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Marine Micropaleontology 66 (2008) 233–246 www.elsevier.com/locate/marmicro

Molecular phylogeny of Rotaliida (Foraminifera) based on complete small subunit rDNA sequences ⁎ Magali Schweizer a,b, , Jan Pawlowski c, Tanja J. Kouwenhoven a, Jackie Guiard c, Bert van der Zwaan a,d

a Department of Earth Sciences, Utrecht University, The Netherlands b Geological Institute, ETH Zurich, Switzerland c Department of Zoology and Animal Biology, University of Geneva, Switzerland d Department of Biogeology, Radboud University Nijmegen, The Netherlands

Received 18 May 2007; received in revised form 8 October 2007; accepted 9 October 2007

Abstract

The traditional morphology-based classification of Rotaliida was recently challenged by molecular phylogenetic studies based on partial small subunit (SSU) rDNA sequences. These studies revealed some unexpected groupings of rotaliid genera. However, the support for the new clades was rather weak, mainly because of the limited length of the analysed fragment. In order to improve the resolution of the phylogeny of the rotaliids, 26 new complete SSU rDNA sequences have been obtained. Phylogenetic analyses of these data, together with seven sequences obtained previously, confirm with stronger statistical support the presence of three major clades among the Rotaliida. The first clade comprises members of the families Uvigerinidae, Cassidulinidae and Bolivinidae. The second clade includes all analysed Discorbidae, Rosalinidae, Planulinidae, Planorbulinidae, Rotaliidae, Elphidiidae, Nummulitidae and one of the Nonionidae. Finally, the third clade comprises the Cibicididae, Pseudoparreliidae, Oridorsalidae, Stainforthiidae, Buliminidae and part of the Nonionidae. The clades 1 and 3 are strongly supported by analyses of the complete SSU rDNA, while the monophyly of clade 2 is less certain, probably due to the rapid evolutionary rates of some lineages included in this clade. These results clearly contradict the classical separation of rotaliid foraminifera into two orders: Rotaliida and Buliminida. Relatively good agreement has been found between molecular data and the morphological definition of the families for which more than one genus was sequenced. However, larger taxon sampling will be necessary for a better definition of the three major clades. © 2007 Elsevier B.V. All rights reserved.

Keywords: Benthic foraminifera; Rotaliida; Molecular phylogeny; Classification; SSU rDNA

1. Introduction the chemical composition of their fossil tests are exten- sively used as proxies for environmental changes in the Benthic foraminifera are important elements of the geological past (Van der Zwaan et al., 1999). Together meiofaunal community; their abundance data as well as with their potential use for monitoring recent environments, this has led to an increasing interest in their ⁎ Corresponding author. Geological Institute, ETH Zurich, biological functioning. Since the second half of the last Switzerland. century research has focused on the use of foraminifera E-mail address: [email protected] (M. Schweizer). for environmental and pollution issues (e.g. Bandy et al.,

