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Taxonomic Identification of Foraminifera Using Ribosomal DNA Sequences

1994; Micropaleontology Press; Volume: 40; Issue: 4 Linguagem: Inglês

10.2307/1485942

1937-2795

Jan Pawłowski, Ignacio Bolívar, José F. Fahrni, Louisette Zaninetti,

Geology and Paleoclimatology Research

DNA was extracted from seven different morphotypes of Glabratella spp. collected at the French littoral zone of the Mediterranean Sea. A fragment of 400 bp situated at the 5' terminal region of the large subunit ribosomal RNA gene was amplified and sequenced. Two different sequences were obtained grouping respectively three and four of the morphotypes, thus allowing distinction of two species. Each of the species included morphologically different agamont and gamont life cycle generations. The species were identified as Glabratella erecta (Sidebottom) and Glabratella elegantissima (Sidebottom) based on differences in size and form of the agamont test. This is a pilot study of the use of molecular taxonomy in foraminifera. This paper is dedicated to the late Professor Paul Bronnimann (1913-1993), for the enthusiasm and encouragement he always expressed towards our research on molecular evolution and taxonomy of foraminifera. INTRODUCTION Systematics of foraminifera is based exclusively on the morphology of their tests (Loeblich and Tappan 1984, 1988). Different morphological traits are used to define various taxonomic categories. Foraminiferal species are distinguished largely on the basis of the number and form of the chambers, form of the test periphery and type of ornament (Haynes 1981). However, the large morphological variability of the foraminiferal test make the species identification particularly difficult. This variability may result from alternation of generation (Lee et al. 1991), from ontogenetic development (Brummer et al. 1986) or from variations in environmental conditions (Murray 1991), including seasonal morphological variations as suggested in this paper. Systematics of foraminifera is a compromise of two tendencies: to allow the presence of large morphological variation within a given species (lumping) or to accept a slight change in morphology as a criterion for the erection of a new taxon (splitting) (Haynes 1992). Hence, the identification of many living and fossil foraminiferal species is arbitrary. One of the possibilities for determining the intraspecific variation in foraminifera is to study living organisms in laboratory cultures. Observations on reproduction and the life cycle of cultured Heterostegina depressa (Rottger et al. 1986, 1990) and Rotaliella elatiana (Pawlowski and Lee 1992) allowed the identification of morphologically different generations in these species. Study of the growth and development of cultured Cibicides lobatulus led to the identification of taxa, previously considered different, as ontogenetic stages of the same species (Nyholm 1958). Observations of morphological variability of Ammonia beccarii in laboratory ecological experiments allowed the determination of characters which change with abiotic factors and the distinguishing of morphotypes of this species (Schnitker 1974). Interbreeding experiments helped to identify some Glabratella species from Japan (Kitazato, pers. comm.). Here, we propose to use the ribosomal RNA gene sequences to examine genetic similarity among different morphotypes of littoral foraminifera belonging to the genus Glabratella. Sequences of both large subunit (LSU) and small subunit (SSU) ribosomal genes are characterized by the presence of highly conserved and divergent domains (Hillis and Moritz 1990). The sequences of conserved regions are largely used for inferring origins and phylogenetic relationships between distant taxonomic groups (Baroin et al. 1988, Perasso et al. 1989, Sogin 1991, Schlegel 1991), while those of divergent regions are used for phylogenetic study of closely related species (Lenaers et al. 1991). The ribosomal sequences also play an important role in identification of bacterial species, which cannot be determined using other taxonomical criteria (Giovannoni et al. 1990; Stackebrandt and Goodfellow 1991). Application of molecular techniques in foraminiferal systematics was considerably delayed because of the difficulties in maintaining foraminifera in axenic conditions (Anderson et al. 1991). Only recently the successful extraction of foraminiferal DNA out of a few collected cells and the amplification of the SSU rRNA gene, using polymerase chain reaction (PCR), has been claimed by Langer et al. (1993) and Wray et al. (1993). The phylogenetic position of foraminifera among the eukaryotes, as inferred from partial LSU rDNA sequences, has recently been presented by Pawlowski et al.