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FRONTIER RESEARCH ON EARTH EVOLUTION, VOL. 1 Phylogenetic relationships among the species of genus Bulimina (Foraminifera) inferred from ribosomal DNA sequences Hiroshi Kitazato1, Masatoshi Tazume2 and Masashi Tsuchiya3 Research Program for Paleoenvironment, Institute for Frontier Research on Earth Evolution (IFREE) Department of Life and Earth Sciences, Shizuoka University 3 Marine Biosystems Research Center, Chiba University 1 2 Introduction Materials and methods Because their distribution patterns are related to environmental parameters such as temperature and salinity, benthic foraminifera have long been used as excellent proxies for reconstructing paleoceanographic parameters. Among foraminiferal genera, species of the genus Bulimina distribute in a wide range of oceanic environments from sublittoral to bathyal depths and have clear habitat preferences. Thus, Bulimina species are thought to be useful foraminiferal taxa for paleoceanographic application. However, there has been considerable discussion concerning the definition of species that are mainly defined only on the basis of morphological test characteristics. One major discussion has focused on whether or not the morphology-based systematics of the genus Bulimina is reliable. Within this genus, two groups have been recognised, one with costate and the other with non-costate test surface ornamentation. Verhallen (1986) considered that costate surface ornamentation, together with the presence of an internal tooth plate, is a diagnostic character for discriminating one group from the other. He suggested that these morphological characteristics are very important taxonomically, and might even represent family-level differences. On the other hand, gradual morphological changes also exist among several species of Bulimina. Both Bulimina marginata and Bulimina aculeata were described on the basis of modern specimens collected in the Adriatic Sea by d’Orbigny (1826). He described both forms as distinct species on the basis of chamber angularity, the arrangement and length of the spines, and test dimensions. Hoeglund (1947) made precise morphological analyses of both species and found that morphological characterics changed gradually from one species to the other. He concluded that they were not separate species but morphological variants of the same species. However, both Collins (1989) and Burgess and Schnitker (1990) concluded that B. marginata could be distinguished from B. aculeata based on morphological characteristics. These conflicting views may arise due to a lack of knowledge on the biological significance of the morphologies. Biological approaches, such as ecological analysis and molecular analysis, may help to solve the problem. In particular, molecular phylogenetic analysis of Bulimina species provide an adequate approach to evaluate the significance of morphological characters for distinguishing taxa. The aim of this study is to analyze molecular phylogenetic relationships among the species of genus Bulimina using nucleotide sequences, and to discuss the relations between molecular- and morphologybased classifications. Samples Eight morphospecies were used for DNA study. Living individuals for study were collected at different localities from sublittoral to bathyal depths around the Japanese Islands (Fig. 1). Sublittoral sediments were collected from Kominato Fishing Port at Uchiura Cove of Chiba Prefecture, Shiogama Fisherman’s Warf at Matsushima Bay of Miyagi Prefecture, and Shimoda Bay of Shizuoka Prefecture. Bathyal sediments were collected from Stns. SB, G and EO in Sagami Bay. The samples with living Bulimina species were maintained in aerated buckets in the laboratory at about 20°C for shallow water species, and 2.5°C for deep-sea species, until DNA analyses were conducted. Molecular phylogenetic analysis The specimens for DNA analysis were picked out from the cultured sediments. Individuals with cytoplasm inside the test, and/or extruded pseudopodia from the aperture, were judged as alive. To remove any associated microorganisms, individual foraminifera were cultured without food at 18°C in a petri dish filled with 0.2µm filtered and sterilized seawater (35‰). After 2 or 3 days, a period by which most ingested food material should have been digested, individuals were dried at 30°C for 30min. (Holzmann and Pawlowski, 1996). Prior to extracting DNA, dried specimens were photographed using a low vacuum SEM (JEOL) in order to compare DNA sequence data with morphological information. DNA extraction Total DNA was extracted from a single individual. A single dried specimen was transferred into a 1.5ml microfuge tube with extraction buffer containing 50µl of 1x DOC (Pawlowski et al., 1994). The specimen was crushed with a siliconized and tip-closed Pasteur pipette, and then incubated at 60°C for 1 hour. Insoluble materials were separated by centrifugation. Supernatant was collected and used for PCR amplifications. Amplification and purification A DNA fragment of about 1,000 base pairs (bp), situated at the 3’ terminal region of the SSU rDNA, was amplified by polymerase chain reaction (PCR) using synthesized primer pairs. Primer pairs of s14f1 (5’-AAGGGCACCACAAGAACGC) and