SSU rDNA Divergence in Planktonic Foraminifera: Molecular Taxonomy and Biogeographic Implications

The use of planktonic foraminifera in paleoceanography requires taxonomic consistency and precise assessment of the species biogeography. Yet, ribosomal small subunit (SSUr) DNA analyses have revealed that most of the modern morpho-species of planktonic foraminifera are composed of a complex of several distinct genetic types that may correspond to cryptic or pseudo-cryptic species. These genetic types are usually delimitated using partial sequences located at the 3′end of the SSUrDNA, but typically based on empirical delimitation. Here, we first use patristic genetic distances calculated within and among genetic types of the most common morpho-species to show that intra-type and inter-type genetic distances within morpho-species may significantly overlap, suggesting that genetic types have been sometimes inconsistently defined. We further apply two quantitative and independent methods, ABGD (Automatic Barcode Gap Detection) and GMYC (General Mixed Yule Coalescent) to a dataset of published and newly obtained partial SSU rDNA for a more objective assessment of the species status of these genetic types. Results of these complementary approaches are highly congruent and lead to a molecular taxonomy that ranks 49 genetic types of planktonic foraminifera as genuine (pseudo)cryptic species. Our results advocate for a standardized sequencing procedure allowing homogenous delimitations of (pseudo)cryptic species. On the ground of this revised taxonomic framework, we finally provide an integrative taxonomy synthesizing geographic, ecological and morphological differentiations that can occur among the genuine (pseudo)cryptic species. Due to molecular, environmental or morphological data scarcities, many aspects of our proposed integrative taxonomy are not yet fully resolved. On the other hand, our study opens up the potential for a correct interpretation of environmental sequence datasets.

IIe is apparently restricted to the North Pacific (Darling and Wade, 2008). Darling et al. (2003) evidenced different timings of reproduction which may explain genetic isolation (Norris, 2000) of the otherwise sympatric Types IIa and IIb-d-f. Unfortunately, an integrative taxonomy for G. bulloides genetic types still remains limited because of the scarcity of the SSU rDNA data. For example, environmental data and sampling through different seasons are insufficient to detect potential ecological differences between the apparently sympatric Types IIc, Type IIb-d-f and Type IIa.
Our data show that only one single species of Beela digitata has been sampled so far.
On the other hand, the distribution patterns of this rare morpho-species (Hemleben et al., 1989) cannot be discussed in this paper, since the studied sequences originate from specimens collected at two stations only, both located in the South Pacific (Fig. II).
All methods also agree on the species status of the previously defined Caribbean, Sargasso and Mediterranean genetic types of Orbulina universa (de Vargas et al., 1999), respectively re-designated as Types I, II and III by de Vargas et al. (2004). The ABGD method clearly delimitates the Types I and III as independent species, while sequences of the Type II were too short to be included in the ABGD dataset. The species status of the Type II is however supported on the basis of the GMYC method. In the world oceans, the three species of O. universa have distributions that are apparently correlated with the productivity of the surface waters (Fig. I;de Vargas et al., 1999;Morard et al., 2009). Types I and II inhabit stratified and nutrient-depleted subtropical waters (the Type II occurring in extreme oligotrophic environments), whereas the Type III favors vertically-mixed and nutrient-rich environments of the tropical to temperate water masses. Biometric analyses by Morard et al. (2009) have shown that the Types I, II and III of O. universa are pseudo-cryptic species.
Shells of the Type I species exhibit larger pore area and higher porosity values than those of the Types II and III, whereas the two later species can be further distinguished on the basis of shell thickness, which is significantly thinner in the case of the Type II species.
Our data confirm that the morphologically diverse plexus Globigerinoides sacculifer, including the G. trilobus, G. immaturus, G. quadrilobatus and G. sacculifer s.s. morphotypes, constitutes a single species (André et al., 2013). Globigerinoides sacculifer is so far a unique example in planktonic foraminifera where the morphological variability exceeds the rDNA genetic variability. Considering the worlwide sampling representativity of the available genetic data, G. sacculifer should be considered as a true cosmopolitan and morphologically diverse (sub)tropical species (Fig. I).
Finally, our data show that only one single species of Sphaeroidinella dehiscens has been sampled so far. Since the studied sequences originate from specimens collected at stations located in the tropical Indo-Pacific (Fig. II), the distribution pattern of this rare morpho-species cannot be discussed further (Hemleben et al., 1989).