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1964; Vénec-Peyré, 1981; Van der Zwaan and Jorissen, perforate foraminifera in the suborder Rotaliina which 1991; Alve, 1995; Mojtahid et al., 2006). Proxy re- included ten superfamilies (i.e. Nodosariacea, Bulimi- search, relevant to applications in the fossil record nacea, Discorbacea, Spirillinacea, Rotaliacea, Globiger- addresses, for instance, the response to environmental inacea, Orbitoidacea, Cassidulinacea, Carterinacea and factors such as organic carbon flux (e.g. Smart et al., Robertinacea) distinguished on the basis of the wall 1994; Murray, 2001; Ernst et al., 2005). The preserva- microstructure as well as coiling and aperture character- tion of foraminiferal tests is important in this type of istics. In 1981, Haynes retained the wall structure as the research (e.g. Berger, 1979; Mackensen and Douglas, primary basis of subdivision; however, more emphasis 1989; Murray, 1989), and so are the vital effects as- was given to the shape of the aperture. In his clas- sociated with incorporation into calcareous tests of sification, the superfamilies were raised to orders, and elements (e.g. Bentov and Erez, 2006) and stable iso- the Nodosariida, Robertinida, Buliminida and Globiger- topes (e.g. Schmiedl et al., 2004). In addition, research inida were separated from the Rotaliida. The Buliminida into the role of benthic foraminifera in biogeochemical comprised the hyaline perforate foraminifers with a cycling has recently resulted in the discovery that some toothplate and included the following superfamilies: species of foraminifera are able to perform complete Buliminacea, Bolivinitacea and Cassidulinacea, where- denitrification (Risgaard-Petersen et al., 2006)andin as the Rotaliida contained the Spirillinacea, Discorba- doing so could play an important, though as yet not cea, Asterigerinacea and Orbitoidacea (Haynes, 1981). quantified, role in the marine nitrogen cycle. In view of In their monumental work, Loeblich and Tappan foraminiferal evolution this raises the question whether (1988) defined supplementary suborders for the hyaline traits like nitrate reduction developed separately in perforate calcareous foraminifers in addition to their different clades, and when. Finally, cryptic speciation former classification (1964): Involutinina, Spirillinina, occurs in planktic (e.g., Darling et al., 2006) as well as in Carterinina, Silicoloculinina, Lagenina, Robertinina and benthic foraminifera (e.g. Hayward et al., 2002); Globigerinina. The suborder Rotaliina was divided into although in Uvigerina the opposite has been described 24 superfamilies; the criteria used were the number of recently (Schweizer et al., 2005). Either way, the use of chambers, the presence or absence of perforations, canals foraminifera as (paleo-) environmental proxies needs to and cavities in the test, and the aperture. In 1992, to solve be reconsidered. In view of these developments, phylo- some of the inconsistencies reported by Haynes (1981, genetic relationships between foraminifera, which were 1990), Loeblich and Tappan raised the foraminifera from previously predominantly based on morphology, need a an order to a class (the foraminiferal suborders were thus re-evaluation. given order status) and recognized the order Buliminida The order Rotaliida comprises calcareous hyaline Fursenko, 1958. In the most recent classification, Sen perforate species and represents one third of the extant, Gupta (2002) followed the classification of Loeblich and described genera of foraminifers (based on data from Tappan (1992) with slight modifications (Fig. 1). Decrouez, 1989). The taxon was introduced by Delage According to this view, Buliminida are distinguished and Hérouard in 1896 as the suborder Rotalidae from Rotaliida by the presence of a toothplate, a loop- (reference in Loeblich and Tappan, 1964). The hyaline shaped aperture and a high trochospiral coil. foraminifers were recognized earlier as a group by Since the mid-90s, the molecular approach has shed Williamson (1858), who was the first author to use the new light on foraminiferal phylogeny. The first molecular wall composition in foraminiferal classification (Cifelli results showed a clade combining the Textulariida and the and Richardson, 1990). However, this distinction was Rotaliida (Pawlowski et al., 1997; Pawlowski, 2000). not always adhered to in early classifications. For This mixed clade was explained either by a radiation example, Reuss (1861), Carpenter et al. (1862), Brady occurring in a relatively short time, or by slow rates of (1884), Rhumbler (1895) and Cushman (1922) placed evolution of both groups (Pawlowski et al., 1997, 2003). arenaceous textulariids and hyaline bolivinids (some- More recent analyses were able to separate the rotaliids times with buliminids and cassidulinids) within the and textulariids, albeit without strong statistical support same group; this was also the case in some more recent (Holzmann et al., 2003; Ertan et al., 2004; Bowser et al., publications (Hofker, 1951, 1956; Mikhalevich and 2006). Within the rotaliids, Holzmann et al. (2003) Debenay, 2001). examined the links between the Nummulitidae and seven The classifications of Cushman (1928) and Galloway other rotaliid families, Ertan et al. (2004) explored the (1933) separated the hyaline genera from other relationships of eleven genera of Rotallida, whereas foraminifera and placed them in several families. Schweizer et al. (2005) investigated the position of In 1964, Loeblich and Tappan grouped all hyaline uvigerinids. Author's personal copy

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Fig. 1. Diagram showing the taxonomic positions of the genera studied in this paper and in Schweizer et al. (2005) with the classifications of Sen Gupta (2002). The first column represents the 22 extant superfamilies with the ones represented in phylogenetic analyses in bold. The second column represents the studied families and the third one the studied genera.

All these studies were based on a fragment of about and Schweizer et al. (2005). For this reason, we decided 1000 base pairs (bp) long, and situated at the 3′ end of to sequence the complete SSU of selected rotaliid spe- the SSU rDNA. The phylogenetic information contained cies which would result in a three-fold increase in the in this fragment seems to be insufficient to resolve number of analysed sites. Therefore, 26 new complete rotaliid phylogeny, as shown by low statistical support sequences belonging to 12 of the 22 extant superfamilies for the deep nodes in the analyses of Ertan et al. (2004) of Rotaliida (according to the classification of Sen Author's personal copy

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Gupta, 2002) were obtained. These new data, along with imaged and extracted as explained in Schweizer et al. seven previously obtained sequences, were analysed (2005). together to investigate the phylogeny of the rotaliids. 2.2. DNA extraction, PCR amplification, cloning and 2. Materials and methods sequencing

