Phylogeography of the Freshwater Raphidophyte Gonyostomum Semen Confirms a Recent Expansion in Northern Europe by a Single Haplotype1
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J. Phycol. 51, 768–781 (2015) © 2015 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and dis- tribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. DOI: 10.1111/jpy.12317 PHYLOGEOGRAPHY OF THE FRESHWATER RAPHIDOPHYTE GONYOSTOMUM SEMEN CONFIRMS A RECENT EXPANSION IN NORTHERN EUROPE BY A SINGLE HAPLOTYPE1 Karen Lebret,2 Sylvie V. M. Tesson, Emma S. Kritzberg Department of Biology, Lund University, Ecology Building, Lund SE-22362, Sweden Carmelo Tomas University of North Carolina at Wilmington, Center for Marine Science, Myrtle Grove 2336, Wilmington, North Carolina, USA and Karin Rengefors Department of Biology, Lund University, Ecology Building, Lund SE-22362, Sweden Gonyostmum semen is a freshwater raphidophyte DNA region; SSU, small subunit of the ribosomal that has increased in occurrence and abundance in DNA region several countries in northern Europe since the 1980s. More recently, the species has expanded rapidly also in north-eastern Europe, and it is frequently referred to as invasive. To better Raphidophytes are planktonic autotrophic protists, understand the species history, we have explored which form blooms in both marine and limnic envi- the phylogeography of G. semen using strains from ronments. Many raphidophyte species are consid- northern Europe, United States, and Japan. Three ered harmful, causing for instance mass mortality in regions of the ribosomal RNA gene (small subunit fish due to toxin production (Edvardsen and Imai [SSU], internal transcribed spacer [ITS] and large 2006). Gonyostomum semen (Ehrenberg) Diesing is the subunit [LSU]) and one mitochondrial DNA marker most common raphidophyte species in freshwater (cox1) were analyzed. The SSU and partial LSU environments, and is considered to have a cosmopol- sequences were identical in all strains, confirming itan distribution (Bourrelly 1985) based on micro- that they belong to the same species. The ITS scopic identification. Nevertheless, this species is region differentiated the American from the other usually rare in phytoplankton communities world- strains, but showed high intra-strain variability. In wide. Interest in G. semen has risen in the scientific contrast, the mitochondrial marker cox1 showed community recently, mainly because this species has distinct differences between the European, increased in abundance and occurrence in northern Europe since the 1980s and more recently in Central American, and Japanese strains. Interestingly, only € one cox1 haplotype was detected in European Europe (Cronberg et al. 1988, Lepisto et al. 1994, Poniewozik et al. 2011, Rengefors et al. 2012, Lebret strains. The overall low diversity and weak _ geographic structure within northern European et al. 2013, Karosiene et al. 2014, Hagman et al. strains supported the hypothesis of a recent 2015). In 2002, G. semen was observed in as much as invasion of new lakes by G. semen. Our data also 27% of all monitored Swedish lakes (Rengefors et al. show that the invasive northern European lineage is 2012). In addition, this raphidophyte is a nuisance genetically distinct from the lineages from the other microalga, and forms dense blooms during extended continents. Finally, we concluded that the periods of time (Lebret et al. 2012b). Although mitochondrial cox1 was the most useful marker in G. semen is not known to be toxic, it expels slimy determining large-scale biogeographic patterns in threads from trichocysts, inducing skin irritation in this species. bathers and thereby affecting recreational activities (Sorensen€ 1954, Cronberg et al. 1988). Key index words: cox1; Gonyostomum semen; ITS; LSU; Although Cronberg et al. (1988) first suggested phylogeography; raphidophyte SSU that G. semen may have increased since the late Abbreviations: cox1, mitochondrial gene of the cyto- 1950s, the expansion of G. semen was first described – chrome c oxidase subunit 1; ITS, internal tran- in a Finnish study during a period of 10 years (1978 € € scribed spacer; LSU, large subunit of the ribosomal 1989; Lepisto et al. 1994). Lepisto et al. (1994) found that the number of studied lakes with G. semen 1Received 8 January 2015. Accepted 30 April 2015. increased from 11 to 42 of 110 sampled, and that it 2Author for correspondence: e-mail [email protected]. was found in a wider range of lake types than Editorial Responsibility: L. Graham (Associate Editor) 768 PHYLOGEOGRAPHY OF GONYOSTOMUM SEMEN 769 observed in the past. Previously, reports of G. semen Despite the increasing number of studies concern- were restricted to humic lakes and ponds (reviewed ing G. semen, almost no genetic information is cur- in Cronberg et al. 1988 and Willen 2003). However, rently available. Only two sequences of the small dense populations have more recently been observed subunit (SSU) of the ribosomal DNA (rDNA) were in nonhumic oligotrophic lakes (Laugaste 1992), available on GenBank at the