PALEVO 1631-0683 Elsevier S1631-0683(11)00005-4 10.1016/j.crpv.2011.01.003 Research article General palaeontology Contributions of molecular phylogenetics to foraminiferal taxonomy: General overview and example of Pseudoeponides falsobeccarii Rouvillois, 1974 Contributions de la phylogénie moléculaire à la taxonomie des foraminifères : aperçu général et cas pratique avec Pseudoeponides falsobeccarii Rouvillois, 1974 Néraudeau Didier Schweizer Magali Magali.Schweizer@ed.ac.uk a Jorissen Frans b c Geslin Emmanuelle b c Geological Institute, ETHZ, Sonneggstrasse 5, 8092 Zurich, Switzerland Laboratory of Recent and Fossil Bio-Indicators, UPRES EA 2644 BIAF, Angers University, 2, boulevard Lavoisier, 49045 Angers cedex, France LEBIM, Ker Chalon, Port-Joinville, Île d’Yeu, France 10 2-3 S1631-0683(11)X0003-9 La notion d'espèce en paléontologie : ontogenèse, variabilité, évolution The species concept in palaeontology: ontogeny, variability, evolution 95 105 © 2011 Académie des sciences. Published by Elsevier B.V. All rights reserved. 2011 Académie des sciences Full (PDF)

Molecular phylogenetics gives new insights into the taxonomy of foraminifera, independent of their morphology. After a survey of the present knowledge on how molecular phylogeny can contribute to foraminiferal taxonomy, we present an applied example. The comparison of ribosomal DNA (rDNA) sequences belonging to the SSU (Small Subunit) and LSU (Large Subunit) genes of Pseudoeponides falsobeccarii with other similar sequences of rotaliids available in GenBank shows that this species actually belongs to the genus Ammonia, because it groups inside the other Ammonia sequences instead of forming a distinct clade. Moreover, Ammonia falsobeccarii forms a clade well separated from other Ammonia phylotypes, meaning that it can be considered as a distinct species, and not as an ecophenotype of one of the other Ammonia species.

Grâce à la phylogénie moléculaire, il est possible d’aborder la taxonomie des foraminifères sous un nouvel angle, indépendant de la morphologie. Après un aperçu général des apports de la phylogénie moléculaire à la taxonomie des foraminifères, nous présentons un cas pratique. Des séquences appartenant aux gènes de la petite (18S) et de la grande sous-unités (28S) de l’ADN ribosomique (ADNr) de Pseudoeponides falsobeccarii ont été comparées à des séquences semblables de rotaliides disponibles dans GenBank. Leur analyse phylogénétique démontre que P. falsobeccarii appartient au genre Ammonia, parce que les séquences d’ADN de cette espèce se retrouvent à l’intérieur du clade formé par toutes les séquences d’Ammonia. À l’intérieur de ce clade, Ammonia falsobeccarii forme un groupe bien distinct des autres phylotypes décrits pour Ammonia, ce qui signifie que ce taxon peut être considéré comme une espèce séparée, plutôt qu’un écophénotype d’une autre espèce d’Ammonia.

 Benthic foraminifers, Rotaliida, SSU and LSU rDNA, Classification Foraminifères benthiques, Rotaliida, ADNr 18S et 28S, Classification presented Written on invitation of the Editorial Board. Introduction Particulars of foraminifera from a phylogenetic point of view

The first foraminiferal DNA sequences were published in the middle of the 1990s (Pawlowski, 2000, and references therein). Compared to other Eukaryotes, there was and still is some delay in the development of foraminiferal phylogenetics. This is due to the difficulty in culturingforaminifera in axenic conditions as well as the peculiarity of their genome.

It is rather easy to maintain foraminifera alive under controlled conditions, but it is much more difficult to actually culture them and make them reproduce (Bowser et al., 2006). The absence of foraminifera from “strain catalogues” means that acquiring foraminiferal DNA is not yet a routine task, and that a better knowledge of how to sample them is still needed.

