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The occurrence of two genotypes of the planktonic foraminifer Globigerinoides ruber (white) and paleo-environmental implications
Marine Micropaleontology 68 (2008) 236–243
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Marine Micropaleontology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m a r m i c r o
The occurrence of two genotypes of the planktonic foraminifer Globigerinoides ruber (white) and paleo-environmental implications Azumi Kuroyanagi a,⁎, Masashi Tsuchiya b, Hodaka Kawahata a,c, Hiroshi Kitazato b a
Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan Institute for Research on Earth Evolution, Research Program for Paleoenvironment, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan c Graduate School of Frontier Sciences, The University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan b
a r t i c l e
i n f o
Article history: Received 24 October 2007 Received in revised form 10 April 2008 Accepted 19 April 2008 Keywords: Planktonic foraminifera Genotype Phylogenetic analysis Morphotype Globigerinoides ruber
a b s t r a c t Planktonic foraminifera provide a record of the upper ocean environment in the chemical and isotopic composition of individual shells. Globigerinoides ruber is a common tropical– subtropical planktonic foraminifer, and this species is used extensively for reconstruction of the paleo-environment. The different stable isotopic compositions of two morphotypes, G. ruber sensu stricto (s.s.) and G. ruber sensu lato (s.l.), first identified in sediments, suggested that G. ruber s.s. was dwelling in the upper 30 m of the water column and G. ruber s.l. at greater depths. Plankton tows and sediment trap experiments provided additional evidence distinguishing the two morphotypes and their habitats and invited the question as to whether the two morphotypes could be distinguished genetically. In this study, using phylogenetic analysis of nuclear partial small subunit ribosomal DNA (SSU rDNA) gene sequences representing 12 new and 16 known sequences, we identified four genotypes within G. ruber white variation; one of which is a sister group of Globigerinoides conglobatus, whereas the three others were sister groups of the G. ruber pink variation. Moreover, these two major groups corresponded to morphological differences described as G. ruber s.l. and s.s., respectively. This genetic evidence corroborates differences between the two morphotypes in the isotope record, and it will contribute to a more precise reconstruction of the thermal structure of the water column. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Planktonic foraminifera can provide a record of some environmental conditions through their assemblage composition and in individual shells. To reconstruct the paleoenvironment, numerous studies have focused on the relationships between the foraminiferal record and environmental parameters such as water temperature, salinity, and nutrient levels (i.e., primary production) in water masses. Recently, molecular investigations have reported multiple cases of cryptic speciation of planktonic foraminifera and illustrated the different geographic distributions among foraminiferal ⁎ Corresponding author. Tel.: +81 3 5351 6442; fax: +81 3 5351 6445. E-mail address: [email protected] (A. Kuroyanagi). 0377-8398/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.marmicro.2008.04.004
genotypes (e.g., Huber et al., 1997; de Vargas et al., 1999; de Vargas et al., 2002). For example, de Vargas et al. (2002) reported that each genotype of Globigerinella siphonifera was adapted to different primary production environments in the Atlantic Ocean. Globigerinoides ruber inhabits warm surface waters (temperature range, 14–32 °C; optimum temperature, 23.0 °C by culture experiment [Bijma et al., 1990]) and is used extensively for paleoceanography through its test chemistry and stable isotopic composition (e.g., de Garidel-Thoron et al., 2005). Wang (2000) was the first to recognize the different stable isotopic compositions of two morphotypes of G. ruber (sensu stricto [s.s.] and sensu lato [s.l.]) in sediments in the South China Sea, which suggested that G. ruber s.s. was dwelling in the upper 30 m of the water column and G. ruber s.l. at deeper depths. Plankton tow and sediment trap studies
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Fig. 1. Locations of the pump sampling stations (stations 1–6) and sites of previous studies reporting two morphotypes of Globigerinoides ruber (Wang, 2000; Kuroyanagi and Kawahata, 2004; Kawahata, 2005).
corresponds to the more compact and higher trochospiral form previously described as Globigerinoides elongates (d'Orbigny, 1826), Globigerinoides pyramidalis (Van den Broeck, 1876), and Globigerinoides cyclostomus (Galloway and Wissler, 1927). In the present study, the taxonomic criteria for morphologic identification of G. ruber (G. ruber s.s. and G. ruber s.l.) followed those of Wang (2000) and Kawahata (2005) (Plate 1). Although it was sometimes difficult to distinguish juvenile forms of G. ruber in our study because of underdeveloped morphological characteristics such as features of the apertures and sutures, we distinguished most of the two morphotypes by using some of features described in the caption of Plate 1.
confirmed this idea (Kuroyanagi and Kawahata, 2004; Kawahata, 2005) and raised the question as to whether the two morphotypes could be distinguished genetically. Darling et al. (1999) reported the phylogenetic relationships within G. ruber, and a sister group relationship between G. ruber and Globigerinoides conglobatus. However, the morphological differences between these genotypes have remained unknown. In this study, we collected G. ruber (white) specimens from surface water in the central Pacific Ocean and examined the relationship between genetic variation and morphology by using phylogenetic analysis of nuclear partial small subunit ribosomal DNA (SSU rDNA) gene sequences. 2. Materials and methods
2.2. DNA extraction 2.1. Sampling and identification Total DNA was extracted from 29 G. ruber specimens by the sodium deoxycholate (DOC) method (Pawlowski et al., 1994). Each specimen was placed in 50 µl of 1 × DOC and crushed with a closed-tip Pasteur pipette. Mixtures were incubated at 60 °C for 1 h. Insoluble materials were separated by centrifugation (5 min, 9000 rpm). The supernatant was collected and used for polymerase chain reaction (PCR) amplifications.
