Phylogenetic relationship of Alexandrium monilatum (Dinophyceae) to other Alexandrium species based on 18S ribosomal RNA gene sequences

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1 Harmful Algae 5 (2006) A. monilatum was first identified and described from waters collected near Melbourne on the east coast of Florida (Howell, 1953). Because many of these blooms are associated with fish mortality, a number of research efforts have been initiated to examine the toxicity of A. monilatum to fish and a number of other organisms. A. monilatum cultures were toxic to mullet, Mugil cephalus (Gates and Wilson, 1960) and guppy, Lebistes reticulates (Aldrich et al., 1967); crude cell extracts were toxic to mice (Erker et al., 1982). Clemons et al. (1980) found that crude cell extracts were hemolytic, and Bass and Kuvshinoff (1982) observed a neurotoxic response. The toxic components were later shown by Erker et al. (1985) not to be saxitoxin, the gonyautoxins, and related toxins known to occur in other toxic Alexandrium spp., such as Alexandrium tamarensis, Alexandrium catenella, or Alexandrium minutum. In addition, filterwww.elsevier.com/locate/hal Phylogenetic relationship of Alexandrium monilatum (Dinophyceae) to other Alexandrium species based on 18S ribosomal RNA gene sequences John E. Rogers a, *, Jeffrey D. Leblond b, Cynthia A. Moncreiff c a United States Environmental Protection Agency, Gulf Ecology Division, National Health Effects and Environmental Research Laboratory, 1 Sabine Island Dr., Gulf Breeze, FL 32561, USA b Department of Biology, P.O. Box 60, Middle Tennessee State University, Murfreesboro, TN 37132, USA c Gulf Coast Research Laboratory, Department of Coastal Sciences, The University of Southern Mississippi, Ocean Springs, MS 39566, USA Received 18 February 2005; received in revised form 16 August 2005; accepted 18 August 2005 Abstract The phylogenetic relationship of Alexandrium monilatum to other Alexandrium spp. was explored using 18S rdna sequences. Maximum likelihood phylogenetic analysis of the combined rdna sequences established that A. monilatum paired with Alexandrium taylori and that the pair was the first of the Alexandrium taxa to diverge, followed by Alexandrium margalefii. All three are members of the Alexandrium subgenus Gessnerium Halim nov. comb. # 2005 Elsevier B.V. All rights reserved. Keywords: Alexandrium monilatum; Phylogeny; 18S rdna 1. Introduction Alexandrium monilatum (Howell) (Balech, 1995) is a chain forming dinoflagellate that periodically blooms in coastal and estuarine waters of the Gulf of Mexico. Blooms have been reported for areas near Galveston Bay, Texas (Connell and Cross, 1950; Gates and Wilson, 1960), in the coastal waters of Mississippi, and as Far East as Mobile Bay, Alabama and Pensacola Bay, Florida (Perry et al., 1979). Not limited to the Gulf of Mexico, A. monilatum has also been documented in the Western Atlantic (Williams and Ingle, 1972; Owen and Norris, 1982) and Caribbean (Halim, 1967). * Corresponding author. Tel.: ; fax: address: rogers.johne@epa.gov (J.E. Rogers) /$ see front matter # 2005 Elsevier B.V. All rights reserved. doi: /j.hal