2.1. Collection of the samples Extraction of DNA from single or multiple specimens was done with DOC lysis buffer, CTAB or Live specimens of rotaliids were collected during guanidine buffer (Pawlowski, 2000), and occasionally several different expeditions between 1995 and 2006 with DNeasy Plant Mini Kit (Qiagen) for multi- (see Table 1 for details). Sediment samples were either specimen samples. The SSU was amplified in three taken by hand with a scraper at the shallow sites or steps with fragments between 800 and 1600 bp long. collected by boxcoring and multicoring at the deeper The primers sA10 and s13 were used to amplify the 5′ sites. The top few centimetres of sediment were col- end fragment (sA-s6), the primers s6F and s17 for the lected from the cores with a spoon and immediately middle fragment (s6-s14) and the primers s14F3 and sB sieved using water from the same environment as the for the 3′ end fragment (s14-sB). A reamplification of sampling site (fractions 500/250/125 μm). The different the PCR products (nested PCR) was often necessary and fractions were stored at temperatures close to that of was performed using the following primers: sA10-s6rA the collection site. Specimens were selected alive, for the sA-s6 fragment, s6F-s15rot for the s6-s14

Table 1 List of new complete and partial SSU sequences with the origin of DNA samples, the SSU length (nt= nucleotides) and the accession numbers Superfamily Species Locality DNA isolate SSU length Access number Cassidulinacea Islandiella sp. Svalbard, Norway 2643 3278 nt DQ408638 Cassidulinoides parkerianus Terranova Bay, Antarctica 3924 3348 nt DQ408639 Turrilinacea Stainforthia fusiformis Oslo Fjord, Norway 3965 sA-s6 DQ205387 Dunstaffnage, Scotland 3979 s6-s14 DQ452714 Buliminacea Bulimina marginata Oslo Fjord, Norway 3599 3462 nt DQ408646 Uvigerina phlegeri Setúbal Canyon, Portgual U239 3579 nt DQ408641 Uvigerina earlandi McMurdo Sound, Antarctica 2187 3571 nt DQ408640 Uvigerina peregrina Oslo Fjord, Norway U27 3517 nt DQ408642 Discorbacea Discorbis rosea Florida, USA 753 3507 nt DQ408644 Rosalina sp. Culture 3675 3402 nt DQ408643 Discorbinellacea Epistominella vitrea Cape Evans, Antartica 2060 3463 nt DQ408647 Planorbulinacea Hyalinea balthica Oslo Fjord, Norway 3604 3631 nt DQ408645 Cibicides pachyderma Nazaré Canyon, Portugal C86 3409 nt DQ408652 Cibicides pachyderma Nazaré Canyon, Portugal C196 3431 nt DQ408653 Cibicides lobatulus Oslo Fjord, Norway C24 3526 nt DQ408649 Cibicides lobatulus Skagerrak, Sweden C120 3632 nt DQ408650 Cibicides lobatulus Marseille, France C170 3596 nt DQ408648 Cibicides sp. North Atlantic 2524 3536 nt DQ408651 Planorbulinella sp. Elat, Israel 358 3365 nt DQ452687 Nonionacea Melonis pompilioides Skagerrak, Sweden 1400 3556 nt DQ408657 Pullenia subcarinata McMurdo Sound, Antarctica 1148 3471 nt DQ408656 Pullenia subcarinata McMurdo Sound, Antarctica 1850 3472 nt DQ408655 Nonionella labradorica Svalbard, Norway 4836 sA-s6 EF534076 Skagerrak, Sweden 3966 s6-s14 DQ452713 Haynesina germanica Den Oever, The Netherlands 6008 3027 nt EF534074 Chilostomellacea Oridorsalis umbonatus Fram Strait, Arctic 5410 3373 nt EF534075 Rotaliacea Elphidium williamsoni Den Oever, The Netherlands 6009 2772 nt EF534073 Ammonia sp. 3015 nt EF534072 S. fusiformis and N. labradorica SSU sequences are hybrids of two genetically close samples. Concerning the uvigerinids, it was shown earlier (Schweizer et al., 2005; Schweizer, 2006) that the two species attributed to the genera Rectuvigerina (R. phlegeri) and Trifarina (T. earlandi) are in fact closer to Uvigerina peregrina than U. mediterranea and U. elongatastriata. It was therefore decided to give all these species the same genus name (see discussion in Schweizer, 2006, Ch. 6). Author's personal copy