Samples were collected from on board the R/V Mirai (cruise MR02-K01), by using a pump, at six stations (numbered 1 through 6) in the tropical and subtropical central Pacific Ocean (Fig. 1 and Table 1). Seawater from 5 m water depth was first passed through a 1/20 in. (1270 µm) sieve. The samples were sieved further through a 63 µm sieve, and live individual foraminifera were picked from the N63 µm fraction by using a Pasteur pipette, identified, and stored at −70 °C. In the laboratory, 29 specimens were examined with a scanning electron microscope (SEM) (JEOL, JSM-5800LV) under low-vacuum conditions and without any coating. Wang (2000) was the first to report that G. ruber s.l. generally
2.3. PCR amplification, cloning, and sequencing PCR amplifications of SSU rDNA were performed as described by Pawlowski (2000). The region of SSU rDNA, consisting of approximately 1000 base pairs (bp), was
Table 1 Locations of pump sampling stations; dates, times, volumes of seawater filtered; and genotypes of Globigerinoides ruber collected Pump st. no.
Position (start)
1 2 3 4 5 6
25°19′N 14°37′N 8°19′N 3°02′N 0°00′N 0°00′N
Position (end) 165°59′W 158°34′W 159°11′W 159°12′W 163°30′W 169°55′W
24°59′N 13°18′N 7°26′N 1°38′N 0°00′S 0°01′S
165°13′W 158°42′W 159°16′W 159°51′W 164°45′W 170°00′W
The numbers in parentheses indicate the numbers of individuals found.
Date
Time (local)
15 Jan 2002 20 Jan 2002 21 Jan 2002 22 Jan 2002 24 Jan 2002 25 Jan 2002
12:43–16:28 12:49–17:59 14:00–17:29 11:46–17:29 12:54–18:26 13:07–18:02
Volume filtered (m3) 3.00 3.72 2.51 4.02 3.19 2.95
Type of Globigerinoides ruber (white) I I I (2), II I (2), II II I (1), II
(1) (8) (3) (7) (1) (4)
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amplified with primers s14f1#1 (5′-aagggcaccacaag(a/c) gcgtgga-3′), which was modified from Pawlowski (2000), and sB (5′-tgatccttctgcaggttcacctac-3′). The amplification consisted of 40 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min. The purified products were ligated into the pGEM-T vector system (Promega, Madison, WI, USA) and used to transform XL-2 blue ultracompetent cells (Stratagene, La Jolla, CA, USA). Plasmids were isolated from cell cultures by using a QIAprep Spin Miniprep Kit (QIAGEN, Tokyo, Japan). The sequencing reaction was carried out by using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The samples were sequenced by using an ABI PRISM 3100 DNA Analyzer (Applied Biosystems). Nucleotide sequences were deposited in the GenBank database (accession numbers, EU012457–EU012487). 2.4. Phylogenetic analysis We used Globigerinoides sacculifer (accession numbers, U65633, Z83963, and Z83964) and Orbulina universa (U65632, U80791, and Z83962) as outgroup species that are closely related to the G. ruber and G. conglobatus morphogroups, based on the phylogenetic analysis of SSU rDNA sequences (de Vargas et al., 1997; Darling et al., 1999, 2000). The DNA sequences were aligned by using Clustal W (Thompson et al., 1994) and corrected manually in the Se–Al ver 2.0a11 Sequence Alignment Editor (Rambaut, 1996). All major gaps were excluded from the analysis. Evolutionary models were selected by Modeltest 3.5 (Posada and Crondall, 1998), which evaluates the best model for DNA evolution by using maximum likelihood (ML) analysis. The model determined to be the best for our results was TrN (Tamura and Nei, 1993), with variable sites assumed to follow a gamma distribution (Γ; Yang, 1994). Genetic distances between nucleotide sequences were estimated by ML distance based on the described calculation for the distance method. The unrooted phylogenetic trees were constructed, with the ML method, distance method (ML dist), and maximum parsimony (MP) performed by using the PAUP⁎ package, version 4.0b10 (Swofford, 2002) with heuristic search option. A starting tree was obtained via random stepwise addition with the TBR (tree bisection-reconnection) branch-swapping algorithm. For MP analysis, 409 sites of 875 characters (alignment) were constant. Seventeen variable characters were parsimony-uninformative. The consistency index (ci) and retention index (ri) were 0.846 and 0.960, respectively. Bootstrapping was conducted with 1000 replicates for ML distance and MP and with 100 replicates for ML analysis. The molecular phylogenetic trees are almost identical for
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three methods, and the ML tree was selected as representative (Fig. 2). 2.5. RFLP analysis In order to obtain supplementary data on genotypes, we carried out PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Restriction enzymes digested the nucleotide sequences at specific positions, and the digestion patterns enabled the separation of G. ruber genotypes. Purified PCR products were digested with two restriction enzymes, AluI and NspV. The reactions were performed for 20 µl volumes containing 2.0 µl of template DNA, 1.0 µl of restriction enzyme, and 2.0 µl of 10× buffer. PCR products that were digested with AluI and NspV were run on 3% agarose gel (Agarose 21; Nippon Gene, Tokyo, Japan) for 1 h at 50 V. Digestion patterns were visualized by using ethidium bromide staining and a UV transilluminator. 3. Results We extracted total DNA from 29 G. ruber (white) specimens (12 extracts were used for sequence analysis, 17 for RFLP analysis) (Table 1). Two to four clones were sequenced from each specimen, and 32 sequences from 12 specimens were examined in this study. The phylogenetic relationships of the two morphotypes, incorporating results of G. ruber from other localities and of G. conglobatus (Darling et al., 1996, 1997, 1999; de Vargas et al. 1997; Pawlowski et al., 1997), gave rise to four distinct clades: G. ruber (white) [Type I], G. ruber (pink), G. ruber (white) [Type II], and G. conglobatus (Fig. 2). The Type I genotype formed one distinct cluster with 3 subclusters (Ia–c), whereas Type II formed another cluster (Fig. 2). The Type I and G. ruber (pink) SSU genotypes clustered with 100% bootstrap support, and Type II and G. conglobatus SSU genotypes with 98% bootstrap support. The Type I genotypes, which included G. ruber from Puerto Rico (Pawlowski et al., 1997), the coast of Japan (accession numbers, EU012452– EU012456), and the Great Barrier Reef (Darling et al., 1997), are divided into three closely related types: Ia, Ib, and Ic. In this study, we could not distinguish these three genotypes morphologically. Genotypes Ia, Ib, and Ic are divergent from the G. ruber (pink) genotype with genetic distances of 10%, 10–11%, and 10%, respectively (Table 2). In contrast, the genetic distance between the pink variety and Type II was 24–25%. SSU rDNA sequence analysis clearly showed that G. ruber s.s. and s.l. morphotypes belong to different distinct genotypes, Type I and Type II (Fig. 2), with SSU rDNA lengths of ~1012 and ~ 1031 bp, respectively. To obtain additional data
