Protist, Vol. 170, 8–20, February 2019 http://www.elsevier.de/protis Published online date 16 October 2018 ORIGINAL PAPER Phylogenomics of Thecamoebida (Discosea, Amoebozoa) with the Description of Stratorugosa tubuloviscum gen. nov. sp. nov., a Freshwater Amoeba with a Perinuclear MTOC 1 James T. Melton III , Fiona C. Wood, Jordan Branch, Mandakini Singla, and Yonas I. Tekle Spelman College, 350 Spelman Lane Southwest, Atlanta, GA 30314, USA Submitted March 19, 2018; Accepted September 18, 2018 Monitoring Editor: Sandra L. Baldauf Thecamoebida Smirnov and Cavalier-Smith, 2011 (Discosea, Amoebozoa) has been molecularly under- studied. The group until recently consisted of three genera containing species that live in terrestrial or aquatic environments. Here, we describe a fourth genus, Stratorugosa tubuloviscum gen. nov. sp. nov., which was isolated from a freshwater Amoeba proteus Ward’s Science culture. Although this species most closely morphologically resembles a large, rugose Thecamoeba, S. tubuloviscum gen. nov. sp. nov. can be differentiated from Thecamoeba spp. by the following: 1) the presence of definitive finger-like (lobate-like) subpseudopodia extending at both the anterior and lateral parts of the cell dur- ing locomotion; 2) a peculiar locomotive mechanism with two sections, frontal and back, of the cells moving in a pulling and piggyback movement, respectively; 3) the presence of fibrillar cytoplasmic microtubules (MTs) organized by a prominent, perinuclear microtubule-organizing center (MTOC). A phylogenomic analysis of 511 genes assembled from transcriptomic data showed that this new genus was highly supported as sister to Stenamoeba. Despite the variance in gross morphology, Stenamoeba and S. tubuloviscum gen nov. sp. nov. both have MTOCs unlike two Thecamoeba spp., which display dot-like cytoplasmic MTs and lack an MTOC. © 2018 Elsevier GmbH. All rights reserved. Key words: Amoebozoa; Thecamoebidae; phylogenomics; transcriptome; new genus; new species. 1 Corresponding author. e-mail [email protected] (J.T. Melton III). Abbreviations: ICC, Immunocytochemistry; MT, microtubules; MTOC, microtubule-organizing center; OG, orthologous group; SSU, small subunit rDNA (18S). https://doi.org/10.1016/j.protis.2018.09.002 1434-4610/© 2018 Elsevier GmbH. All rights reserved. Phylogenomics of Thecamoebida 9 Introduction et al. 2007). These three genera of Thecamoebidae are characterized by striate, rugose, and lingu- The amoebozoan family Thecamoebidae was orig- late morphologies (morphotypes sensu Smirnov inally described by Schaeffer (1926) for naked and Goodkov 1999; Smirnov and Brown 2004). amoebae with ovoid to circular shapes that lacked Species in this group mostly have a thin, electron- pseudopodial extensions and for the most part dense cell coat and often have a glycocalyx layer maintained a consistent shape. This group has (Dyková et al. 2010; Geisen et al. 2014; Goodfellow gone through several taxonomic expansions and et al. 1974; Page and Blakey 1979; Smirnov et al. contractions, and has been understudied in molec- 2011; Wylezich et al. 2015) that sometimes has ular studies. Schaeffer (1926) originally included glycostyle-like structures (as observed in Sappinia two genera in this family, Thecamoeba and diploidea; Michel et al. 2006). Rugipes. The type species of the latter (R. bilzi The monophyly of Thecamoeba, Sappinia, and Schaeffer, 1926) is now considered to be a tax- Stenamoeba has been supported by phylogenetic onomic synonym of Thecamoeba (Page 1969, analyses based on SSU-rDNA (18S) (Michel et al. 1971), and other members of this genus, R. vivax 2006; Pawlowski and Burki 2009; Shadwick et al. Schaeffer, 1926 and R. placidus Page, 1968, have 2009; Smirnov et al. 2007; Tekle et al. 2008), now been moved to separate vannellid genera a concatenation of SSU and actin genes (Lahr (i.e., Clydonella in Sawyer (1975) and Vannella et al. 2011), and phylogenomics with transcrip- in Bovee (1965), respectively). More recently, tome data (Kang et al. 2017). This clade has this family used to be comprised of eight gen- been defined using the name of the order, The- era of naked amoebae with a pellicle-like cell camoebida (Adl et al. 2005, 2012; Kang et al. coat that are morphologically diverse (i.e., stri- 2017), which will be used hereafter. Our recent ate, rugose, lingulate, and polytactic morphotypes) phylogenomic studies reported that Thecamoebida (Page 1987, 1988; Smirnov et al. 2011). These forms a strong affiliation to a robust clade Fla- genera included Thecamoeba Fromentel, 1874; bellinea (Dactylopodida + Vannellida) (Tekle et al. Sappinia Dangeard, 