Another difficulty with foraminifera is that the genes whose sequences are known differ from the ones of other Eukaryotes. For example, the ribosomal genes (Small (SSU) and Large (LSU) Subunits of ribosomal DNA (rDNA)) of foraminifera are much longer than those of other Eukaryotes due to typical insertions (Pawlowski, 2000). A few other foraminiferal genes have been successfully amplified such as actin, RNA polymerase II largest subunit, ubiquitin and tubulin (see Bowser et al., 2006 for references). However, despite many attempts, it was not yet possible to amplify mitochondrial DNA, which is widely used for barcoding in many other organisms (Pawlowski and Lecroq, 2010). The fact that foraminiferal DNA is very divergent compared to other Eukaryotes also means that this group does not appear in DNA environmental surveys where biodiversity is targeted with universal eukaryotic primers (Berney et al., 2004).

 Comparison between phylogenetic studies of planktic and benthic foraminifera

Phylogenetic studies on planktic foraminifera showed that genetic variation was usually much higher than the described morphological variation in this group (e.g. Kucera and Darling, 2002, and references therein). Cryptic (species separated by genetics but undistinguishable morphologically) or pseudocryptic (morphological distinction possible after re-examination) species are regularly described in planktic foraminifera (e.g. Aurahs and Grimm, 2009, de Vargas et al., 1999 and Huber et al., 1997).

Among benthic foraminifera, allogromiids (foraminifera with an organic test) also present a high level of genetic diversity compared to a rather low level of morphological diversity (Habura et al., 2008 and Pawlowski et al., 2002). In the group of rotaliids (foraminifera with a calcitic hyaline bilamellar perforate test), Ammonia Brünnich, 1772 was the first genus studied at a large scale. At a first view, this cosmopolitan genus showed a similar tendency, with genetic diversity being much higher than morphological diversity (Hayward et al., 2004 and Holzmann, 2000). However, when looking in more detail, it appears that Ammonia morphotypes may have been subject to extreme lumping; only one to three species are routinely used by most foraminiferologists, although over 40 species have been described on the basis of small morphological differences (Hayward et al., 2004). In this context, the discovery of 13 different phylotypes in a worldwide study of Ammonia (Pawlowski and Holzmann, 2008) was not really a surprise. Further studies on rotaliids confirmed that there is a fairly good correspondence between phylotypes and morphospecies (e.g. Schweizer et al., 2005, Schweizer et al., 2009, Tsuchiya et al., 2000 and Tsuchiya et al., 2008).

In many other taxonomic groups it is possible to fix a threshold in the divergence between sequences beyond which they will be considered as belonging to different species (see Hayward et al., 2004 for references). In foraminifera, the evolutionary rates are so heterogeneous that it is not possible to define any general threshold (Pawlowski and Lecroq, 2010). However, there are other methods to investigate species boundaries. Some variable regions of the SSU rDNA such as 37/f are rather accurate to discriminate the different morphospecies of foraminifera (Pawlowski and Lecroq, 2010). Moreover, a general observation of the phylogenetic tree will usually show phyloclades well separated by branches longer than the branches inside them. In the case of rotaliids, these phyloclades correspond to known morphospecies most of the time.

 Contributions of phylogenetic studies for enhancing the taxonomy of foraminifera Taxonomy and classification of higher levels

For the time being the classification and taxonomy of foraminifera at suprageneric levels are still exclusively based on the wall structure and the morphology of the test (Sen Gupta, 2002), without taking into account biological aspects such as ecology (except for the group of planktic foraminifera), life cycle or DNA. However, phylogenetic studies were able to shed new light and/or to confirm some aspects of the classification of foraminifera previously based on the characteristics of the test.