Plate 1. Globigerinoides ruber s.s. (1a–c and 2a–c) and G. ruber s.l. (3a–c and 4a–c) collected by pump sampling. a, umbilical side view; b, side view; c, dorsal side view. Scale bar is 100 µm. 1a–c, 2a–c, and 4a–c specimens are 10-2⁎, 13-6⁎, and 55-26⁎ samples in Fig. 2, respectively. 3a–c was identified by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. The taxonomic criteria for morphologic identification of G. ruber (G. ruber s.s. and G. ruber s.l.) follows Wang (2000) and Kawahata (2005). Globigerinoides ruber s.s.: test medium trochospiral coil with three spherical chambers in the final whorl, increasing rapidly in size, symmetrical over the previous sutures; sutures radial and deep; surface coarsely perforate; primary aperture large, wide, rounded high-arched opening, symmetric over the previous suture; secondary supplementary apertures on spiral side, wide and highly arched rounded opening at opposite sutures of previous chambers (Plate 1; 1a–c and 2a–c). Globigerinoides ruber s.l.: test medium to high trochospiral coil with three compressed subspherical chambers in the final whorl, increasing moderately, sometimes with a relatively small last chamber, asymmetrical over the previous sutures; sutures radial and depressed; surface coarsely to medium densely perforate; primary aperture small round or medium arched opening, sometimes symmetric over the earlier suture; secondary supplementary apertures on spiral side small to medium size, rounded opening at opposite sutures of previous chambers (Plate 1; 3a–c and 4a–c).
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Fig. 2. Molecular phylogenetic tree for Globigerinoides ruber and related species. The tree was reconstructed using the maximum likelihood method based on the small subunit ribosomal DNA (SSU rDNA) sequences. Bootstrap values are given next to each branch of the tree. We used Globigerinoides sacculifer (U65633, Z83963, and Z83964) and O. universa (U65632, U80791, and Z83962) as outgroup species (de Vargas et al., 1997; Darling et al., 1999, 2000), and incorporated results from other localities and G. conglobatus (Darling et al., 1996, 1997, 1999; de Vargas et al. 1997; Pawlowski et al., 1997) in the phylogeny.
about the relationship between genetics and morphology, we performed RFLP analysis of the SSU rDNA using the two enzymes NspV and AluI, by which we were able to identify the two different genotypes. Based on the base pair sequences of these genotypes, the NspV enzyme should divide the Type II SSU rDNA into two fragments (length, ~366 and ~665 bp). AluI is expected to cleave Type I either at one site (yielding fragments of ~77 and ~935 bp) or leave it uncut (~ 1012 bp band). AluI should divide Type II SSU rDNA into three fragments (~ 130, ~ 415, and ~ 486 bp) (Fig. 3). RFLP patterns from 7 of 17 G. ruber specimens show the first Type I genotype, and 10 specimens show the Type II pattern. 4. Discussion 4.1. Phylogenetic tree of G. ruber Darling et al. (1999) presented the phylogenetic relationships of G. ruber, including G. conglobatus. In this study, we add our 12 new sequences and morphological information to their results. Molecular phylogenetic analysis of foraminiferal SSU rDNA demonstrated that G. ruber s.s. is genetically highly distinct from G. ruber s.l. (Fig. 2), because the genetic distance
of SSU rDNA between Type I and Type II is 23–24%, whereas Type II differs from G. conglobatus by 11–12% (Table 2). Darling et al. (1999) showed that pink specimens of G. ruber, collected from the Caribbean, are divergent from another G. ruber cluster, including the white variation from the Coral Sea, with an average genetic distance of 5.5%. Moreover, G. ruber specimens from the Southern California Bight were highly divergent from those in the Caribbean and Coral Sea clusters, with a mean genetic distance of 10.8%, and the Southern California Bight cluster was a sister group of G. conglobatus (Darling et al., 1999). Therefore, we speculate that the specimen collected from the Coral Sea could be G. ruber s.s. and that from the Southern California Bight is G. ruber s.l. If true, this finding suggests that G. ruber s.s. inhabits both the Pacific and Atlantic Oceans. In contrast, G. ruber s.l. is known at present to occur only in the Pacific Ocean (Fig. 2). 4.2. Horizontal distribution of two genotypes of G. ruber (white) in the central Pacific Ocean Both G. ruber (white) genotypes often occurred at the same sites, except for at our station 2 (Fig. 1 and Table 1). Although the number of individuals that we sampled is not sufficient for
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Table 2 Pairwise genetic distances, as a percentage difference of nucleotides, between Globigerinoides ruber (Types Ia–c, pink variation, and Type II) and Globigerinoides conglobatus
1 2 3 4 5 6 7 8
Type Ia Type Ib Type Ic Pink Type II conglo. sacculifer universa
1
2
3
4
5
6
7
8
0.6–1.0 1.5–1.7 3.7–4.2 9.7–10.4 22.7–23.9 20.7–21.5 36.7–37.0 33.1–33.4
0 3.7–3.8 10.4–10.6 23.2–23.7 21.3–21.4 37.0–37.3 32.4–32.6
0–0.5 9.5–10.2 22.9–23.5 20.7–20.9 35.9–36.3 32.6–33.0
0.3 23.8–24.6 21.0–21.6 35.9–36.3 32.9–33.2
0–0.8 11.1–11.9 34.6–35.5 33.3–34.0
1.6 34.4–34.9 32.0–32.3
0.2–0.8 30.7–31.0
0–0.2
Globigerinoides sacculifer and Orbulina universa are used as outgroup species.
