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. References Carlton, J.T. and J.B. Geller. 1992. 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