M. Schweizer et al. / Marine Micropaleontology 66 (2008) 233–246 237 fragment and s14F1-sB for the s14-sB fragment. The from the EMBL/GenBank database have been added positions and sequences of the primers are summarized (deposited by M. Holzmann (unpublished) and Paw- in Fig. 2. The PCR conditions were the following: total lowski et al. (1996, 1999)). Out-group sequences have volume of 50 μl, denaturation at 94 °C for 30 s, been chosen among textulariids, the closest relatives of annealing for 30 s at 50 °C for the amplification and at rotaliids (Bowser et al., 2006). Presently, there are only 52 °C for the reamplification, extension at 72 °C for two sequences available for the complete SSU and they 2 min with 40 cycles for the amplification and 35 cycles are used in our analyses. Sequences were aligned manual- for the reamplification, final elongation for 5 min at ly by using Seaview software (Galtier et al., 1996), and the 72 °C. The positive PCR products were purified using regions which were too variable to be properly aligned High Pure PCR Purification Kit (Roche Diagnostics). A were removed. From an alignment of 5565 sites, 1766 few amplifications of the s14-sB fragment were sites were removed to obtain a final alignment of 3799 sequenced directly; all the others were cloned. Purified sites, 1745 of them containing no gap. products were ligated in the pGEM-T Vector system The GTR+I+Γ model was chosen by Modeltest (Promega) or the Topo Cloning vector (Invitro Gene), (Posada and Crandall, 1998). However, we preferred to and cloned using ultracompetent cells XL2-Blue MRF' also use another model (HKY +I+Γ) to make compar- (Stratagene). Sequencing reactions were prepared using isons, as Modeltest tends to favour complex models an ABI-PRISM Big Dye Terminator Cycle Sequencing (Kelchner and Thomas, 2007). Therefore, the maximum Kit and analysed with an ABI-377 DNA sequencer or an likelihood (ML) trees were obtained using PhyML 2.4.4 ABI-PRISM 3100 (Applied Biosystems), all according (Guidon and Gascuel, 2003) with the HKY (Hasegawa, to the manufacturer's instructions. Kishino, Yano) model (Hasegawa et al., 1985) allowing transitions and transversions to have potentially differ- 2.3. Phylogenetic analysis ent rates, and the GTR (General Time Reversible) model allowing all types of substitution to have different The new sequences presented here were deposited in rates (Lanave et al., 1984; Rodriguez et al., 1990). To the EMBL/GenBank Nucleotide Sequence Database; correct for the among-site rate variations, the proportion their accession numbers are reported in Table 1.To of invariable sites (I) and the alpha parameter of gam- extend the data set, other complete SSU sequences ma distribution (Γ), with eight rate categories, were

Fig. 2. Localization and sequences of the primers used to amplify the three fragments of the complete SSU rDNA. Author's personal copy

238 M. Schweizer et al. / Marine Micropaleontology 66 (2008) 233–246 estimated by the program and taken into account in all GTR+I+Γ model. Two independent analyses were analyses. Non-parametric bootstrapping (BS) (Felsen- performed at the same time with four simultaneous stein, 1985) was performed with 100 replicates to assess chains (one cold and three heated) run for 1,000,000 the reliability of internal branches. generations, and sampled every 100 generations with The Bayesian analysis was performed with MrBayes 2500 of the initial trees discarded as burn-in. The posterior 3.1.2 (Huelsenbeck and Ronquist, 2001), using the probabilities (PP) were calculated at the same time.

Fig. 3. Localization of the inserts found in Rotaliida (in black) compared to the schematised SSU secondary structure of Chlamydomonas reinhardtii (in grey, redrawn from Wuyts et al., 2004) with the names of the helices (in grey, Wuyts et al., 2001). F1–F6 are inserts found only in foraminifers, whereas V1–V9 are variable regions recognized in all eukaryotes (Wuyts et al., 2000). The minimum and maximum numbers of nucleotides (=nt) observed in rotaliids for each insert are indicated, with the number found in C. reinhardtii between brackets for the variable regions (V2–V9). Author's personal copy

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3. Results The GC content of the studied rotaliids is between 37 and 46%, with the lowest value observed in Cassiduli- 3.1. Sequence data noides and the highest one in Ammonia. These values are higher than those recorded for Miliolida and The 26 complete SSU sequences presented here have Astrorhizida (29–32%), but comparable to the value a length ranging from 2772 (Elphidium) to 3768 of 45% found in Globigerinida (Pawlowski, 2000). nucleotides (Nonionella); the four shortest sequences The typical insertions observed in other foraminifera of which belong to the fast evolving taxa Elphidium, (e.g. Pawlowski et al., 1999) are also present in the Ammonia and Haynesina (see Table 1 for details). rotaliid sequences (Fig. 3). Some of these insertions are