a statistical discussion, we could carry out the continuous pump sampling along the cruise route (up to ~50 km per sample) (Fig 1 and Table 1). It is known that different genotypes of Globorotalia truncatulinoides can adapt to particular hydrographic conditions (de Vargas et al., 2001). In this study, station 2 had the most oligotrophic surface conditions (Fig. 4; McClain et al., 2004), and only Type I (G. ruber s.s.) was found there in high numbers (Table 1). The vertical distribution of G. ruber morphotypes in the modern ocean (Kuroyanagi and Kawahata, 2004) suggests that the different habitats of each can be determined by different controlling factors; G. ruber s.s. distributions are mainly dependent upon light intensity and G. ruber s.l. distributions on food availability. Therefore, low surface chlorophyll-a content could prevent Type II (G. ruber s.l.) from dwelling in the surface ocean (~ 0–5 m water depth) at station 2. Differences in oxygen isotopic ratios between the two morphotypes also seem to be related to thermal stratification of the water column (Kawahata, 2005). Thus, strong stratification (low concentration of surface chlorophyll-a) may result in the high numbers of Type I (G. ruber s.s.) at the surface, much like at station 2, and would lead to a large difference in the isotopic record from the two genotypes. 4.3. Paleo-environmental implications of two genotypes of G. ruber The differences in isotopic records between the two genotypes can be applied to paleoceanography. Sediment samples have demonstrated the differences in the isotopic record between G. ruber s.s. and s.l., and the mean difference in δ18O values of 0.21 ± 0.21‰ is likely due to their different habitats (Wang, 2000). Sediment trap experiments investigating the seasonal variation of the oxygen isotopic composition of the two morphotypes in the subtropical North Pacific showed that the mean difference was 0.25‰ from August to October under stratified conditions, corresponding to a difference of 1 °C in water temperature (the temperature difference between water at the surface and at 30–50 m) (Kawahata, 2005). Some recent studies have focused on the water column conditions in the tropical region related to the estimation of El Niño- or La Niña-like conditions (e.g., de Garidel-Thoron et al., 2005; Rickaby and Halloran, 2005). Because the δ18O values for G. ruber have a glacial–interglacial variation that ranges from approximately 0.6‰ (from 1.75 to 0.85 Ma) to 1.4‰ (terminations I, II, and IV) in the western equatorial Pacific Ocean (de Garidel-Thoron et al., 2005), the
interpretation of isotopic data from G. ruber genotypes may not require extensive modification. However, the two genotypes of G. ruber can be very useful tools in reconstructing the detailed thermal stratification structure in the tropical and subtropical oceans with high-resolution samples, especially in the Holocene. The chemical composition of foraminiferal tests of the two genotypes (e.g., the Mg/Ca ratio) might also provide insights into ocean stratification, although there is a little information currently available for this application (Steinke et al., 2005). If more information can be extracted from the chemical and isotopic data from the two genotypes, more detailed reconstructions of past changes in stratification and climate in tropical areas could be achieved. 5. Conclusion We have examined genetic variation in the SSU rDNA sequences of a representative tropical–subtropical species of foraminifera, G. ruber, and found four genotypes within the G. ruber white variation, one of which is a sister group of G. conglobatus whereas the other three are sisters to the G. ruber pink variation. Moreover, two major genotypes (Types I and II) are identical to morphotypes “s.s.” and “s.l.”, respectively. Because the genetic evidence corroborates the existence of two morphotypes, the differences in the isotopic record of the
Fig. 3. Restriction patterns resulting from restriction fragment length polymorphism (RFLP) analysis using two restriction enzymes, NspV and AluI. Two RFLP samples, #11 and #17, show the Type I pattern; #32 and #39 represent the Type II pattern.
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Fig. 4. Locations of the sampling stations (stations 1–6) along with surface chlorophyll-a concentrations. Dark color indicates low concentration and light color indicates high (McClain et al., 2004).
two genotypes can be applied to a reconstruction of historic thermal stratification using information about their different habitation depths. Acknowledgments We thank E. Thomas and an anonymous reviewer for their helpful comments to improve the manuscript. We express our sincere appreciation to the captain and crew of the R/V Mirai and our colleagues who were on board during the cruise. We are also grateful to M. Kawato (JAMSTEC) for his support in sequencing and Y. Ujiie (ORI) and the staff members at ORI, JAMSTEC, and AIST for their helpful discussions and suggestions. Appendix A. (Taxonomic list) The taxonomy follows Parker (1962) and Saito et al. (1981). Globigerinoides cyclostomus (Galloway and Wissler) = Globigerina cyclostoma Galloway and Wissler, 1927, p42, pl. 7, figs. 8–9. Globigerinoides elongatus (d'Orbigny) = Globigerina elongata d'Orbigny, 1826, p. 277, list no. 4, (no figs.). Globigerinoides pyramidalis (van den Broeck) = Globigerina bulloides d'Orbigny var. rubra d'Orbigny subvar. pyramidalis Van den Broeck, 1876, p. 127, pl. 3, figs. 9–10. Globigerinoides ruber (d'Orbigny)=Globigerina rubra d'Orbigny, 1839, pp. 82–83, (plates published separately), pl. 4, figs. 12–14. References Bijma, J., Faber Jr., W.W., Hemleben, Ch., 1990. Temperature and salinity limits for growth and survival of some planktonic foraminifers in laboratory cultures. J. Foraminifer. Res. 20, 95–116. Darling, K.F., Kroon, D., Wade, C.M., Leigh Brown, A.J., 1996. Molecular phylogeny of the planktic foraminifera. J. Foraminiferal Res. 26, 324–330.
