicellular rhizoids. The rhizoids (20–40 mm in diameter)
arise as outgrowths of pericentral cells without septation.
Ultimate branches arise alternately and have a sharply
pointed apex. The main axis is 50–160 mm thick in lower
branches and 50–100 mm in upper branches. At the sterile stage, trichoblasts rarely occur near the apex of sterile
branches. Tetrasporangia are 50–60 mm in diameter and
are found in short rows on the ultimate branchlets. No
fertile axillary adventitious endogenous branchlets were
observed in the axils of branches (Figures 2–3). Cystocarpic and spermatangial thalli were not found in our
collections.
Interspecific p-distances of rbcL sequence differences
among Polysiphonia species ranged from 3.07%
between P. morrowii and P. pacifica Hollenberg to
14.12% between P. brodiaei (Dillwyn) Sprengel and P.
scopulorum Harvey.
All Polysiphonia morrowii specimens formed a strongly
supported clade (BSs95% for ML, 100% for both ME
and MP) in our rbcL tree (Figure 4). The specimens were
clearly separated from Polysiphonia sp. and P. paniculata
from Chile. The Polysiphonia sp. was related to P. scopulorum with strong support (BSs96% for ML, 99% for
ME, and 97% for MP).
Molecular analyses
We determined a total of 15 rbcL sequences from Polysiphonia morrowii (eight specimens) and putative relatives (seven specimens) in the present study. In all,
1379 bp of the rbcL were aligned; 443 sites (32.1%) were
variable and 341 sites (24.7%) were phylogenetically
informative. The base composition was slightly AT biased
(63.4%). Of the P. morrowii specimens that were compared, four sequences were identical: specimens from
Anin and Mireukdo, Korea and from Fuerte Bulnes, Chile.
Intraspecific rbcL sequence differences within Polysiphonia morrowii were minimal. For example, most specimens of P. morrowii from Chile and Korea differed by
1–3 bp (99.8–99.9% sequence identity). The sample
from Namhaekyo, Korea, had one unique base relative to
others (position 200), which was a synonymous substitution (T instead of C). Specimens from Hansuri, Hoidong, and Dolsando, Korea, shared two bases, T at
position 470 and C at position 851, which separated
them from the other five P. morrowii specimens that had
silent mutations. Three events for rbcL sequences of
eight P. morrowii specimens occurred at the third codon
position.
Discussion
Our collections of Polysiphonia morrowii from Punta Arenas, Chile, corresponded, in their habit, number of pericentral cells, and type of tetrasporangial branchlets, to
the lectotype designated by Masuda et al. (1995) and
detailed descriptions by Kudo and Masuda (1992) and
Kim et al. (1994). Male and female thalli were not collected, and it is not known whether they occur in other
seasons. The Chilean thalli are more delicate, soft, and
abundantly branched than those from Korea and Japan
(Kudo and Masuda 1992, Kim et al. 1994). The height of
P. morrowii was approximately 20 cm in Punta Arenas,
whereas it was 16–25 cm in Korea (Kim et al. 1994),
35 cm in Japan (Kudo and Masuda 1992), and up to
50 cm in the Mediterranean Sea (Curiel et al. 2002). The
paucity or complete lack of endogenous axillary branchlets in the Chilean specimens may have been due to the
early life history stage at which they were collected. Thus,
the Chilean materials have all the vegetative characteristics associated with P. morrowii from the Northwest
Pacific Ocean (Yoon 1986, Kudo and Masuda 1992, Kim
392 M.-S. Kim et al.: Introduction of Polysiphonia morrowii to Chile
Figure 4 Polysiphonia morrowii: maximum likelihood tree including putative relatives estimated from the plastid encoded rbcL
sequences.
Bootstrap values shown above or under the branches are ML (100), ME (1000), and MP (1000).
et al. 1994) and the Mediterranean Sea (Curiel et al. 2001,
Verlaque 2001), except for the endogenous axillary
branches. This is the first reported occurrence of P. morrowii in extreme southern Chile.