Some authors have considered the composition and ultrastructure of the test as subsidiary features compared to the overall form of the test and the chamber arrangement (e.g. Hofker, 1956 and Mikhalevich and Debenay, 2001), but for the majority of recent authors, the wall composition remains the most important criterion to separate the different foraminiferal orders (Haynes, 1981, Loeblich and Tappan, 1987 and Sen Gupta, 2002). However, phylogenetic studies have shown that the absence of shell was sometimes due to a secondary loss instead of being a primary state (Pawlowski et al., 1999) and that allogromiids and textulariids (foraminifera with an agglutinated test) formed paraphyletic clades (Bowser et al., 2006, Pawlowski, 2000 and Pawlowski et al., 2003). A very interesting example, showing that the chamber arrangement could be a more important criterion than wall composition, is Miliammina fusca (Brady, 1870). This foraminifer has an agglutinated test, but with a chamber arrangement similar to the one found in most miliolids (foraminifera with a porcellaneous, non perforate test). Traditionally, M. fusca is placed in the order Textulariida on the basis of the wall composition, which is agglutinated (Loeblich and Tappan, 1987). However, DNA analyses clearly show that this species groups with porcelaneous foraminifera and therefore belongs to the order Miliolida (Fahrni et al., 1997 and Habura et al., 2006).

Foraminifera with a calcitic hyaline bilamellar perforate test (rotaliids sensu lato, Bowser et al., 2006) have been separated in three different orders in some classifications based on the test morphology (e.g. Haynes, 1981, Loeblich and Tappan, 1992 and Sen Gupta, 2002). The order Globigerinida includes all planktic foraminifera, whereas benthic rotaliids are separated into Rotaliida and Buliminida on the basis of the coiling of the test (low/high), the toothplate (absence/presence) and the shape of the aperture (arched/loop-shaped). Phylogenetic studies based on the SSU rDNA showed that rotaliids sensu lato form three main phyloclades mixing Buliminida and Rotaliida (Schweizer et al., 2008) and that planktic foraminifera are polyphyletic (Darling et al., 1997, Darling et al., 2009 and Ujiié et al., 2008), originating from different phyloclades of benthic rotaliids (Bowser et al., 2006).

Surprisingly, the wall structure, which was considered a main criterion to distinguish superfamilies in rotaliids in a former classification (Loeblich and Tappan, 1964), was still used to separate closely related genera such as Cibicides de Montfort, 1808 and Cibicidoides Thalmann, 1939 in different superfamilies in a later classification (Loeblich and Tappan, 1987), although this criterion had already been dismissed some years before (Deutsch Conger et al., 1977 and Towe and Cifelli, 1967). Phylogenetic studies confirmed that the wall structure was not an important criterion because genera such as Cibicides and Cibicidoides are closely related genetically (Schweizer et al., 2009). Moreover, the effort to separate plano- and biconvex cibicidids into Cibicides and Cibicidoides, respectively, appears to be incorrect, since the shape of the test varies in function of the form of the substrate to which the foraminifer is fixed (Schweizer et al., 2009). The coiling mode has also been considered as a major morphological criterion to distinguish superfamilies and families (Haynes, 1981 and Loeblich and Tappan, 1987). However, phylogenetic analyses (Schweizer et al., 2008 and Schweizer et al., 2009) have shown that some genera with very different coiling modes grouped together in phyloclades (e.g. Melonis de Montfort, 1808 (planospiral) with Cibicides, Cibicidoides and Hanzawaia Asano, 1944 (trochospiral); Ammonia (trochospiral) with Elphidium de Montfort, 1808 and Haynesina Banner and Culver, 1978 (planospiral)).

Conversely, other morphological criteria, which were previously considered less important for taxonomy, gained new weight after re-examination of the morphology in the light of phylogenetic results. The coiling direction in planktic foraminifera, which was usually thought to be influenced by ecological factors, could have a genetic origin (Darling et al., 2006), and also the size and number of pores seem, at least in some benthic and planktic foraminifera, to be determined genetically rather than influenced by environmental factors (Holzmann and Pawlowski, 1997, Huber et al., 1997, Morard et al., 2009 and Schweizer et al., 2009).