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implications for reconstructing past tropical/subtropical surface water conditions. Geochem. Geophys. Geosyst. 6 (11). doi:10.1029/2005GC000926. Swofford, D.L., 2002. PAUP⁎. Phylogenetic analysis using parsimony (⁎and other methods). Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts. Tamura, K., Nei, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Bio. Evol. 10, 512–526. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Van den Broeck, E., 1876. Etude sur les Foraminiferes de la Barbade (Antilles). Soc. Belge Microsc. An. 1, 55–152. Wang, L., 2000. Isotopic signals in two morphotypes of Globigerinoides ruber (white) from the South China Sea: implications for monsoon climate change during the last glacial cycle. Palaeogeogr., Palaeoclimatol., Palaeoecol. 161, 381–394. Yang, Z., 1994. Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J. Mol. Evol. 39, 306–341.
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Marine Micropaleontology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m a r m i c r o
The occurrence of two genotypes of the planktonic foraminifer Globigerinoides ruber (white) and paleo-environmental implications Azumi Kuroyanagi a,⁎, Masashi Tsuchiya b, Hodaka Kawahata a,c, Hiroshi Kitazato b a
Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan Institute for Research on Earth Evolution, Research Program for Paleoenvironment, Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan c Graduate School of Frontier Sciences, The University of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan b
a r t i c l e
i n f o
Article history: Received 24 October 2007 Received in revised form 10 April 2008 Accepted 19 April 2008 Keywords: Planktonic foraminifera Genotype Phylogenetic analysis Morphotype Globigerinoides ruber
a b s t r a c t Planktonic foraminifera provide a record of the upper ocean environment in the chemical and isotopic composition of individual shells. Globigerinoides ruber is a common tropical– subtropical planktonic foraminifer, and this species is used extensively for reconstruction of the paleo-environment. The different stable isotopic compositions of two morphotypes, G. ruber sensu stricto (s.s.) and G. ruber sensu lato (s.l.), first identified in sediments, suggested that G. ruber s.s. was dwelling in the upper 30 m of the water column and G. ruber s.l. at greater depths. Plankton tows and sediment trap experiments provided additional evidence distinguishing the two morphotypes and their habitats and invited the question as to whether the two morphotypes could be distinguished genetically. In this study, using phylogenetic analysis of nuclear partial small subunit ribosomal DNA (SSU rDNA) gene sequences representing 12 new and 16 known sequences, we identified four genotypes within G. ruber white variation; one of which is a sister group of Globigerinoides conglobatus, whereas the three others were sister groups of the G. ruber pink variation. Moreover, these two major groups corresponded to morphological differences described as G. ruber s.l. and s.s., respectively. This genetic evidence corroborates differences between the two morphotypes in the isotope record, and it will contribute to a more precise reconstruction of the thermal structure of the water column. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Planktonic foraminifera can provide a record of some environmental conditions through their assemblage composition and in individual shells. To reconstruct the paleoenvironment, numerous studies have focused on the relationships between the foraminiferal record and environmental parameters such as water temperature, salinity, and nutrient levels (i.e., primary production) in water masses. Recently, molecular investigations have reported multiple cases of cryptic speciation of planktonic foraminifera and illustrated the different geographic distributions among foraminiferal ⁎ Corresponding author. Tel.: +81 3 5351 6442; fax: +81 3 5351 6445. E-mail address: [email protected] (A. Kuroyanagi). 0377-8398/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.marmicro.2008.04.004
genotypes (e.g., Huber et al., 1997; de Vargas et al., 1999; de Vargas et al., 2002). For example, de Vargas et al. (2002) reported that each genotype of Globigerinella siphonifera was adapted to different primary production environments in the Atlantic Ocean. Globigerinoides ruber inhabits warm surface waters (temperature range, 14–32 °C; optimum temperature, 23.0 °C by culture experiment [Bijma et al., 1990]) and is used extensively for paleoceanography through its test chemistry and stable isotopic composition (e.g., de Garidel-Thoron et al., 2005). Wang (2000) was the first to recognize the different stable isotopic compositions of two morphotypes of G. ruber (sensu stricto [s.s.] and sensu lato [s.l.]) in sediments in the South China Sea, which suggested that G. ruber s.s. was dwelling in the upper 30 m of the water column and G. ruber s.l. at deeper depths. Plankton tow and sediment trap studies
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Fig. 1. Locations of the pump sampling stations (stations 1–6) and sites of previous studies reporting two morphotypes of Globigerinoides ruber (Wang, 2000; Kuroyanagi and Kawahata, 2004; Kawahata, 2005).
corresponds to the more compact and higher trochospiral form previously described as Globigerinoides elongates (d'Orbigny, 1826), Globigerinoides pyramidalis (Van den Broeck, 1876), and Globigerinoides cyclostomus (Galloway and Wissler, 1927). In the present study, the taxonomic criteria for morphologic identification of G. ruber (G. ruber s.s. and G. ruber s.l.) followed those of Wang (2000) and Kawahata (2005) (Plate 1). Although it was sometimes difficult to distinguish juvenile forms of G. ruber in our study because of underdeveloped morphological characteristics such as features of the apertures and sutures, we distinguished most of the two morphotypes by using some of features described in the caption of Plate 1.
confirmed this idea (Kuroyanagi and Kawahata, 2004; Kawahata, 2005) and raised the question as to whether the two morphotypes could be distinguished genetically. Darling et al. (1999) reported the phylogenetic relationships within G. ruber, and a sister group relationship between G. ruber and Globigerinoides conglobatus. However, the morphological differences between these genotypes have remained unknown. In this study, we collected G. ruber (white) specimens from surface water in the central Pacific Ocean and examined the relationship between genetic variation and morphology by using phylogenetic analysis of nuclear partial small subunit ribosomal DNA (SSU rDNA) gene sequences. 2. Materials and methods
2.2. DNA extraction 2.1. Sampling and identification Total DNA was extracted from 29 G. ruber specimens by the sodium deoxycholate (DOC) method (Pawlowski et al., 1994). Each specimen was placed in 50 µl of 1 × DOC and crushed with a closed-tip Pasteur pipette. Mixtures were incubated at 60 °C for 1 h. Insoluble materials were separated by centrifugation (5 min, 9000 rpm). The supernatant was collected and used for polymerase chain reaction (PCR) amplifications.
