Published in Protist 166, issue 1, 122-130, 2015 1 which should be used for any reference to this work Morphological and Molecular Diversification of Asian Endemic Difflugia tuberspinifera (Amoebozoa, Arcellinida): A Case of Fast Morphological Evolution in Protists? a,b,1,2 c,1,2 a,d e Fatma Gomaa , Jun Yang , Edward A.D. Mitchell , Wen-Jing Zhang , c,f g a Zheng Yu , Milcho Todorov , and Enrique Lara aLaboratory of Soil Biology, University of Neuchâtel, Rue Emile-Argand 11, 2000 Neuchâtel, Switzerland bZoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt cAquatic Ecohealth Group, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China dJardin Botanique de Neuchâtel, Chemin du Perthuis-du-Sault 58, CH-2000 Neuchâtel, Switzerland eMarine Biodiversity and Global Change Center, College of Ocean and Earth Sciences, Xiamen University, Xiamen 361102, China fGraduate University of Chinese Academy of Sciences, Beijing 100049, China gInstitute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin St., 1113 Sofia, Bulgaria Planktonic arcellinid testate amoebae exhibit a broad-range of morphological variability but it is cur- rently unclear to what extent this variability represents phenotypic plasticity or if it is genetically determined. We investigated the morphology and phylogenetic relationships of three endemic east- asian Difflugia taxa 1) the vase-shaped D. mulanensis, 2) and a spinose and a spineless morphotypes of D. tuberspinifera using scanning electron microscopy and two ribosomal genetic markers (SSU rDNA and ITS sequences). Our phylogenetic analyses shows that all three taxa are genetically distinct and closely related to D. achlora and Netzelia oviformis. The genetic variations between the spineless and spinose morphotypes of D. tuberspinifera were low at the SSU rRNA level (0.4%), but ten times higher 1Both authors contributed equally to this work. 2Corresponding authors; e-mail [email protected] (F. Gomaa), [email protected] (J. Yang). 2 at the ITS level (4.5-6%). Our data suggest that the two forms of D. tuberspinifera are sufficiently dif- ferentiated in terms of morphology and genetic characteristics to constitute two separate entities and that the presence of spines does not result from phenotypic plasticity due to environmental selective pressure. However further observational and experimental data are needed to determine if these two forms constitute different biological species. Key words: Molecular phylogeny; SSU rRNA gene; ITS; Difflugia tuberspinifera; morphological variation; fast evolution; China. Introduction bacterivores share the same habitat and har-bour almost identical SSU rRNA gene sequences Recent molecular phylogenetic studies have con- despite their extremely different sizes, morpholo- siderably expanded our knowledge of the phy- gies and lifestyles (Foissner et al. 2003). Similarly logenetic relationships and diversity of microbial the dinoflagellates Scripsiella hangoei and Peri- eukaryotes (Cuvelier et al. 2008; Lara et al. 2011; dinium aciculatum were described as separate López-García et al. 2001; Nikolaev et al. 2004). genera based on their morphology, but their ribo- However, diversification and speciation processes somal DNA sequences were shown to be identical in free-living protists remain major questions in (Logares et al. 2008). Likewise, the ITS sequence evolutionary biology (Coyne and Orr 2004; Lahr et of the tank bromeliad-inhabiting aplanosporic al. 2011b; Logares et al. 2007, 2008). In partic- oomycete Geolegnia helicoides appeared to be ular, the pace at which new morphologies appear nested within the flagellate genus Leptolegnia, has remained largely understudied, which may, for showing an incipient loss of the whole flagellar example, be a serious problem when interpreting apparatus (Steciow et al. 2013). fossil records. However, understanding trait evolution can be Protist phenotypes are believed to be extremely impaired by phenotypic plasticity, which has been old, because of a supposed stabilizing selection demonstrated or suggested in Arcellinida, espe- that acts on large asexual populations (Civetta and cially among members of genus Difflugia, leading Singh 1999; Fenchel and Finlay 2006). In line with to much confusion in species identification and this, Arcellinid morphotypes are thought to remain differentiation (Chardez 1974; Lahr and Lopes very stable in time, as illustrated by the resem- 2006; Medioli et al. 1987; Meisterfeld 2002; Ogden blance observed between extant forms and 742 1983; Ogden and Meisterfeld 1989; Todorov and Mya old vase-shaped microfossils. For example, Golemansky 2007). The current systematic the fossil Palaeoarcella athanata shares a striking scheme of genus Difflugia is inconsistent