Non-spinose species
For Hirsutella hirsuta and Pulleniatina obliquiloculata, see body-text. Genetic sequencing studies have identified five genetic types within Truncorotalia truncatulinoides (de Vargas et al., 2001;Ujiié and Lipps, 2009), but applications of the ABGD and GMYC methods suggest that this morpho-species harbors only 3 or 4 species, respectively (Table 4).
All methods agree on the species status of the right-coiled Type V (Ujiié and Lipps, 2009), which is apparently restricted to the NW Pacific where no other putative cryptic species occurs (Fig. III). ABGD is the only approach that significantly reduces intra-genetic type and inter-genetic type patristic distances overlapping (Fig. 2B). It clusters Types I and II on the one hand, and Types III and IV on the other hand (de Vargas et al., 2001), into two distinct species with contrasted environmental affinities. Type I-II is a warm-water species restricted to the (sub)tropical oceans, whereas Type III-IV appears to be restricted to the productive subtropical and the colder subpolar frontal zones of the Southern Ocean (Fig. III). Both species are pseudo-cryptic, since they can be differentiated on the basis of shell morphology: Type I-II specimens exhibit large, highly conical left or right-coiled shells, and Type III-IV specimens typically show smaller, axially-compressed and biconvex left-coiled shells (Quillévéré et al., 2013).
Our data show that to date, no cryptic species have been sampled in the oceans for the morpho-species Hirsutella hirsuta, Menardella menardii, Globorotalia tumida, Globorotalia ungulata and Globoquadrina conglomerata ( Table 4). The sequences available for M. menardii, G. tumida, G. ungulata and G. conglomerata originate from too scarce and isolated locations of the world oceans (Fig. III), then precluding any further discussion about their cryptic diversity and biogeography.
Previous studies of the ITS genes have identified two genetic types of Globoconella inflata (Morard et al., 2011). ABGD and patristic distance methods agree on the species status of the Type I. However, due to the shortness of available sequences, these methods cannot be used to test whether the Type II belongs to the same species as the Type I or is indeed an independent species. Application of the GYMC method confirms the species status of the Type I, which has been found to inhabit transitional to subtropical waters of both hemispheres, and of the Type II, which is restricted to the Antarctic subpolar waters (Fig. IV).  (Table 1; Darling and Wade, 2008). Patristic distances are compatible with the species status of these genetic types ( Fig. 2A), although the distance gap is much reduced.
The distribution pattern of the genetic types defined in the literature tends to show that most of them are allopatric and related to specific water masses (Fig. V). The Type I was found in polar waters of the North Atlantic (Darling et al., 2004). The Types II and III were found in subpolar and transitional waters of the southern Ocean. The Type IV was found in Antarctic polar waters (Darling et al., 2004). The Types V and VI were only collected from the South Atlantic, in the vicinity of the Benguela upwelling (Darling and Wade, 2008). Finally, specimens of the Type VII seem to be restricted to transitional waters of the North Pacific (Darling et al., 2007). However, contrary to (Darling et al., 2004), the ABGD method leads to an alternative delimitation hypothesis which is also cross-validated by patristic distances (Fig.   2B; Table 1), clustering the genetic types from sub-antarctic and Benguela upwelling waters (i.e., Types II, III, V, VI) into a single species (Fig. V).
Regarding Neogloboquadrina incompta, all methods agree on the species status of the Types I and II of Darling et al. (2006). These two species have distinct distributions, the Type I inhabiting the Southern Ocean and the North Atlantic, and the Type II inhabiting the North Pacific (Fig. V). Sequence AY453130, isolated as a possible third species on the basis of GMYC analysis, is characterized by numerous substitutions that were not found in any other N. incompta specimens from the NW Pacific (Kimoto and Tsuchiya, unpublished), making the taxonomic status of this sequence unclear until further sampling.
Our study suggests that Neogloboquadrina dutertrei constitutes a unique species that is cosmopolitan in (sub)tropical waters of the world oceans ( Fig. V; Table 4). The GMYC method isolated the sequence AY241708 as a putative second species (Fig. 5). Because of its short length, isolation of this sequence as a putative species may be an artifact. On the other hand, we speculate that this sequence may represent another cryptic species restricted to the North Pacific Ocean, a region that is known for harboring endemic cryptic species of planktonic foraminifera (Darling and Wade, 2008).    Darling et al. (1996;2000;2003;2004;2007); de Vargas et al. (1997); Darling and Wade (2008)