Samples were collected from on board the R/V Mirai (cruise MR02-K01), by using a pump, at six stations (numbered 1 through 6) in the tropical and subtropical central Pacific Ocean (Fig. 1 and Table 1). Seawater from 5 m water depth was first passed through a 1/20 in. (1270 µm) sieve. The samples were sieved further through a 63 µm sieve, and live individual foraminifera were picked from the N63 µm fraction by using a Pasteur pipette, identified, and stored at −70 °C. In the laboratory, 29 specimens were examined with a scanning electron microscope (SEM) (JEOL, JSM-5800LV) under low-vacuum conditions and without any coating. Wang (2000) was the first to report that G. ruber s.l. generally
2.3. PCR amplification, cloning, and sequencing PCR amplifications of SSU rDNA were performed as described by Pawlowski (2000). The region of SSU rDNA, consisting of approximately 1000 base pairs (bp), was
Table 1 Locations of pump sampling stations; dates, times, volumes of seawater filtered; and genotypes of Globigerinoides ruber collected Pump st. no.
Position (start)
1 2 3 4 5 6
25°19′N 14°37′N 8°19′N 3°02′N 0°00′N 0°00′N
Position (end) 165°59′W 158°34′W 159°11′W 159°12′W 163°30′W 169°55′W
24°59′N 13°18′N 7°26′N 1°38′N 0°00′S 0°01′S
165°13′W 158°42′W 159°16′W 159°51′W 164°45′W 170°00′W
The numbers in parentheses indicate the numbers of individuals found.
Date
Time (local)
15 Jan 2002 20 Jan 2002 21 Jan 2002 22 Jan 2002 24 Jan 2002 25 Jan 2002
12:43–16:28 12:49–17:59 14:00–17:29 11:46–17:29 12:54–18:26 13:07–18:02
Volume filtered (m3) 3.00 3.72 2.51 4.02 3.19 2.95
Type of Globigerinoides ruber (white) I I I (2), II I (2), II II I (1), II
(1) (8) (3) (7) (1) (4)
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amplified with primers s14f1#1 (5′-aagggcaccacaag(a/c) gcgtgga-3′), which was modified from Pawlowski (2000), and sB (5′-tgatccttctgcaggttcacctac-3′). The amplification consisted of 40 cycles of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 2 min. The purified products were ligated into the pGEM-T vector system (Promega, Madison, WI, USA) and used to transform XL-2 blue ultracompetent cells (Stratagene, La Jolla, CA, USA). Plasmids were isolated from cell cultures by using a QIAprep Spin Miniprep Kit (QIAGEN, Tokyo, Japan). The sequencing reaction was carried out by using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The samples were sequenced by using an ABI PRISM 3100 DNA Analyzer (Applied Biosystems). Nucleotide sequences were deposited in the GenBank database (accession numbers, EU012457–EU012487). 2.4. Phylogenetic analysis We used Globigerinoides sacculifer (accession numbers, U65633, Z83963, and Z83964) and Orbulina universa (U65632, U80791, and Z83962) as outgroup species that are closely related to the G. ruber and G. conglobatus morphogroups, based on the phylogenetic analysis of SSU rDNA sequences (de Vargas et al., 1997; Darling et al., 1999, 2000). The DNA sequences were aligned by using Clustal W (Thompson et al., 1994) and corrected manually in the Se–Al ver 2.0a11 Sequence Alignment Editor (Rambaut, 1996). All major gaps were excluded from the analysis. Evolutionary models were selected by Modeltest 3.5 (Posada and Crondall, 1998), which evaluates the best model for DNA evolution by using maximum likelihood (ML) analysis. The model determined to be the best for our results was TrN (Tamura and Nei, 1993), with variable sites assumed to follow a gamma distribution (Γ; Yang, 1994). Genetic distances between nucleotide sequences were estimated by ML distance based on the described calculation for the distance method. The unrooted phylogenetic trees were constructed, with the ML method, distance method (ML dist), and maximum parsimony (MP) performed by using the PAUP⁎ package, version 4.0b10 (Swofford, 2002) with heuristic search option. A starting tree was obtained via random stepwise addition with the TBR (tree bisection-reconnection) branch-swapping algorithm. For MP analysis, 409 sites of 875 characters (alignment) were constant. Seventeen variable characters were parsimony-uninformative. The consistency index (ci) and retention index (ri) were 0.846 and 0.960, respectively. Bootstrapping was conducted with 1000 replicates for ML distance and MP and with 100 replicates for ML analysis. The molecular phylogenetic trees are almost identical for
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three methods, and the ML tree was selected as representative (Fig. 2). 2.5. RFLP analysis In order to obtain supplementary data on genotypes, we carried out PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Restriction enzymes digested the nucleotide sequences at specific positions, and the digestion patterns enabled the separation of G. ruber genotypes. Purified PCR products were digested with two restriction enzymes, AluI and NspV. The reactions were performed for 20 µl volumes containing 2.0 µl of template DNA, 1.0 µl of restriction enzyme, and 2.0 µl of 10× buffer. PCR products that were digested with AluI and NspV were run on 3% agarose gel (Agarose 21; Nippon Gene, Tokyo, Japan) for 1 h at 50 V. Digestion patterns were visualized by using ethidium bromide staining and a UV transilluminator. 3. Results We extracted total DNA from 29 G. ruber (white) specimens (12 extracts were used for sequence analysis, 17 for RFLP analysis) (Table 1). Two to four clones were sequenced from each specimen, and 32 sequences from 12 specimens were examined in this study. The phylogenetic relationships of the two morphotypes, incorporating results of G. ruber from other localities and of G. conglobatus (Darling et al., 1996, 1997, 1999; de Vargas et al. 1997; Pawlowski et al., 1997), gave rise to four distinct clades: G. ruber (white) [Type I], G. ruber (pink), G. ruber (white) [Type II], and G. conglobatus (Fig. 2). The Type I genotype formed one distinct cluster with 3 subclusters (Ia–c), whereas Type II formed another cluster (Fig. 2). The Type I and G. ruber (pink) SSU genotypes clustered with 100% bootstrap support, and Type II and G. conglobatus SSU genotypes with 98% bootstrap support. The Type I genotypes, which included G. ruber from Puerto Rico (Pawlowski et al., 1997), the coast of Japan (accession numbers, EU012452– EU012456), and the Great Barrier Reef (Darling et al., 1997), are divided into three closely related types: Ia, Ib, and Ic. In this study, we could not distinguish these three genotypes morphologically. Genotypes Ia, Ib, and Ic are divergent from the G. ruber (pink) genotype with genetic distances of 10%, 10–11%, and 10%, respectively (Table 2). In contrast, the genetic distance between the pink variety and Type II was 24–25%. SSU rDNA sequence analysis clearly showed that G. ruber s.s. and s.l. morphotypes belong to different distinct genotypes, Type I and Type II (Fig. 2), with SSU rDNA lengths of ~1012 and ~ 1031 bp, respectively. To obtain additional data