All rbcL sequences for samples of Polysiphonia morrowii from Punta Arenas were identical to those from
three locations in Korea. The divergence between
Chilean and Korean (Hansuri) individuals was extremely
low (0.08% on average). All analyses of the rbcL dataset
strongly supported the monophyly of specimens from
Korea and Chile. Analyses of our rbcL data suggest that
P. morrowii from Punta Arenas is not a Gondwanan relic,
but has been introduced recently from the Northwest
Pacific, including Korea and Japan. Two studies have
reported the recent introduction of P. morrowii from the
Northwest Pacific Ocean to the Mediterranean Sea (Verlaque 2001, Curiel et al. 2002). Our study provides the
first molecular evidence for the recent introduction of the
species from native waters to a distant and isolated area.
Polysiphonia morrowii from Punta Arenas is clearly different from other Polysiphonia species from Chile on the
basis of morphology as well as the rbcL phylogeny. For
example, Polysiphonia paniculata from Punta Arenas is
distinguished by its having 10–12 pericentral cells and
rhizoids cut off by cross walls, and is related to P. brodiaei. Since Polysiphonia sp. from Punta Arenas and
Central Chile is related to P. scopulorum from Oregon,
USA in rbcL data and is also similar to P. subtilissima
Montagne in morphology, detailed morphological and
molecular studies are needed for the taxonomic identification of the Polysiphonia sp. from Punta Areans.
Polysiphonia morrowii occurs in the intertidal zones of
Punta Arenas, Korea, Japan, and the Mediterranean Sea
(Kudo and Masuda 1992, Kim et al. 1994, Curiel et al.
2002). It inhabits a variety of substrata, such as rocks,
wooden piles, ropes, mussels, crab, and tunicate shells,
and other large algae, such as Sargassum and Undaria
(Kudo and Masuda 1992, Kim et al. 1994, Curiel et al.
2002). All reproductive stages occur during the same
period, and the time to complete one life cycle is just
four months in the field (Kim et al. 1994). Thus, P. morrowii possesses a number of biological features, such as
the ability to colonize a variety of substrata in intertidal
habitats, rapid growth, and a short life history, which
M.-S. Kim et al. Introduction of Polysiphonia morrowii to Chile
might give it a competitive edge over some native algal
species.
The occurrence of Polysiphonia morrowii in Punta Arenas may result from importation of molluscs and seaweeds, or from ship fouling, ballast water, or rafting.
Based on the fact that P. morrowii was abundant in the
oyster ponds of Yerseke Oesterbank, Maggs and Stegenga (1996) hypothesized the invasion of P. senticulosa
Harvey, which is very similar to P. morrowii (Kim et al.
1994), with Asian oysters or indirectly via other sites of
shellfish aquaculture. Since P. morrowii occurs in the
Mediterranean Sea (Verlaque 2001, Curiel et al. 2002),
there is a possibility that this species, which originated
from the Northwest Pacific Ocean, may have been secondarily transported from Europe to Chile with European
oysters. However, there are no reports of importation of
oysters from Japan, Korea, or Europe to Punta Arenas.
An edible brown alga, Undaria pinnatifida, which is native
to the Northwest Pacific Ocean but now grows at a depth
of approximately 6.5 m in Golfo Nuevo, Argentina (Casas
and Piriz 1996), may also be a vector of P. morrowii.
However, again there are no reports on the occurrence
of U. pinnatifida in the Punta Arenas area.
Shipping constitutes a possible means for the introduction of the species. As a free harbour, Punta Arenas
Harbour is a center for ships visiting from Japan, Korea,
and Europe. However, it is not likely that a delicate coldwater species such as P. morrowii (Kim et al. 1994, Curiel
et al. 2002) could survive the long journey via equatorial
tropical waters across approximately 10,000 km.
Despite increased boat transportation and fishery
imports between Chile and Japan, Polysiphonia morrowii
seems to have a restricted range in Chile, given that the
species was not found in Ancud, approximately 1,300 km
north of Punta Arenas, nor in Central Chile, about
3,500 km north, during the same period. However, since
the species is competitively dominant over native species, there should be concern about the possible detrimental consequences of the introduction of P. morrowii
into Punta Arenas.
Acknowledgements
This work was supported by a Korea Research Foundation Grant
(KRF-2002-075-C00026) to MSK, Mineduc-Chile to AM, and
KRF 2002-070-C00083 to SMB.
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Received 19 March, 2004; accepted 3 September, 2004