 Choice of generic names

Many generic names have been described for rotaliids (about 640 retained by Loeblich and Tappan, 1987). For the time being, intrageneric phylogenetic variability has only been studied in some detail for a handful of rotaliid genera: Ammonia (Hayward et al., 2004), Bulimina d’Orbigny, 1826 (Tsuchiya et al., 2008), Cibicides/Cibicidoides (Schweizer et al., 2009), Glabratella Dorreen, 1948 and related genera (Tsuchiya et al., 2000), Nummulites Lamarck, 1801 and related genera (Holzmann et al., 2003), Uvigerina d’Orbigny, 1826 and related genera (Schweizer et al., 2005).

Among the three genera of uvigerinids studied by Schweizer et al. (2005), Trifarina Cushman, 1923 is traditionally distinguished from Uvigerina by having a triangular cross section instead of a round one, whereas Rectuvigerina Mathews, 1945 has a uniserial arrangement of the final chambers compared to a triserial one throughout for Uvigerina. However, the species Trifarina earlandi (Parr, 1950) and Rectuvigerina phlegeri Le Calvez, 1959 form a clade with Uvigerina peregrina Cushman, 1923, which is the sister-group (the closest clade) of the clade formed by Uvigerina mediterranea Hofker, 1932 and U. elongatastriata (Colom, 1952) in partial SSU rDNA analyses (Schweizer et al., 2005). Therefore, Trifarina and Rectuvigerina should rather be included in the genus Uvigerina, demonstrating that generic distinctions based on the section and the seriality of the test are probably not relevant in that case.

For cibicidids, the phylogenetic results were less clear, although six morphospecies have been sequenced for the SSU rDNA gene. In a first study with partial SSU sequences, the cibicidids were forming a clade and it was decided to call them all Cibicides, which is the senior generic name, although there were two clearly separated subgroups in the clade (Schweizer, 2006). However, in a later study with more complete SSU sequences (Schweizer et al., 2009), some doubt was shed on this monophyly by the position of Melonis, which was separating the two cibicidid subgroups in some analyses. More sequences of Melonis and Cibicides/Cibicidoides are needed to elucidate this problem. For the time being, Schweizer et al. (2009) decided to give different names to the two subgroups of cibicidids (Cibicides and Cibicidoides) in order to avoid future problems if the clade turns out to be polyphyletic with Melonis positioned in the middle.

 Endemism and cosmopolitanism in rotaliids

In benthic foraminifera, very similar morphotypes are often determined using different species names in different areas. In such cases, an important question is whether taxonomical differences between such morphologically close specimens from different regions reflect true endemism or are just the consequence of a poorly constrained taxonomy and regional traditions.

Phylogenetic analyses show that in some cases endemism may be substantial. For instance, Uvigerina akitaensis Asano, 1950, a species described from Japan, has been considered by Scott et al. (2000) as a synonym of the cosmopolitan species U. peregrina. Although further investigations are needed with more variable parts of the DNA, the SSU sequences of U. akitaensis (Ertan et al., 2004) seem slightly different from comparable ones of U. peregrina (Schweizer, 2006). Moreover, many shallow water cibicidid species, such as Cibicides refulgens de Montfort, 1808 or Cibicidoides dispars (d’Orbigny, 1839) are often considered synonymous with Cibicidoides lobatulus (Walker and Jacob, 1798) (e.g. Hageman, 1979 and Heron-Allen and Earland, 1932). However, these species are well separated genetically from C. lobatulus, and even the morphospecies C. lobatulus itself could be a mosaic of different species (Schweizer et al., 2009, Schweizer et al., submitted). On the other hand, Virgulinella fragilis Grindell and Collen, 1976 and some deep-sea species such as Cibicidoides wuellerstorfi (Schwager, 1866) and Epistominella exigua (Weltner, 1895) have genetically very homogeneous populations throughout large geographical spaces, making them cosmopolitan species (Pawlowski et al., 2007 and Tsuchiya et al., 2009).

 Ecophenotypic versus genetic variations in rotaliids

When DNA is extracted from single specimens for which the morphology is known (either by taking SEM pictures prior to the destruction of the test during DNA extraction or by using extraction buffers which can preserve the test (e.g. Morard et al., 2009)), it is possible to directly compare morphological and genetic variations. In the case of ecophenotypy, morphological variation will be higher than genetic variability and influenced by the environment, whereas genetic variation will be higher than morphological for cryptic species.