Plate 1. Globigerinoides ruber s.s. (1a–c and 2a–c) and G. ruber s.l. (3a–c and 4a–c) collected by pump sampling. a, umbilical side view; b, side view; c, dorsal side view. Scale bar is 100 µm. 1a–c, 2a–c, and 4a–c specimens are 10-2⁎, 13-6⁎, and 55-26⁎ samples in Fig. 2, respectively. 3a–c was identified by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. The taxonomic criteria for morphologic identification of G. ruber (G. ruber s.s. and G. ruber s.l.) follows Wang (2000) and Kawahata (2005). Globigerinoides ruber s.s.: test medium trochospiral coil with three spherical chambers in the final whorl, increasing rapidly in size, symmetrical over the previous sutures; sutures radial and deep; surface coarsely perforate; primary aperture large, wide, rounded high-arched opening, symmetric over the previous suture; secondary supplementary apertures on spiral side, wide and highly arched rounded opening at opposite sutures of previous chambers (Plate 1; 1a–c and 2a–c). Globigerinoides ruber s.l.: test medium to high trochospiral coil with three compressed subspherical chambers in the final whorl, increasing moderately, sometimes with a relatively small last chamber, asymmetrical over the previous sutures; sutures radial and depressed; surface coarsely to medium densely perforate; primary aperture small round or medium arched opening, sometimes symmetric over the earlier suture; secondary supplementary apertures on spiral side small to medium size, rounded opening at opposite sutures of previous chambers (Plate 1; 3a–c and 4a–c).
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Fig. 2. Molecular phylogenetic tree for Globigerinoides ruber and related species. The tree was reconstructed using the maximum likelihood method based on the small subunit ribosomal DNA (SSU rDNA) sequences. Bootstrap values are given next to each branch of the tree. We used Globigerinoides sacculifer (U65633, Z83963, and Z83964) and O. universa (U65632, U80791, and Z83962) as outgroup species (de Vargas et al., 1997; Darling et al., 1999, 2000), and incorporated results from other localities and G. conglobatus (Darling et al., 1996, 1997, 1999; de Vargas et al. 1997; Pawlowski et al., 1997) in the phylogeny.
about the relationship between genetics and morphology, we performed RFLP analysis of the SSU rDNA using the two enzymes NspV and AluI, by which we were able to identify the two different genotypes. Based on the base pair sequences of these genotypes, the NspV enzyme should divide the Type II SSU rDNA into two fragments (length, ~366 and ~665 bp). AluI is expected to cleave Type I either at one site (yielding fragments of ~77 and ~935 bp) or leave it uncut (~ 1012 bp band). AluI should divide Type II SSU rDNA into three fragments (~ 130, ~ 415, and ~ 486 bp) (Fig. 3). RFLP patterns from 7 of 17 G. ruber specimens show the first Type I genotype, and 10 specimens show the Type II pattern. 4. Discussion 4.1. Phylogenetic tree of G. ruber Darling et al. (1999) presented the phylogenetic relationships of G. ruber, including G. conglobatus. In this study, we add our 12 new sequences and morphological information to their results. Molecular phylogenetic analysis of foraminiferal SSU rDNA demonstrated that G. ruber s.s. is genetically highly distinct from G. ruber s.l. (Fig. 2), because the genetic distance
of SSU rDNA between Type I and Type II is 23–24%, whereas Type II differs from G. conglobatus by 11–12% (Table 2). Darling et al. (1999) showed that pink specimens of G. ruber, collected from the Caribbean, are divergent from another G. ruber cluster, including the white variation from the Coral Sea, with an average genetic distance of 5.5%. Moreover, G. ruber specimens from the Southern California Bight were highly divergent from those in the Caribbean and Coral Sea clusters, with a mean genetic distance of 10.8%, and the Southern California Bight cluster was a sister group of G. conglobatus (Darling et al., 1999). Therefore, we speculate that the specimen collected from the Coral Sea could be G. ruber s.s. and that from the Southern California Bight is G. ruber s.l. If true, this finding suggests that G. ruber s.s. inhabits both the Pacific and Atlantic Oceans. In contrast, G. ruber s.l. is known at present to occur only in the Pacific Ocean (Fig. 2). 4.2. Horizontal distribution of two genotypes of G. ruber (white) in the central Pacific Ocean Both G. ruber (white) genotypes often occurred at the same sites, except for at our station 2 (Fig. 1 and Table 1). Although the number of individuals that we sampled is not sufficient for
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Table 2 Pairwise genetic distances, as a percentage difference of nucleotides, between Globigerinoides ruber (Types Ia–c, pink variation, and Type II) and Globigerinoides conglobatus
1 2 3 4 5 6 7 8
Type Ia Type Ib Type Ic Pink Type II conglo. sacculifer universa
1
2
3
4
5
6
7
8
0.6–1.0 1.5–1.7 3.7–4.2 9.7–10.4 22.7–23.9 20.7–21.5 36.7–37.0 33.1–33.4
0 3.7–3.8 10.4–10.6 23.2–23.7 21.3–21.4 37.0–37.3 32.4–32.6
0–0.5 9.5–10.2 22.9–23.5 20.7–20.9 35.9–36.3 32.6–33.0
0.3 23.8–24.6 21.0–21.6 35.9–36.3 32.9–33.2
0–0.8 11.1–11.9 34.6–35.5 33.3–34.0
1.6 34.4–34.9 32.0–32.3
0.2–0.8 30.7–31.0
0–0.2
Globigerinoides sacculifer and Orbulina universa are used as outgroup species.