2 276 J.E. Rogers et al. / Harmful Algae 5 (2006) feeding shellfish did not accumulate the toxins of A. monilatum. A. monilatum is a chain former similar to A. catenella. However, A. monilatum is generally larger (Balech, 1995) with a protoplasmic continuum between cells in chains (Walker and Steidinger, 1979). Balech (1995) placed A. monilatum in the subgenus Gessnerium Halim nov. comb. of the genus Alexandrium; A. catenella was placed in the subgenus Alexandrium. A more recent morphotype grouping scheme (Dr. Makoto Yoshida, personal communication in Usup et al., 2002) identifies A. catenella as a Type C along with other Alexandrium spp. producing toxins that cause paralytic shellfish poisoning. A. monilatum was not included in this scheme nor has it been included in the many phylogenetic analyses of the Alexandrium genus (Destombe et al., 1992; Adachi et al., 1994, 1996; Scholin et al., 1994; Scholin and Anderson, 1994; Hirashita et al., 2000; Sako, 2000; Usup et al., 2002; Hansen et al., 2003; John et al., 2003; Montresor et al., 2004). Exclusion in the latter analyses occurred because sequence data was not available. Here we report the phylogenetic relationship of A. monilatum to other Alexandrium spp. based on 18S rdna analysis. 2. Materials and methods 2.1. Dinoflagellate cultures and growth conditions Nuclear 18S rdna sequences of 12 taxa were included in the phylogenetic analysis. The sequences for A. monilatum JR07 (AY883005), Alexandrium minutum CCMP 113 (AY883006), and Alexandrium tamarense UTEX 2521 (AY883004) were determined from unialgal cultures maintained at the Gulf Ecology Division. The sequences were deposited in GenBank. A. monilatum JR07 was isolated from a bloom sample collected on 12 September 2000, just south of the Mississippi barrier islands ( N, W). At the time of collection the surface was visibly discolored, the A. monilatum cell count was 550 ml 1, salinity was 35, and the temperature was approximately 28 8C. A clonal culture was developed from a single cell isolated from a mixed culture maintained on L1 medium. The clonal culture was unialgal but not axenic. A. minutum CCMP 113 (AY883006) was obtained from the Provasoli Guillard Center for the Culture of Marine Phytoplankton (CCMP), and A. tamarense (AY883004), listed as Gonyaulax tamarensis UTEX 2521, was obtained from The Culture Collection of Algae at the University of Texas at Austin (UTEX). Both organisms were cultured in f/2 medium at 21 8C and a 14-h light:10-h dark cycle, with a light intensity of 45 me m 2 s 1. In addition, the following 18S rdna sequences (with GenBank accession numbers) were used: Alexandrium affine (AJ535375), A. catenella (AJ535392), Alexandrium fundyense (U09048), Alexandrium margalefii (U27498), A. minutum (U27499), Alexandrium ostenfeldii (AJ and U27500), A. tamarense (AF and X54946), Alexandrium tamutum (AJ535376), Alexandrium tamiyavanichi (AF113935), Crypthecodinium cohnii (M64245), Gonyaulax spinifera (AF022155), Pyrocystis noctiluca (AF022156), and Sarcocystis muris (M64244) DNA extraction and PCR conditions DNA was extracted from cell pellets derived from 15 ml of culture according to the method outlined by Amann et al. (1992). Polymerase chain reaction (PCR) amplification of the gene for 18S rrna was amplified using a minor variation of the primers specified by Medlin et al. (1998) and Ready-To-Go PCR Beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The forward (1F) and reverse (1800R) primers were AACCTGGTTGATCCTGCCAGT-3 0 and 5 0 -TCCTT CTGCAGGTTCACCTAC-3 0, respectively. PCR was initiated by holding at 94 8C for 4 min. The initial hold was followed by 36 cycles that included for each cycle: denaturation at 94 8C (1 min), annealing at 60 8C (1 min), and elongation at 72 8C (1 min). Each dinoflagellate DNA sample yielded one PCR product (approximately 1800 bp), that was purified using Ultrafree-MC 1 centrifugal filter units (Millipore, Bedford, MA) Sequencing and alignment Sequencing of purified PCR products was performed at the University of Florida ICBR Core Lab using the fluorescent dideoxy terminator method of McCombie et al. (1992). In addition to the external primers mentioned above, six internal primers were used: 5 0 -CGGTAATTCCAGCTCC-3 0 (560F), GGAGCTGGAATTACCG-3 0 (560R), 5 0 -TTTGACT- CAACACGGG-3 0 (1170F), 5 0 -CCCGTGTTGAGT- CAAA-3 0 (1170R), 5 0 -CAGGTCTGTGATGCCC-3 0 (1420F), and 5 0 -TCCTTCTGCAGGTTCACCTAC-3 0 (1420R). The resulting partial sequences were assembled into contiguous sequences, edited and aligned using the Lasergene99 software package (DNASTAR Inc., Madison, WI). External primer sequences were excluded from the analysis.