Fig. 4. Phylogeny of Rotaliida inferred from complete SSU rDNA sequences using the ML method (HKY+I+Γ). Tree is rooted on textulariids. Bootstrap values for ML analyses (HKY and GTR) and PP values for Bayesian analysis are indicated at the nodes. Species names written in bold designate new sequences, the others were taken from GenBank (accession numbers are added). Author's personal copy

240 M. Schweizer et al. / Marine Micropaleontology 66 (2008) 233–246 in regions of the SSU which show variations among Clade 3 includes Nonionella, Bulimina, Stainforthia, eukaryotes (V1–V9), whereas others are only present in Epistominella, Oridorsalis, Pullenia, Melonis and Ci- certain eukaryotes (8e/1, 23e/15–23e/17, 45e/1), or bicides, and combines species with elongate and exclusively in foraminifers (F1–F6). The insertions F4– lenticular tests. This clade has a high statistical support F6 belong to the s14-sB fragment and have been (ML/HKY+ML/GTR: 99% BS, BI: 1.00 PP). A first described elsewhere with different names (e.g. Darling subgroup comprises elongate triserial tests with a loop- et al., 1997 (regions F1–F3); de Vargas et al., 1997 shaped aperture (Bulimina, Stainforthia)andlow (regions I, II and V)). trochospiral ones with an interiomarginal arch-like aperture (Nonionella, Epistominella). The statistical 3.2. Phylogenetic analyses support of this subgroup is at least 83% BS. A second subgroup includes species with a planispiral (Pullenia, The phylogenetic analysis of the 26 complete SSU Melonis) or low trochospiral (Oridorsalis, Cibicides) sequences of rotaliid foraminifera and the seven test and a slit-like interiomarginal aperture. The support sequences from GenBank is presented in Fig. 4.Two of this subgroup is even better with 97% BS or higher. textulariids (Trochammina sp. and Eggerelloides scabrum) are used as an outgroup. Analyses performed with 4. Discussion PhyML (ML/HKY and ML/GTR) and MrBayes (GTR) give exactly the same topology. Three major clades 4.1. Ribosomal RNA secondary structure and emerge within the Rotaliida. foraminiferal insertions Clade 1 comprises Bolivina, Cassidulinoides, Islan- diella and Uvigerina. These genera commonly have an The secondary structure of the s14-sB fragment has elongate test (Islandiella being an exception) and an been studied by Ertan et al. (2004) and Habura et al. aperture with a toothplate. The statistical support is 89% (2004). There is evidence that the three foraminiferal BS (ML/HKY) or more (96% BS for ML/GTR and insertions found in this fragment (here referred to as F4, 1.00 PP for Bayesian inference (BI)). F5 and F6, see Fig. 3) are not introns and are retained in Clade 2 includes Rosalina, Discorbis, Planorbuli- the final rRNA inside the ribosome (Habura et al., nella, Hyalinea, Pararotalia, Cycloclypeus, Heteroste- 2004). However, at the same time it has been suggested gina, Operculina, Nummulites, Ammonia, Elphidium by Pawlowski et al. (1996) that about 1000 nt are re- and Haynesina, genera with a trochospiral to planispiral moved from the SSU of Ammonia during the maturation test. The statistical support of this clade is 77% BS (ML/ process of RNA. Ertan et al. (2004) have used the HKY) or higher (ML/GTR: 83% BS, BI: 1.00 PP). This variable regions (here referred to as V7–V9, see Fig. 3) second clade can be divided in three subgroups. The and the foraminiferal insertions (see above) to improve first subgroup comprises species with a trochospiral to the alignment and for taxonomic purposes, but it was planispiral test and an interiomarginal arch-shaped noticed that these regions were not always diagnostic aperture, sometimes with the presence of a second and that convergent evolution could occur. identical aperture (Planorbulinella) or secondary In the data presented here, representing the complete openings (Discorbis). Genera included in this first SSU, half of the insertions present similar patterns in subgroup are Rosalina, Discorbis, Planorbulinella rotaliids, textulariids (Trochammina sp. and Eggerel- and Hyalinea. The statistical support is rather good loides scabrum) and globigerinids (Globorotalia inflata, (88% BS or higher and 1.00 PP). The second subgroup G. hirsuta, Neogloboquadrina dutertrei) showing that includes Pararotalia, a genus with a low trochospiral these groups are closely related. test and an imperforate toothplate and the Nummuli- If all the foraminiferal insertions are retained in the tidae, a family with planispiral tests and a complex rRNA, as could be the case for F4–F6 (Habura et al., canal system. This subgroup is less well supported 2004), there are certainly evolutionary constraints to statistically: 77% for ML/HKY, 83% for ML/GTR and modelling their DNA sequences. Therefore, the use of 0.93 PP. The third subgroup is composed of three these insertions for taxonomic purposes, as was initiated shallow-water and fast evolving genera: Ammonia, by Ertan et al. (2004), is promising. However, con- Elphidium and Haynesina. The test is a low trochos- structing the secondary structure of foraminiferal rRNA piral (Ammonia)orplanispiralcoil(Elphidium and is rather difficult, because of all the original insertions Haynesina) with primary and secondary apertures. and the rate heterogeneity, and therefore any conclu- This is the best-supported subgroup of clade 2 (95% sions based on rRNA structure should be treated with BS or higher). caution. Author's personal copy