(in press), elsewhere. MATERIAL AND METHODS Living specimens of Glabratella were isolated from samples of algal material collected in May, July and October 1993, at the French coast of the Mediterranean Sea, in three localities situated near Toulon: La Faviere (Baie du Gau), La Nartelle (Baie de Bougnon) and Ile de Porquerolles. The samples were rapidly transferred to the laboratory in Geneva where the foraminifera were isolated. Different morphological forms were identified and sorted. 10-20 individuals of each morphotype were cleaned by washing in sterile sea water to eliminate any associated microorganisms. Later the specimens were ground in 1.5 ml microfuge tubes containing 50gl of TE buffer. Samples containing a single specimen were also prepared. DNA was extracted using the CTAB method (Clark 1992) and stored at -20?C. A few specimens of each morphotype were dried and retained for measuring and SEM studies. micropaleontology, vol. 40, no. 4, pp. 373-377, text-figures 1-2, table 1, 1994 373 This content downloaded from 207.46.13.75 on Fri, 08 Jul 2016 06:17:54 UTC All use subject to http://about.jstor.org/terms Jan Pawlowski, Ignacio Bolivar; Jose Fahrni, Louisette Zaninetti: Taxonomic identification offoraminifera using ribosomal DNA sequences TABLE 1 Measurements of the test diameter and height of seven morphotypes of Glabratella examined in this study. Diameter (jim) Height (gim) Diameter/Hei Morphotype Origins Date Generation Average (min-max) Average (min-max) GLA 1 Porquerolles May Agamont 294.0 (250-330) 136.8 (120-150) 2.2 GLA 2 Porquerolles Oct. Agamont 235.3 (190-250) 165.5 (140-200) 1.4 GLA 3 La Faviere May Agamont 241.7 (200-260) 218.0 (180-240 1.1 GLA 4 Porquerolles Oct. Agamont 201.3 (160-240) 283.8 (270-300) 0.7 GLA 5 La Nartelle July Agamont 253.3 (190-310) 194.7 (170-250) 1.3 GLA 6 Porquerolles May Gamont 128.4 (80-150) 107.4 (60-140) 1.2 GLA7 LaFaviere May Gamont 125.1 (110-150) 105.7 (60-180) 1.4 A DNA fragment of about 1200 bp located at 5' terminal region of LSU rRNA gene was amplified by PCR using standard conditions (35 sec. at 93.5?C, 35 sec. at 52?C and 2 min. at 72?C, 40 cycles). A 5' terminal primer (Rib 2TA) specific for foraminifera and a universal consensus primer (Rib 0) constructed from the database of eukaryotic LSU rDNA sequences were used for amplification. Amplified DNA was purified using Spin-Bind DNA extraction units (FMC) and stored at -20?. The region of about 400 bp, which covers a part of conserved region C1 and the divergent domain D1 of the LSU rRNA gene (Hassouna et al. 1984), was directly sequenced using finol DNA Sequencing System (Promega). The oligonucleotides used for amplification and sequencing are described in Pawlowski et al. (in press). The obtained sequences were assemblaged using PC/GENE (Bairoch 1989). Clustal V multiple sequence alignment program (Higgins et al. 1991) was used to align the sequences. Most foraminiferal samples are polyxenic beacause the foraminifera themselves can harbour diverse symbiotic microorganisms. It is most important to ascertain that the amplified sequences belong to the foraminifera and not to another, even unnoticed, contaminant. We are confident this to be the case for several reasons: 1) The specific foraminiferal primer was constructed from the RNA sequence of Ammonia sp. obtained by reverse transciptase sequencing of total Ammonia RNA (Pawlowski et al., in press). For RNA extraction, the specimens of Ammonia were thoroughly cleaned and were obviously the major organisms in the sample. Northern blot analysis using universal primers showed that the LSU rRNA is nicked or spliced in at least four fragments of different sizes. If the samples were heterogeneous, it is highly unlikely that the LSU rRNA's from different organisms would show the same fragmentation pattern, hence one or more primers would have hybridized with several fragments of different sizes. Since this was never found, we concluded that our samples contained a single LSU rRNA species that belonged to the foraminifera. 2) Fragments amplified with at least one specific foraminiferal primer were sequenced and matched perfectly the RNA sequences. DNA from more than 20 foraminiferal species collected in different localities were successfully amplified using the specific primers. Amplifications of the LSU rRNA 5' terminal region performed on a variety of other organisms with corresponding but universal primers, usually produced fragments visibly shorter than those of foraminifera. Amplification of foraminiferal samples with universal primers produced both long and short fragments. When cloned, long fragments were always homologous to fragments amplified with specific foraminiferal primers, while short fragments showed homology to fungi, algae, etc. Therefore we concluded that we can amplify foraminiferal ribosomal DNA selectively from a heterogeneous mix. 3) In the improbable event that fragments from other organisms would be amplified by specific, well designed foraminiferal primers, the sequences of foraminifera are different enough from those of other eukaryotes to make us confident that we will distinguish them from spurious ones.