We have seen already that genetic variation is apparently high in comparison with the rather restrictive commonly used taxonomic concepts in Ammonia and C. lobatulus. In these cases, only a limited number of morphospecies (Ammonia) or just one (C. lobatulus) are routinely used, whereas extensive morphological variability is observed within them. For Ammonia, a re-examination of the material following genetic analyses allowed us to distinguish a larger morphological variation correlated with genetic variation (Hayward et al., 2004). A morphological re-examination of the material for C. lobatulus connected with a phylogenetic study could also help to determine the limits of morphological intraspecific variation, but it has yet to be done. The Elphidium excavatum (Terquem, 1876) plexus is another example where the traditionally distinguished subspecies or ecophenotypes (e.g. Feyling-Hanssen, 1972, Goubert, 1997 and Miller et al., 1982) correspond to different phylotypes, and are the results of genetic variation instead of environmental influence (Schweizer et al., 2010).

At present, there are very few well-documented examples of ecophenotypy in foraminifera confirmed by DNA analyses. A population of U. peregrina from the Oslo Fjord showed some morphological variation although very variable DNA regions (Internal Transcribed Spacer (ITS) rDNA) were similar (Schweizer et al., 2005). Uv. peregrina sampled in the Bay of Biscay are morphologically distinct from the Oslo Fjord populations, but have identical SSU sequences (unpublished data). Further studies with more variable regions of DNA (e.g. ITS) are needed to check if these populations are indeed identical genetically. Another example of possible ecophenotypy is provided by Virgulinella fragilis, which tests vary in the size and density of pores among genetically similar populations (Tsuchiya et al., 2009).

It is evident that genetic analyses can unearth new evidence in longstanding taxonomic problems. One of these problems, which has intrigued us for a long time, is the status of the genus Pseudoeponides Uchio, 1950, and of the species Pseudoeponides falsobeccarii Rouvillois, 1974 in particular. In this paper, we will use this species as an example to show how phylogenetic studies can contribute to the solution of complex taxonomic questions.

 The enigmatic case of <italic>P. falsobeccarii</italic>

The genus Pseudoeponides was established by Uchio (1950), with Pseudoeponides japonica (Uchio, 1950) as the type species. According to the original description, the genus is a basic trochospiral form resembling Eponides, and is characterised by secondary openings on both sides. The original description mentions elongate slits parallel to the sutures on the dorsal side and loop-shaped secondary openings, positioned along each suture radiating from the umbilicus on the ventral side.

This generic description has been emended by Hofker (1958) who demonstrated that Uchio wrongly described and figured the secondary openings on the dorsal side. In fact, these openings are not areal (parallel to the sutures), as figured by Uchio (figure reproduced on Pl. 667, Fig. 12 in Loeblich and Tappan, 1987), but are entirely sutural, positioned at the junctions of spiral and chamber sutures (Fig. 1). In the earlier part of the test, the openings are rather circular; they become more or less triserial toward later ontogenetic stages. Hofker (1958) also mentioned that the dorsal supplementary openings are surrounded by a poreless area. He presented very precise drawings of the ventral secondary openings, which are positioned at the external side of triangular chamber extensions pointing into the umbilical area. Hofker remarked that these umbilical features were similar to those found in the genus Streblus (now Ammonia). He showed that the only difference between Pseudoeponides and Ammonia was the position of the toothplate, which extends to the chamber wall, and is connected to the dorsal secondary openings in Pseudoeponides, but does not reach the chamber wall in Ammonia. In all other aspects, Pseudoeponides is similar to Ammonia. Therefore, Hofker concluded that Pseudoeponides is a Streblus (now Ammonia) in which the chambers are slightly turned towards the ventral side, so that the toothplate reaches the dorsal wall, the poreless area becomes very prominent and a secondary opening is formed (in fact, this turning of the chambers towards the ventral side causes the spiral to be higher in Pseudoeponides than in Ammonia). For Hofker (1958), there is not enough difference to justify a separate genus, and the species Pseudoeponides japonica should be included in the genus Streblus. Unfortunately, the very judicious emendation of Hofker (1958) has been largely overlooked, and the description and figures of the genus Pseudoeponides in Loeblich and Tappan, 1964 and Loeblich and Tappan, 1987 faithfully reproduce the error of Uchio.