and resemblance with the extant genus Arcella (Porter unsatisfactory due to the lack of good diagnos-tic et al. 2003). This conservatism contrasts with the characters, often inadequate descriptions and a derived position of this genus in molecular phy- general lack of molecular data (Gomaa et al. logenies of genus Arcella with respect to other 2012). It is thus difficult to determine which traits Arcellinida (Gomaa et al. 2012; Lahr et al. 2013) are genetically fixed and which represent variable and would thus suggest that Arcellinida as a whole characters, and this makes the evaluation of their are much older than 742 Mya (Fiz-Palacios et al. stability throughout evolutionary times even more 2013). challenging. Given the paleontological significance However, such morphological conservatism is of Arcellinida it is crucial to better understand the not the rule in all protist groups. Recent spe- relationships between morphological and molecu- ciation events have been demonstrated in coc- lar diversity within this group, both overall and colithophorids, where the morphs Calcidiscus within groups of closely-related taxa. leptoporus ssp. leptoporus and quadriperforatus To address these questions, we studied the diverged only 0.3 Mya ago as demonstrated both spinose and spineless morphotypes of Difflugia by the fossil record and molecular clock approaches tuberspinifera, a freshwater planktonic raptorial (Saez et al. 2003). Instances of rapid evolutionary species described from China (Han et al. 2008; radiations can also be expected in ciliates inhabit- Yang et al. 2004). D. tuberspinifera was described ing tank bromeliads in the neotropics, where large as a spherical agglutinated shell, ornamented by predatory forms and closely related diminutive two to eight conical hollow spines at the upper 3 equatorial region with a short collar and an aper- the D. tuberspinifera insertion, but differing in both ture ornamented with 6-11 tooth-like structures. sequence and length. Spineless individuals that were otherwise highly We observed no intra-morphotype sequence similar in shape and structure to D. tuberspinifera polymorphism in the sequenced portion of the SSU were also often observed in the same bodies of rRNA gene among the D. tuberspinifera sequences. water (Yu et al. 2014). It remained unclear if the The sequences of the SSU rRNA exon regions in absence of spines was genetically determined, spinose and spineless morphotypes of D. tuber- and, more generally, if the presence or absence of spinifera were highly similar to each other (99.6% spines is a valid taxonomical criterion for Arcellinid identity). testate amoebae in general (e.g. including also The general topologies of the SSU rRNA gene other genera such as Centropyxis or Argynnia) or phylogenetic trees inferred from maximum if it is just an expression of phenotypic plasticity as likelihood and Bayesian inference were similar was suggested in the case of the Centropyxis (Fig. 2) and correspond to previously published aculeata (but with no molecular data existing for data by Gomaa et al. (2012). Most members of that group of taxa) (Lahr et al. 2008). If this differ- Arcellinida branch together in a single well sup- ence was genetically determined, its high ported clade that receives 93% bootstrap support variability would suggest that this is a fast-evolving (BS), 94% expected-likelihood weights of local charac-ter. If no genetical difference was found rear-rangements edge support (LR-ELW; this would rather suggest the existence of different equivalent to approximate bootstraps) (Strimmer life-phases, such as the benthic-planktonic cycle and Rambaut 2002) and 1.00 Bayesian inference reported for Difflugia limnetica (Schönborn 1962), posterior prob-ability (PP). This main clade divides or an envi-ronmental control on morphology into two major groups; the first group comprises (Wanner 1999). We amplified and sequenced the Hyalospheniidae, Bullinularia spp., Centropyxis SSU rRNA gene of both spinose and spineless laevigata and the cylindrical/elongated Difflugia morphotypes of D. tuberspinifera and placed these spp. (D. acuminata, D. lanceolata, D. bacillariarum, sequences on a large Arcellinid tree together with D. hiraethogii) and receives moderate support another Eastern Asian species, D. mulanensis. In (BS=60%, ELW=85%, PP=0.80). The second addition, we also sequenced the internal group comprises genus Arcella, the globular/ transcribed spacer region (ITS1/5.8S/ITS2) of the spherical species Difflugia achlora, Netzelia two D. tuberspinifera forms and of D. labiosa. oviformis, and the newly obtained sequences of D. mulanensis and D. tuberspinifera (both morphotypes). Physochila griseola, Argyn-nia Results dentistoma