sB (5’-TGATCCTTCTGCAGGTTCACC233 FRONTIER RESEARCH ON EARTH EVOLUTION, VOL. 1 morphospecies ranged from 0.0 to 5.2% (Table 1). As in the case of sequence divergence within an individual, most species show a divergence of less than 1.0%. An exception is Bulimina kochiensis in which sequence divergence within a population ranged from 1.1% to 4.8%. There is also a 3.0% sequence divergence within B. marginata forma denudata. Among species of the genus Bulimina, sequence divergence reached a maximum of 35.0%. The divergence between B. aculeata and B. pagoda was smaller (0.1-0.6%) than between other species. A low degree of divergence was also observed within the group B. aculeata, B. marginata, B. elongata and B. pagoda. However, the divergence between B. kochiensis and B. striata was significant, with a value of 34.5-35.0%. Apart from these extreme values, divergence clustered around 15%. Sequence divergence between B. kochiensis and other morphospecies was around this value; for instance, between B. kochiensis and B. aculeata it ranged from 15.5% to 16.3%. Phylogenetic relationships among species of Bulimina indicate that the genus consists of four different genetic clusters (Fig. 3). The sequence divergences between these clusters are large. Phylogenetic relationships of the four Bulimina clusters with other genera of foraminifera show that three of the four groups are scattered within the phylogenetic tree (Tazume et al., in prep.). Each cluster exhibits characteristic morphological features, such as pore size, pore distribution, density distribution of pores, aperture shapes and test ornamentation. Other characters, such as test chemical composition, overall outline of tests, and the shape of the loop-like aperture, are common to all species of Bulimina. This means that morphological convergence has occurred in the genus Bulimina. The main reason for the large genetic difference between the species clusters was the existence of either insertions or deletions among genetic fragments at the SSU section. This phenomenon, insertion or deletion, is very common in prokaryotic genes but rarely found in eukaryotic metazoans. Further investigations are needed to determine why genetic insertion or deletion often takes place in certain foraminiferal genera. Five morphospecies, Bulimina aculeata, B. pagoda, B. marginata forma marginata, B. marginata forma denudata, and B. elongata, are genetically very close within the Bulimina group. They all belong to a single cluster in a phylogenetic tree (Fig. 3). Four subcluster units could be recognized within the main cluster (Fig. 4). Some morphotypes, such as B. elongata, B. marginata forma denudata and B. marginata forma marginata, corresponded to a single subcluster. Both Bulimina aculeata and B. pagoda, however, belong to different subclusters with B. elongata and B. marginata. Thus, morphological divergence may occur among closely related species of genus Bulimina. TAC) were used for SSU amplification. These primers were designed by Pawlowski et al. (1994; 1996). PCR amplifications were performed in initial denaturation for 2min. at 94°C: 1 cycle, denaturation for 30sec. at 94°C, annealing for 30sec. at 55°C, and extension for 90sec. at 70°C: 40 cycles, annealing for 2min. at 55°C, final extension for 7min. at 72°C: 1 cycle. The reactions were performed with 5.0µl of 10x reaction buffer, 5.0µl of dNTP mixture (contain 2.5mM each of dATP, dCTP, dGTP, and dTTP), 2.5µl each of forward and reverse primers (10pM), and 0.5µl of Taq polymerase (2.5U), for 50µl reactions. The length of the fragment was confirmed by agarose gel electrophoresis. PCR products were purified with High Pure PCR Product Purification Kit (Roche). Purified products were stored at –20°C. Cloning and sequencing The purified products were ligated in the pGEM-T vector System (Promega) and cloned into XL-2 blue ultracompetent cells (Stratagene). Plasmids were isolated from cell cultures by the mini-preparation methods (Sambrook et al., 1989). The sequencing reaction on the DNA fragments was carried out the using Thermo Sequenase pre-mix cycle sequencing kit (Amarsham Pharmacia) with Texas Red labeled primers. The samples were sequenced using an Hitachi SQ-5500 DNA sequencer. Phylogenetic analysis The DNA sequences were aligned using Clustal W (Thompson et al., 1994) and corrected manually. Estimation of sequence divergence was calculated by the two-parameter method of correction of multiple substitutions at a site (Kimura, 1980) (transition/transversion ratio=2.0). The unrooted trees were constructed by neighbor joining (NJ) (Saitou and Nei, 1987) and were performed using Clustal W. Bootstrap was with 1,000 replicates for NJ. Results and discussions The amplified SSU rDNA fragment of species of the genus Bulimina corresponds to the 3’ terminal region of the SSU rDNA of Mus musculus (X00686), starting at position 1,208 and ending at position 1,866. The length of the SSU rDNA fragment varies from 981 to 1,232bp between different morphospecies of Bulimina. The small subunit rDNA fragment length of most morphospecies is 1,000 bp on average, although Bulimina striata has longer sequences (1,232bp). In contrast, fragment length (981bp) is rather short in Bulimina subornata. The diagram