P. falsobeccarii is a common species in continental shelf environments in the Mediterranean and north-eastern Atlantic. It was first described by Rouvillois (1974), who indicated that this species had previously often been determined erroneously as Ammonia beccarii (Linnaeus, 1758), which is morphologically similar, but lacks the dorsal secondary openings (Fig. 1). Unfortunately, confusion between both taxa has continued until today. For example, P. falsobeccarii has been incorrectly determined as A. beccarii or A. batava (Hofker, 1951) by Haake (1977) (Pl. 1, Figs 1-2), Vénec-Peyré, 1983 (ecophenotype 4: Pl. 1, Fig. 6; Pl. 2, Fig. 5; Pl. 5, Fig. 4) and Hayward et al. (2004, Pl. 4, T3S). This list is far from exhaustive, many other examples exist.

P. falsobeccarii has been described in recent sediments (thanatocoenoses) from the Adriatic Sea (Jorissen, 1988), where it accounts for up to 10% of the faunas, at water depths between 20 and 60 m. It is mainly found in the centre of the coast-parallel mud belt, which slightly deepens from 10 to 30 m depth in front of the Po delta to 40 to 60 m depth off Pescara. In live faunas from a sediment core from 35 m depth off Ancona, Pucci et al. (2009) found about 20 specimens per 50 cm2, corresponding to about 8% of the total fauna. Mojtahid et al. (2009) found abundant live populations of P. falsobeccarii between 60 and 90 m depth in the Rhône prodelta. In the Bay of Biscay, Rouvillois (1974) described P. falsobeccarii at water depths between 60 and 130 m. This larger water depth in the Bay of Biscay has recently been confirmed by Fontanier et al. (2002), Duchemin et al. (2005) and Langezaal et al. (2006), who described numerous live (Rose Bengal stained) specimens in the Bay of Biscay, on silty clays between 100 and 140 m depth. In the five previously cited studies on live specimens, P. falsobeccarii shows a clear infaunal tendency; an abundance maximum is systematically observed at a depth of several centimeters, in anoxic sediments. Pucci et al. (2009) incubated several multicores sampled at a 35 m deep site in the Adriatic Sea in oxic as well as strongly dysoxic conditions, for various periods of time, to a maximum of 69 days. In both experimental setups, P. falsobeccarii specimens migrated to the uppermost sediment level. In comparison with the other dominant taxa, standing stocks of P. falsobeccarii decreased rapidly over time, suggesting that this taxon resisted badly to the experimental conditions (without added food), in oxic as well as strongly dysoxic conditions. Summarising, it appears that P. falsobeccarii has a clear preference for organically enriched fine-grained sediments, such as those present in front of major deltaic systems. It appears well adapted to support the phenomena of oxygen deficiency associated with such systems, as shown by its presence in deeper, anoxic, sediment layers.

Jorissen (1988) suggested that P. falsobeccarii could be an ecophenotype of the Ammonia parkinsonia (d’Orbigny, 1839) group (which includes also A. tepida Cushman, 1926, but not A. beccarii, which is characterised by an abundant ornamentation, and deeply incised sutures on the dorsal side). He hypothesised that this morphospecies could have adapted to the stressed conditions of the enriched clay belt environments by a more intensive use of the canal system, leading to the formation of supplementary apertures (outlets of the channel system) on the dorsal side.

The question we want to address in this article is twofold:

is the genus Pseudoeponides sufficiently different from Ammonia from a genetic point of view to justify a separated genus?

is P. falsobeccarii an ecophenotype of one of the Ammonia species, or is it different enough genetically to be considered as a separate species?