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4.2. Complete versus partial SSU phylogeny sented in the s14-sB analysis. Clade 3 includes the same two subgroups in both analyses, but with slightly dif- A question hotly debated is whether adding more ferent topologies. Bulimina and Nonionella were also taxa, or more sites will improve phylogenetic accuracy closely related in the partial SSU analysis. However, (e.g. Hedtke et al., 2006 and references therein). It has Virgulina and Virgulinella, two taxa not represented in been shown that increasing the taxon sampling could the complete SSU phylogeny, branched closer to No- improve the accuracy of phylogenetic inferences, nionella. Epistominella and Stainforthia were sister- particularly in cases of whole genomes sequenced for groups in the partial SSU phylogeny, albeit with a low a few taxa (Geuten et al., 2007 and references therein). statistical support (31% BS with PhyML and HKY). In In the case of foraminifers, however, the situation is the second subgroup, Chilostomella (not included in the rather different. For the moment, there is a wide taxon present analysis) was the closest relative of Pullenia sampling on a portion of the SSU and we are therefore in with a reasonable support (74% BS with PhyML and the situation described by Hillis et al. (2003), where a HKY), and Cibicides was branching with that subgroup few characters are obtained from the taxa analysed. The instead of with Melonis, albeit with no statistical authors argue that in this case, it is better to add more support. characters per taxon and we followed this suggestion by The global topology of the second clade also sequencing the complete SSU for some taxa, for which appeared in an analysis performed by Holzmann et al. the partial SSU sequences were obtained previously. (2003) on the s14-sB fragment and focusing on The 24 rotaliid genera present in the tree based on the Nummulitidae. The Nummulitidae were equally well complete SSU (Fig. 4) represent 65% of the rotaliid supported (87% BS or higher) and branched together genera for which partial SSU rDNA sequences were with Pararotalia and the Calcarinidae, despite a low deposited in GenBank. The general topology of this tree support with the ML analysis (51% BS). The general is relatively congruent with previous studies based on topology of the other subgroup was similar to the one partial SSU sequences (Holzmann et al., 2003; Ertan observed in the present analysis. However, the Cassi- et al., 2004; Schweizer et al., 2005). However, important dulinidae and Bolivinidae (belonging to the first clade in differences exist in relations between higher taxa and the this study) appeared as a sister-group of the sub-clade support for particular groupings. Planorbulina–Rosalina,withHyalinea less closely The three clades found in the complete SSU tree were related. Moreover, the branching of Cassidulinidae– also identified in our previous analysis based on the s14- Bolivinidae inside this subgroup in Holzmann et al. sB fragment (Schweizer et al., 2005, Fig. 7). However, (2003) was not statistically supported. the statistical support of the lower nodes is particularly The main discrepancy between our present study and improved with the analysis of the complete SSU partial SSU analysis of Ertan et al. (2004) concerns the compared to our former analysis, where the clade 1 relations between members of our clade 3. In Fig. 7 of branched as a sister-group of clade 3 instead of clade 2, Ertan et al. (2004), Bulimina and Virgulinella, belonging albeit the relations between these clades were not to the first subgroup in our clade 3 branched as a sister- supported (33% for the grouping of clades 1 and 3 in the group of taxa belonging to our clade 1, whereas Chi- partial SSU). The structure of particular clades is similar lostomella and Melonis formed a clade sister to all the both in the present analyses and those in Schweizer et al. other rotaliid sequences. These relations were inter- (2005). In clade 1, the relations between Bolivina, preted as evidence for partition between the orders Cassidulinidae and Uvigerinidae are the same, but this Rotaliida and Buliminida, however, the support of these clade comprised also Globobulimina in analyses of the two deeper nodes was very weak (respectively 0.19 and partial SSU. In clade 2 the two subgroups are present 0.38 PP). Despite this discrepancy, the relations between with similar topology, except for the first subgroup, other rotaliids were quite congruent with the present where Hyalinea and Planorbulinella grouped together study. The study of Ertan et al. (2004) confirmed with in the partial SSU phylogeny with a good support (75% good support (0.88 PP) the grouping between members of BS with PhyML and HKY). The second subgroup our clade 2 (Rosalina, Haynesina, Ammonia, Elphidium). comprises the Nummulitidae, which always forms a Representatives of clade 1 (Bolivina, Uvigerina, Globo- homogeneous and highly supported clade (N90% BS) bulimina) equally branched together with a good sup- and Pararotalia, which in the partial SSU phylogeny port (0.80 PP), but with a different topology than in branched with a high support close to the Calcarinidae (a Schweizer et al. (2005): Globobulimina branched closer family not included in the complete SSU analysis). to Bolivina instead of Uvigerina in the study of Ertan et al. Ammonia, Elphidium and Haynesina were not repre- (2004). Author's personal copy