start of this study (acces- eutrophic ponds (Pithart et al. 1997) and large deep sion numbers: DQ408616 and AB512123). The aim of reservoirs (Lecohu et al. 1989, Negro et al. 2000). this study was thus to investigate the phylogenetic rela- Several factors have been suggested to favor the tionships among G. semen strains. We analyzed strains development of G. semen in northern European from lakes in northern Europe, Japan, and the United lakes. Cronberg et al. (1988) suggested that mass States. Four DNA markers; the mitochondrial gene blooms were connected to low pH. However, recent cytochrome c oxidase subunit 1 (cox1), the entire studies showed that the species is also able to form small subunit rDNA (SSU), the internal transcribed blooms in more alkaline lakes (Rengefors et al. spacer (ITS) including ITS1, 5.8S and the ITS2, and 2012), and that G. semen strains can grow in a wide the partial large subunit of the rDNA (LSU) were range of pH (Sassenhagen et al. 2015). By analyzing sequenced. We aimed to compare the variability in data from the Swedish national lake inventory pro- the four markers and their potential to identify phylo- gram (Rengefors et al. 2012, Trigal et al. 2013) it geographical patterns in G. semen. In accordance with was found that high water color, high dissolved the hypothesis that G. semen has expanded in the last organic carbon concentration, and low pH were four to five decades in Norway, Sweden, and Finland, strong predictors for G. semen occurrence. Moreover, we expected little difference among sequences of when performing a trend analysis, Rengefors et al. those strains, and larger difference between the (2012) found that temperature was the most impor- northern European (Norway, Sweden, and Finland) tant variable driving biomass temporal variations, and the American and Japanese strains. and suggested this as a driver of G. semen expansion. Thus, the increase in occurrence and abundance METHODS of G. semen is likely due to a changing environment and to more favorable conditions with an interac- Strains and cultivation of G. semen. In total, 62 strains were tion of all these factors. Due to its increase in occur- sequenced, including 60 strains of G. semen (30 from Sweden, rence and expansion of its habitat range, G. semen is 10 from Norway, eight from Finland, 10 from United States and two from Japan), one of Gonyostomum latum (Iwanoff) (Japan) now viewed to be invasive in Norway, Sweden, and and one unknown raphidophyte strain from the United States, Finland (Lebret et al. 2012a, 2013, Rengefors et al. which was a priori identified as G. semen (Table 1). The G. semen 2012, Hagman et al. 2015). According to Valery strains from Finland, Norway, and Sweden (n = 48), were iso- et al. (2008), an invasive species is a species that has lated from 15 locations (14 lakes including lake Helgasjon€ sam- gained competitive advantage after the disappear- pled at two distinct stations; Fig. 1), with one to four strains per ance of a natural obstacle, which can either result location (Table 1). The isolations and cultivation of the strains were performed according to Lebret et al. (2013). The Norwe- in dispersal to a new environment or that the spe- gian strain NIVA-7/05 isolated from Vansjø was obtained from cies has increased its distribution range by adapting the Norwegian Institute for Water Research (NIVA) culture col- to new conditions, allowing it to spread and form lection. The Japanese strains (G. semen and G. latum)were dominant populations. obtained as extracted DNA from the National Institute for Envi- To date, little is known concerning the evolution- ronmental Studies (NIES) culture collection in Japan. The ary and phylogeographic history of the species. American strains were obtained from the Center of Marine Sci- ence in Wilmington, North Carolina (United States). All strains Lebret et al. (2013) showed that the Fenno-Scandina- were grown in modified Wright’s cryptophyte medium (Guillard vian populations belong to a single metapopulation, and Lorenzen 1972) with an addition of selenium to a final con- À suggesting that it has likely expanded recently. How- centration of 1.2 lg Á L 1. The cultures were harvested by cen- À ever, it is unknown how this population is related to trifugation at concentrations of ~2,000 cells Á mL 1 according other lineages of G. semen and when the expansion to Lebret et al. (2012b) and the pellets were stored at À80°C occurred. Phylogenetic analyses using molecular until DNA extraction. tools could provide new insights into the species’ his- DNA extraction. DNA was extracted using a CTAB-based protocol as described by Lebret et al. (2012b). The DNA con- tory and expansion. In macroorganisms, phylogeo- centration of the samples was estimated by measuring the graphic studies have enhanced the understanding of absorbance of a subsample diluted ten times at 260 nm using the evolutionary history, expansion, or invasion pat- a spectrophotometer (Ultraspec 3000; Pharmacia Biotech, terns of species, as well as gene flow between regions Cambridge, England).