Material and methods

Four live specimens of P. falsobeccarii have been sampled, three in the Rhône prodelta at the station 15 (Goineau et al., 2011) and one in the Bay of Biscay at the station D (Fontanier et al., 2002) (Fig. 2, Table 1). The specimens were picked, photographed and sequenced according to the protocol described in Schweizer et al. (2010).

The new sequences (one partial SSU and three partial LSU ones) have been deposited in the EMBL/GenBank database (accession numbers given in Table 1). These sequences were aligned manually with related sequences available from GenBank in two separate datasets (SSU and LSU) using Seaview (Gouy et al., 2010). The genus Elphidium was chosen as the out-group taxon because it is the closest known relative of Ammonia (Schweizer et al., 2008).

The regions, which were impossible to align properly were removed and 847 and 348 sites were respectively used for SSU and LSU analyses. Maximum likelihood (ML) analyses were performed with 100 bootstrap (BS) replicates by PhyML 2.4.4 (Guindon and Gascuel, 2003) under the HKY (Hasegawa, Kishino, Yano) model (Hasegawa et al., 1985) for the SSU and the LSU alignments. In order to correct for among-site rate variations, the proportion of invariable sites (I) and the alpha parameter of gamma distribution (Γ), with six rate categories, were estimated by PhyML (HKY+I+Γ). In addition, BIONJ phylogenetic trees (Gascuel, 1997) were inferred with Seaview under the K2P (Kimura's two parameter) evolution model (Kimura, 1980) with non-parametric bootstrapping (1000 replicates).

 Results and discussion

Two regions of the rDNA gene cluster are investigated here: the 3’ end fragment of the SSU which is widely used in foraminiferal phylogenetic studies (Pawlowski, 2000) and the 5’ end of the LSU which was used to define 13 different phylotypes of Ammonia by Hayward et al. (2004). In the SSU analysis (Fig. 3), only four phylotypes of Ammonia are represented: T1, T2, T3 and T6. The P. falsobeccarii sequence is the sister group of the phylotype T3. The same topology, also well supported, is observed in the LSU analysis with three sequences of P. falsobeccarii and the 13 phylotypes of Ammonia previously defined (Fig. 4).

 Generic assignation and taxonomic status of <italic>Pseudoeponides falsobeccarii</italic>

Both SSU and LSU phylogenetic analyses show that Pseudoeponides falsobeccarii sequences have a central position in the Ammonia sequences (Fig. 3 and Fig. 4). Therefore, it is not justified to give this species a separate generic name, Pseudoeponides. These phylogenetic results confirm the earlier conclusion of Hofker (1958) based on a morphological analysis of both genera.

DNA sequences of Ammonia falsobeccarii form a well-supported clade, closely related but clearly separated from the phylotype T3 in both SSU and LSU analyses (Fig. 3 and Fig. 4).

 Relationships between <italic>Ammonia falsobeccarii</italic> and the phylotype T3

The phylotype T3 has been sampled in Sweden, the Channel, the Bay of Biscay and the Gulf of Lions (Hayward et al., 2004) (Fig. 5). Although this heavily ornamented phylotype was not identified as A. beccarii by Hayward et al. (2004), because it is morphologically slightly different from the topotypes of that species, similar specimens from Vendée (Bay of Biscay) have previously been commonly attributed to A. beccarii (e.g. Debenay et al., 1998). The northern specimens (e.g. Sweden, Channel) are usually identified as Ammonia batava (Hayward et al., 2004). Although specimens genetically identified as T3 have been presented with different species names, they all clearly share a common morphology (Hayward et al., 2004).

Ammonia falsobeccarii and the phylotype T3 can be found in the same locations. However, it is usually relatively easy to morphologically discriminate them. Ammonia falsobeccarii has more lobulated chambers and unfilled secondary openings at the junctions of the radial sutures with the spiral one on the spiral side (Fig. 1 and Fig. 2), whereas the T3 phylotype has deeply incised fissured sutures on the spiral side and a more ornamented umbilical side (Fig. 1).

The fact that A. falsobeccarii is well separated morphologically and genetically from T3 strongly suggests that it can be considered as a new phyloclade.