is illustrating alignment of two distinct species, B. striata and B. subornata (Fig. 2) indicateds that either insertion in B. striata or deletion in B. subornata may occur in any part of the DNA sequences. Results from alignment of all species indicate that the sequenced region consisted of both conserved and variable regions (Fig. 2). Variable regions occupied about 650bp of the total 1,338 aligned sequences. The differences in the length of DNA fragments for each species originated in the variable region. G+C contents are stable within the genus Bulimina, ranging from 42.8% to 46.9%. The sequence divergence within a population of a single Summary DNA analyses were carried out on SSU rDNA for eight morphospecies of the genus Bulimina. The genus consists of four genetic clusters. Large sequence divergences were detected among Bulimina species. The differences may exceed those at the family level, despite the fact that these morphospecies belong to one genus. Morphological convergence may also have taken place within the genus Bulimina. Large sequence 234 FRONTIER RESEARCH ON EARTH EVOLUTION, VOL. 1 differences are mainly due to either the insertion or deletion of gene fragments within the variable region. Five out of the eight morphospecies belong to one genetic cluster. However, each morphospecies constitutes different subcluster within the main cluster. Morphological divergence has taken place in these morphospecies. Thus, both morphological convergence and divergence may have occurred within one genus of foraminifera. Further research is needed to clarify the genetic features of foraminifera using molecular, morphological and ecological information. This kind of approach may reveal why benthic foraminifera are finely attuned to specific environmental factors. Acknowledgments. The authors heartily thank the staff of Misaki Marine Biological Station and R/V Tanseimaru of the Ocean Research Institute, University of Tokyo, Shimoda Marine Research Center of Tsukuba University, and Marine Biosystems Research Center, Chiba University for collecting living Bulimina individuals. Dr. Andrew J. Gooday kindly read the earlier version of the manuscript and gave helpful comments. This research was partly supported by JSPS Research Fellowships (no. 8489) to M.T., and Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (nos. 0645002 and 09554025) to H.K. References Burgess, V. M., and D. Schnitker, Morphometry of Bulimina aculeata d’Orbigny and Bulimina marginata d’Orbigny, J. Foram. Res., 20, 37-49, 1990. Collins, L. S., Regional versus physiographic effects on morphologic variability within Bulimina aculeata and Bulimina marginata, Gulf of Maine Area, J. Foram. Res., 19, 222-234, 1989. Hoeglund, H., Foraminifera in the Gullmar Fjord and the Skagerak, Zoologiska Bidrag Fran Uppsala, 26, 1-328, 1947. Holzmann, M., and J. Pawlowski, Preservation of foraminifera for DNA extraction and PCR amplification, J. Foram. Res., 26, 264267, 1996. Kimura, M., A simple method for estimating evolutionary rates of base substitutions through comparative of nucleotide sequences, J. Molecular Evolution, 16, 111-120, 1980. Orbigny, A. D. d’, Tableau méthodique de la classe des Céphalopodes, Annales des Sciences Naturelle, 7, 245-314, 1826. Pawlowski, J., I. Boliver, J. Guiard-Maffia, and M. Guoy, Phylogenetic position of foraminifera inferred from LSU rRNA gene sequences, Mol. Biol. Evol., 11, 929-938, 1994. Pawlowski, J., I. Boliver, J. F. Fahrni, T. Cavalier-Smith, and M. Guoy, Early origin of foraminifera suggested by SSU rRNA gene sequences, Mol. Biol. Evol., 13, 445-450, 1996. Saitou, N., and M. Nei, The neighbor-joining method: a new method for reconstructing phylogenetic trees, Mol. Biol. Evol., 4, 406425, 1987. Sambrook, J., E. F. Fritsch, and T. Maniatis, Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor laboratory Press, New York, 1-3, 1989. Thompson, J. D., D. G. Higgins, and T. J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice, Nucleic Acids Research, 22, 46734680, 1994. Verhallen, P. J., J. M., Morphology and function of the internal structures of non-costate, Bulimina, Palaeontology, Proceedings, B, 89, 367-385, 1986. 235 FRONTIER RESEARCH ON EARTH EVOLUTION, VOL. 1 Figure 1. Map showing sampling localities. Eight morphospecies were used for DNA analysis. Figure 2. Diagram of aligned sequences of buliminid species within the SSU region. SSU rDNA region consisted of both conservative and variable parts. Shaded area shows conservative part of SSU region. Either insertion or deletion is shown in the variable section. 236 FRONTIER RESEARCH ON EARTH EVOLUTION, VOL. 1 Figure 3. Unrooted phylogenetic tree indicates four major clusters recognized among eight morphospecies of genus Bulimina. SEM photographs are attached in each morphospecies. Scale bar=100µm. Figure 4. Phylogenetic relationships among five morphospecies of genus Bulimina based on SSU rDNA sequences. Each morphospecies roughly corresponds with one subcluster. Both B. aculeata and B. pagoda constitute one cluster. 237 FRONTIER RESEARCH ON EARTH EVOLUTION, VOL. 1 Table 1. Sequence differences among eight morphospecies of genus Bulimina for SSU rDNA. This table also indicates sequence divergences among individuals in one population. 238