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4.3. Molecular phylogeny and morphology-based classifications (Haynes, 1981; Loeblich and Tappan, classification of Rotaliida 1992; Sen Gupta, 2002). Although the two main groups observed in the phylogenetic analyses of the complete Because there is a good congruence between the trees SSU (clade 1 and clades 2+3, see Fig. 4)roughly based on complete and partial SSU sequences, an analysis correspond to these orders, several buliminid genera has been performed including the complete SSU se- (Bulimina, Stainforthia, Virgulina, Virgulinella), branch quences from the present study and the partial sequences within clade 3 together with some of the rotaliids. The (s14F-sB) from Schweizer et al. (2005,Fig.7)(Fig. 5). position of these genera questions the pertinence of the This recapitulative tree is compared to the mainstream criteria used to separate the two orders. Apparently, the morphological classifications, with the one of Sen Gupta presence of a toothplate, which is the main feature to (2002) as a summary (Fig. 1). distinguish the two orders, is not as important as was All genera represented by more than one species previously thought (e.g., Hofker, 1951, 1956; Mikhale- were monophyletic, except Rosalina (Fig. 5). Four vich and Debenay, 2001) and was already questioned by morphologically defined families for which different Haynes (1981, p. 24). Moreover, the fact that Cassidu- taxa could be sampled, i.e. Cassidulinidae, Uvigerini- lina, a genus without a toothplate, is traditionally placed in dae, Calcarinidae and Nummulitidae, received a high the Cassidulinidae (position confirmed by the molecular statistical support in the phylogenetic analyses. On the analyses, see Schweizer et al., 2005), a family where other other hand, the Nonionidae (Melonis, Pullenia, Non- genera represented in this study do possess a toothplate, ionella, Haynesina), the Rotaliidae (Pararotalia, Am- shows that the taxonomic value of this character is monia) and the Buliminidae (Bulimina, Globobulimina) questionable. Another representative feature of the appeared polyphyletic, questioning the morphological Buliminida is the high trochospiral coil. However, this basis of their grouping and asking for further, preferably characterizes only 70% of the members of this order integrated morphological and genetic studies. (Haynes, 1981). At a higher taxonomic level, the superfamilies In view of (paleo-) ecological applications of Buliminacea, Nonionacea and Planorbulinacea appear foraminifera it is interesting to note that Nonionella, polyphyletic. The polyphyly of the first two superfamilies which appears closely related to Bulimina, includes is already clear at the family level. The Planorbulinacea, species that are able to denitrify nitrate, as well as which include Hyalinea, Planorbulinella, Planorbulina, Stainforthia and Globobulimina (Risgaard-Petersen and Cibicides, are split between clades 2 and 3 (Fig. 5). et al., 2006), a process which was formerly recognized This is quite surprising given that Hyalinea has always only in prokaryotes. The results presented here indicate been grouped with Cibicides (e.g. Loeblich and Tappan, that the denitrification pathway exists in at least two 1964, 1988; Haynes, 1981) and that Planorbulina was rotaliid clades: clade 1 with Globobulimina (some thought to be a stage in the life cycle of Cibicides Uvigerina and Bolivina species are also possible (Nyholm, 1961). candidates but further tests need to confirm this (N. Some clades observed in the molecular analyses were Risgaard-Petersen and E. Geslin, pers. comm.)) and proposed by Haynes (1981) based on morphological clade 3 with the pair Nonionella–Stainforthia. studies. The genera Pullenia and Chilostomella were classified in the family Chilostomellidae and the genera 5. Conclusions and perspectives Bulimina, Stainforthia, Virgulina and Virgulinella in the family Buliminidae, superfamily Buliminacea (Haynes, Currently, our results based on complete and partial 1981, 1990). Haynes (1981) also included Epistominella SSU rDNA sequences favour the existence of a unique in the Buliminacea, but in a different family (Turrilinidae). order Rotaliida. This order is subdivided into three clades Our results partially corroborate Haynes' subdivision, which could be considered as suborders. However, further although other Buliminacea (Uvigerina and possibly molecular studies are needed to confirm this partition and Globobulimina) branch well separated from this group. to better define the three clades morphologically. Our data do not support the separation between the Sequencing of the complete SSU rDNA clearly improved orders Buliminida and Rotaliida as proposed in the latest the phylogenetic signal contained in analyses of the s14-