a statistical discussion, we could carry out the continuous pump sampling along the cruise route (up to ~50 km per sample) (Fig 1 and Table 1). It is known that different genotypes of Globorotalia truncatulinoides can adapt to particular hydrographic conditions (de Vargas et al., 2001). In this study, station 2 had the most oligotrophic surface conditions (Fig. 4; McClain et al., 2004), and only Type I (G. ruber s.s.) was found there in high numbers (Table 1). The vertical distribution of G. ruber morphotypes in the modern ocean (Kuroyanagi and Kawahata, 2004) suggests that the different habitats of each can be determined by different controlling factors; G. ruber s.s. distributions are mainly dependent upon light intensity and G. ruber s.l. distributions on food availability. Therefore, low surface chlorophyll-a content could prevent Type II (G. ruber s.l.) from dwelling in the surface ocean (~ 0–5 m water depth) at station 2. Differences in oxygen isotopic ratios between the two morphotypes also seem to be related to thermal stratification of the water column (Kawahata, 2005). Thus, strong stratification (low concentration of surface chlorophyll-a) may result in the high numbers of Type I (G. ruber s.s.) at the surface, much like at station 2, and would lead to a large difference in the isotopic record from the two genotypes. 4.3. Paleo-environmental implications of two genotypes of G. ruber The differences in isotopic records between the two genotypes can be applied to paleoceanography. Sediment samples have demonstrated the differences in the isotopic record between G. ruber s.s. and s.l., and the mean difference in δ18O values of 0.21 ± 0.21‰ is likely due to their different habitats (Wang, 2000). Sediment trap experiments investigating the seasonal variation of the oxygen isotopic composition of the two morphotypes in the subtropical North Pacific showed that the mean difference was 0.25‰ from August to October under stratified conditions, corresponding to a difference of 1 °C in water temperature (the temperature difference between water at the surface and at 30–50 m) (Kawahata, 2005). Some recent studies have focused on the water column conditions in the tropical region related to the estimation of El Niño- or La Niña-like conditions (e.g., de Garidel-Thoron et al., 2005; Rickaby and Halloran, 2005). Because the δ18O values for G. ruber have a glacial–interglacial variation that ranges from approximately 0.6‰ (from 1.75 to 0.85 Ma) to 1.4‰ (terminations I, II, and IV) in the western equatorial Pacific Ocean (de Garidel-Thoron et al., 2005), the
interpretation of isotopic data from G. ruber genotypes may not require extensive modification. However, the two genotypes of G. ruber can be very useful tools in reconstructing the detailed thermal stratification structure in the tropical and subtropical oceans with high-resolution samples, especially in the Holocene. The chemical composition of foraminiferal tests of the two genotypes (e.g., the Mg/Ca ratio) might also provide insights into ocean stratification, although there is a little information currently available for this application (Steinke et al., 2005). If more information can be extracted from the chemical and isotopic data from the two genotypes, more detailed reconstructions of past changes in stratification and climate in tropical areas could be achieved. 5. Conclusion We have examined genetic variation in the SSU rDNA sequences of a representative tropical–subtropical species of foraminifera, G. ruber, and found four genotypes within the G. ruber white variation, one of which is a sister group of G. conglobatus whereas the other three are sisters to the G. ruber pink variation. Moreover, two major genotypes (Types I and II) are identical to morphotypes “s.s.” and “s.l.”, respectively. Because the genetic evidence corroborates the existence of two morphotypes, the differences in the isotopic record of the
Fig. 3. Restriction patterns resulting from restriction fragment length polymorphism (RFLP) analysis using two restriction enzymes, NspV and AluI. Two RFLP samples, #11 and #17, show the Type I pattern; #32 and #39 represent the Type II pattern.
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Fig. 4. Locations of the sampling stations (stations 1–6) along with surface chlorophyll-a concentrations. Dark color indicates low concentration and light color indicates high (McClain et al., 2004).
two genotypes can be applied to a reconstruction of historic thermal stratification using information about their different habitation depths. Acknowledgments We thank E. Thomas and an anonymous reviewer for their helpful comments to improve the manuscript. We express our sincere appreciation to the captain and crew of the R/V Mirai and our colleagues who were on board during the cruise. We are also grateful to M. Kawato (JAMSTEC) for his support in sequencing and Y. Ujiie (ORI) and the staff members at ORI, JAMSTEC, and AIST for their helpful discussions and suggestions. Appendix A. (Taxonomic list) The taxonomy follows Parker (1962) and Saito et al. (1981). Globigerinoides cyclostomus (Galloway and Wissler) = Globigerina cyclostoma Galloway and Wissler, 1927, p42, pl. 7, figs. 8–9. Globigerinoides elongatus (d'Orbigny) = Globigerina elongata d'Orbigny, 1826, p. 277, list no. 4, (no figs.). Globigerinoides pyramidalis (van den Broeck) = Globigerina bulloides d'Orbigny var. rubra d'Orbigny subvar. pyramidalis Van den Broeck, 1876, p. 127, pl. 3, figs. 9–10. Globigerinoides ruber (d'Orbigny)=Globigerina rubra d'Orbigny, 1839, pp. 82–83, (plates published separately), pl. 4, figs. 12–14. References Bijma, J., Faber Jr., W.W., Hemleben, Ch., 1990. Temperature and salinity limits for growth and survival of some planktonic foraminifers in laboratory cultures. J. Foraminifer. Res. 20, 95–116. Darling, K.F., Kroon, D., Wade, C.M., Leigh Brown, A.J., 1996. Molecular phylogeny of the planktic foraminifera. J. Foraminiferal Res. 26, 324–330.
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