 Acknowledgments

We would like to thank the crews of the “Côte de la Manche” for sampling in the Bay of Biscay and the “Tethys” for sampling in the Rhône prodelta. We also thank Kate Darling for fruitful discussions and the Swiss NSF for financial support (grant 200020-109639/1 to M.S.).

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SEM pictures of specimens identified as Ammonia beccarii (F431, F432) and Pseudoeponides falsobeccarii (F433, F443). F431, F432, F433 come from the Rhône prodelta and F443 from the Bay of Biscay. Scale bar: 200 μm.

Photos au MEB de spécimens identifiés comme Ammonia beccarii (F431, F432) et Pseudoeponides falsobeccarii (F433, F443). F431, F432, F433 proviennent du prodelta du Rhône et F443 du Golfe de Gascogne. Échelle : 200 μm.

SEM pictures of the specimens sequenced and DNA identification numbers. Scale bar: 100 μm. N.B. No picture was taken for specimen F187 and DNA amplification was negative for specimen F276.

Photos au MEB des spécimens séquencés, avec leur numéro d’identification ADN. Échelle : 100 μm. N.B. Il n’y a pas de photo pour le spécimen F187 et l’amplification d’ADN n’a rien donné pour le spécimen F276.

Molecular phylogeny of selected Ammonia species based on partial SSU rDNA sequences inferred using the ML method (model HKY+I). Tree is rooted on Elphidium williamsoni and E. aculeatum and bootstrap values for ML and BIONJ analyses are indicated at the nodes.

Phylogénie moléculaire de représentants du genre Ammonia, basée sur des séquences partielles d’ADNr 18S et calculée d’après la méthode du maximum de vraisemblance (ML) (modèle HKY+I). L’arbre est enraciné sur Elphidium williamsoni et E. aculeatum et les valeurs de bootstrap pour les analyses ML et BIONJ sont indiquées aux nœuds.

Molecular phylogeny of selected Ammonia species based on partial LSU rDNA sequences inferred using the ML method (model HKY+I+Γ). Tree is rooted on N. calcar, P. nipponica and H. germanica and bootstrap values for ML and BIONJ analyses are indicated at the nodes.

Phylogénie moléculaire de représentants du genre Ammonia, basée sur des séquences partielles d’ADNr 28S et calculée d’après la méthode ML (modèle HKY+I+Γ). L’arbre est enraciné sur N. calcar, P. nipponica et H. germanica et les valeurs de bootstrap pour les analyses ML (100 réplicats) et BIONJ sont indiquées aux nœuds.

Map of Europe with sampling sites for specimens T3 analysed by Hayward et al. (2004) (grey diamonds) and A. falsobeccarii from the present study (white stars).

Carte d’Europe avec indication des sites d’échantillonnage pour les spécimens T3 analysés par Hayward et al. (2004) (losanges gris) et les A. falsobeccarii de la présente étude (étoiles blanches).

List of new partial rDNa SSU and LSU sequences with the location of DNA samples, the water depth and the GenBank accession numbers for both genes. The samples of the Rhône prodelta were taken from station 15 (43°17’055 N and 04°45’148 E, Goineau et al., 2011) and the one from the Bay of Biscay from station D (43°42’ N and 01°34’ W, Fontanier et al., 2002).

Liste des nouvelles séquences partielles d’ADNr 18S et 28S, avec la localisation des échantillons, la profondeur d’eau et les numéros d’accès GenBank pour les deux gènes. Les échantillons du prodelta du Rhône proviennent de la station 15 (43°17’055 N et 04°45’148 E, Goineau et al., 2011) et celui du Golfe de Gascogne de la station D (43°42’ N et 01°34’ W, Fontanier et al., 2002).

DNA number Location Water depth (m) SSU sequence LSU sequence F187 Rhône delta 60 Negative HM448840 F276 Rhône delta 60 Negative Negative F277 Rhône delta 60 HM448841 HM448838 F300 Bay of Biscay 140 Negative HM448839