Fig. 5. Phylogeny of Rotaliida based on complete (this study, genera written in black) and partial (Schweizer et al., 2005, genera written in grey) sequences using the ML method (HKY+I+Γ). The number of sequences is indicated between brackets for genera represented by more than one sequence. Tree is rooted on textulariids. Bootstrap values (100 replications) are indicated at the nodes by white dots when they are 60% or higher and by black dots when they are 95% or higher. Author's personal copy

244 M. Schweizer et al. / Marine Micropaleontology 66 (2008) 233–246 sB fragment. By multiplying the number of analysed sites Hyalinea balthica (Schroeter): Nautilus balthicus by a factor three, very strong statistical support was Schroeter, 1783, Einleitung in die Conchylienkenntnis obtained for most of the clades. Still, problems persist nach Linné, 1, p. 20, pl. 1, Fig. 2. with some clades composed of fast evolving species (i.e. Melonis pompilioides (Fichtel and Moll): Nautilus Ammonia, Elphidium, Haynesina), which are more pompilioides Fichtel and Moll, 1798, Test. Micr. Arg. sensitive to the long branch attraction (LBA) phenome- Naut., p. 31, pl. 2, figs a–c. non. Analysis of protein-coding genes could resolve these Nonionella labradorica (Dawson): Nonionina lab- problems. However, it is already known that at least two radorica Dawson, 1860, Canadian Nat. Geol., 5, p. 192, genes for which data are available (actin, β-tubulin), are Fig. 4. too conserved to resolve the phylogeny of Rotaliida Oridorsalis umbonatus (Reuss): Rotalina umbonata (Flakowski et al., 2005; Ujiie and Pawlowski, in pre- Reuss, 1851, Deutsch. Geol. Ges. Zeitschr., 3, p. 75, pl. 5, paration). Although it is expected that some mitochondrial Fig. 35. genes could be helpful, the data are not yet available. At Pullenia subcarinata (d'Orbigny): Nonionina subcar- the same time, to revise the classification of Rotallida it inata d'Orbigny, 1839, Voyage dans l'Amérique Mér- will be compulsory to obtain complete SSU sequences for idionale; Foraminifères, 5, pt. 5, p. 28, pl. 5, Figs. 23–24. taxa belonging to the families that have not yet been Stainforthia fusiformis (Williamson): Bulimina examined. pupoides d'Orbigny 1846 var. fusiformis Williamson, 1858, Rec. foram. G. B., p. 63, pl. 5, Figs. 129–130. Acknowledgements Uvigerina earlandi (Parr): Angulogerina earlandi Parr 1950, Brit. N. Z. Ant. Res. Exp., 5, p. 341, pl. 12, We thank Elisabeth Alve, Stefan Agrenius, Henko de Fig. 21. Stigter, Christoffer Schander, the crews of the R/V Uvigerina peregrina Cushman 1923, U. S. Nat. Mus. Trygve Braarud, Arne Tiselius, Pelagia and Hans Bul., 104, pt. 4, p. 166, pl. 42, figs 7–10. Brattström, Sam Bowser, Sergei Korsun, Tomas Cedha- Uvigerina phlegeri (Le Calvez): Rectuvigerina gen and Maria Holzmann for their help in sampling, and phlegeri Le Calvez 1959, Rec. Trav. Inst. Pêches José Fahrni and Delphine Berger for their technical Maritimes, 23, p. 263, pl. 1, Fig. 11. assistance. This study was supported by the Dutch NWO/ALW grant 811.32.001 and Swiss NSF grants References 3100A0-100415 and 200020-109639/1. Alve, E., 1995. Benthic foraminiferal response to estuarine pollution: a review. Journal of Foraminiferal Research 25, 190–203. Appendix A. 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