3 J.E. Rogers et al. / Harmful Algae 5 (2006) Phylogenetic analysis The PAUP (version 4.0b8, Swofford, 2000) program was used with default parameters in maximum likelihood phylogenetic analysis of the combined rdna sequences. Robustness of the resulting trees was estimated by bootstrap analysis with 100 replicates. To improve the speed of the analyses, the data for taxa with identical sequences were removed before analysis. Phylogenetic trees were visualized using TREEVIEW by Page (1996). 3. Results and discussion A. monilatum JR07 was isolated from an A. monilatum bloom containing large numbers of chains (Fig. 1A). In culture, A. monilatum JR07 was commonly observed as chains of 4 and 8 cells (Fig. 1B), and chains of 64 cells were observed in some cultures. Late-log cultures produced smooth-walled hypnozygotes containing one or two golden-yellow globules (Fig. 1C). Hypnozygote production followed gamete fusion. Walker and Steidinger (1979) described similar hypnozygotes for an A. monilatum culture isolated from Tampa Bay, Florida in Maximum likelihood phylogenetic analysis of the partial SSU rdna sequences, which included A. monilatum and other representative species available for the genus Alexandrium, produced a tree (Fig. 2). Estimated likelihood parameters were LN likelihood ( ), transition/transversion ratio (2.58), base frequencies (A = 0.271, C = 0.181, G = 0.254, T = 0.293), and shape parameter (a = 0.266). With the exception of new SSU rdna sequences included in this work, the tree-topology was basically the same as the tree recently reported by Montresor et al. (2004). The Alexandrium subgenera Gessnerium taxa were the first to diverge followed by the Alexandrium subgenera Alexandrium taxa. Of the subgenera Gessnerium taxa in this study, A. monilatum JR07 and A. taylori formed a pair with strong bootstrap support and were the first to diverge, followed by A. margalefii. The remaining subgenus Alexandrium taxa divided between two clusters: one containing A. minutum, A. tamutum, and A. ostenfeldii, and second containing A. affine, A. tamiyavanichii, A. fundyense, and A. tamarense species complex. A. affine was the first to diverge in this latter cluster. The previous placement of the two chain formers, A. monilatum and A. catenella, in separate subgenera was confirmed. A. monilatum had been placed in the Alexandrium subgenus Gessnerium and A. catenella in the subgenus Alexandrium as a result of previous morphological analyses (Walker and Steidinger, 1979; Balech, 1995). A. monilatum was clearly one of the first of the Alexandrium to diverge whereas A. catenella embedded within the A. tamarense species complex. In recent years, the phylogenetic relationships of the Alexandrium subgenus Gessnerium taxa to the subgenus Alexandrium have changed as new SSU rdna sequences have become available. Previous phylogenetic analyses using fewer representative Alexandrium species placed A. margalefii either as a sister group to a clade containing A. tamarense, A. fundyense and A. tamiyavanichii (Usup et al., 2002), or in a cluster with A. ostenfeldii and an A. minutum/lusitanicum species complex (John et al., 2003). With the addition of several new Alexandrium species including A. taylori, Montresor et al. (2004) found that A. margalefii diverged after A. taylori, but before the remaining Alexandrium species. The inclusion of A. monilatum in our analyses also supports the early divergence of A. taylori and A. margalefii. In addition to A. taylori and A. margalefii, A. monilatum represents the third member Fig. 1. Photomicrographs of Alexandrium monilatum cells. (A) A field sample with chains of various lengths and single cell; (B) a single chain of 12 cells in a laboratory culture; (C) a hypnozygote produced in a late-log culture.

4 278 J.E. Rogers et al. / Harmful Algae 5 (2006) Fig. 2. Maximum likelihoodtreeobtainedfrompartial18srdnasequences.thepercentageoftimesthatabranchoccurredinthe100bootstrap replications is shown in parentheses at branch intersects. Only bootstrap values >50% are shown. The scale bar indicates the number of substitutions per site. of the subgenus Gessnerium to diverge early within the genus Alexandrium. Phylogenetic analyses using the D1/D2 region of LSU rdna have produced similar results. Usup et al. (2002) found that Alexandrium split into two major groups. A. margalefii fell within the group containing A. ostenfeldii, A. andersoni, A. lusitanicum and A. minutum, forming a sister group to an A. minutum/lusitanicum species complex. Hansen et al. (2003) divided the Alexandrium taxa into two clades. Clade 1 contained A. concavum, A. affine, A. excavatum and A. tamarense. Clade 2 contained A. andersoni, A. ostenfeldii, A. insuetum, A. minutum and subgenus Gessnerium taxa, A. margalefii and Alexandrium pseudogonyaulax. A. pseudogonyaulax was the first of the Clade 2 taxa to diverge, followed by A. margalefii. That same year, John et al. (2003) established a phylogenetic profile that included a third subgenus Gessnerium taxon, A. taylori. In this case, A. taylori was the first of the Alexandrium spp. to diverge, followed by A. margalefii. A. pseudogonyaulax, however, grouped with an A. minutum/lusitanicum species complex. More recent phylogenetic analyses (Montresor et al., 2004) show A. taylori and A. margalefii diverging as a pair before the remaining Alexandrium taxa.

5 J.E. Rogers et al. / Harmful Algae 5 (2006) Although the number of Alexandrium subgenus Gessnerium SSU and LSU sequences is limited, the majority of these taxa generally diverge early, if not first in the phylogenetic analyses of the Alexandrium. John et al. (2003) used fossil records to develop a SSU rdna molecularclockthatplacestheappearanceofa.monilatum and A. taylori within the late to early Cretaceous ( MYA). As more sequences of subgenus Gessnerium taxa becomeavailable,thephylogeneticrelationshipsofthetwo subgenera will become clearer. The results of this study clearly indicate that A. monilatum is not a member of the PSP-toxin producing species of the genus Alexandrium. 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