1896; Stenamoeba Smirnov, 2016; Tekle and Wood 2017); however, The- Nassonova, Chao, et Cavalier-Smith, 2007; Der- camoebida in these studies also included the mamoeba Page et Blakey, 1979; Paradermamoeba unstable taxon Vermistella with poor support and Smirnov et Goodkov, 1996; Parvamoeba Roger- lacked Sappinia in the phylogenetic analysis. This son, 1993; Pseudothecamoeba Page, 1988; and finding is contrary to previous classifications of Thecochaos Page, 1981. Thecamoebida within Longamoebia (Dermamoe- Increasing amounts of molecular data and mor- bida + Thecamoebida + Centramoebida) – a lin- phological characters are continuing to shape eage that included flat amoebae with pointed sub- our knowledge of this group. A closer examina- pseudopodia and elongated cell shape (Smirnov tion of this taxon resulted in major taxonomic et al. 2011). The monophyly of Longamoebia was revisions (see Smirnov et al. 2011). These revi- questioned in our most recent phylogenomic stud- sions include the transfer of genera previously ies (Tekle and Wood 2017). Similarly, another placed within the Thecamoebidae into other phylogenomic analysis with increased taxon sam- taxonomic groups (i.e., Dermamoeba and Para- pling also corroborates our findings and placed the dermamoeba to Dermamoebidae; Parvamoeba to Thecamoebida within the Flabellinia (Kang et al. Himatismenida- taxonomic revision made based on 2017). SSU and actin trees from Kudryavtsev et al. (2011)) Thecamoebida contains species that live in or have been placed as incertae sedis due to the freshwater, marine, and terrestrial environments high degree of variation in morphology from the (e.g., Brown et al. 2007; Geisen et al. 2014; core thecamoebids and lack of molecular data (i.e., Kudryavtsev and Hausmann 2009; Michel et al. the polytactic genera Pseudothecamoeba and The- 2012; Page 1977, 1983, 1988). Some species are cochaos placed as incertae sedis in Smirnov et al. potentially pathogenic to humans causing amoebic 2005) (Smirnov et al. 2011). Only three core gen- encephalitis (Sappinia diploidea in Gelman et al. era (i.e., Thecamoeba, Sappinia, and Stenamoeba) 2001; Gelman et al. 2003) and to other animals have remained in the family Thecamoebidae prior including fishes (Thecamoeba hoffmani in Sawyer to this study (Smirnov et al. 2011). Stenamoeba et al. 1974; two species of Stenamoeba, S. ama- stenopodia (previously Platyamoeba stenopodia in zonica and S. limacina, in Dyková et al. 2010) Page 1969) was originally identified as a vannellid and horses (Stenamoeba polymorpha in Peglar amoeba; however, SSU data showed that this iso- et al. 2016). Historically, over 20 Thecamoeba late clustered within the Thecamoebidae (Smirnov species have been described. This genus currently 10 J.T. Melton III et al. contains 11 species that are accepted taxonomi- the uroidal part of the cell is dragged forward. cally (combined from Page 1983, 1988; Smirnov Contains a perinuclear MTOC, and microtubules and Goodkov 1999; Kudryavtsev and Hausmann wrap around the nucleus. 2009). These species have either a rugose or a TYPE SPECIES: Stratorugosa tubuloviscum striated morphology and a pellicle-like cell coat Melton et Tekle (Page and Blakey 1979; Page 1988; Smirnov and Goodkov 1999; Smirnov et al. 2011). The- ETYMOLOGY: The name was derived from Latin in camoeba species range in length from as small two parts; “stratum” meaning “layer” and “rugosa” ␮ as 11 m (T. orbis Schaeffer, 1926 (Page 1977)) to meaning “wrinkled”. as large as over 150 ␮m (T. terricola Greeff, 1866 Stratorugosa tubuloviscum Melton et Tekle, n. sp. (Page 1977)). There are currently only three known DIAGNOSIS: Cells rugose and sometimes striate. species of Sappinia Dangaerd, 1986 that exhibit Cells broadly fan-shaped, triangular, to spatulate rugose, striate (Smirnov et al. 2011), and lingu- during locomotion. A knob-shaped or elongated late (Wylezich et al. 2015) morphologies and are uroid frequently present while moving. Cells chang- bi- to tetranucleate (Brown et al. 2007; Michel et al. ing direction bi- to tri-lobate. Lobose or finger-like 2006; Wylezich et al. 2015). These cells range in subpseudopodia sometimes present at anterior length from 35–85 ␮m as observed in S. pedata or posterior positions. Average locomotive cell (Brown et al. 2007). Six lingulate and sometimes sizes 89.8 ␮m (range from 47.5 ␮m to 134.9 ␮m) striate species are currently accepted taxonomi- in length and 72.6 ␮m (range from 34.7 ␮m to cally for Stenamoeba Smirnov, Nassova, Chao et 122.1 ␮m) in width. Floating cells with many Cavalier-Smith, 2007 (Page 1969 as Platyamoeba radiating pseudopodia up to 60 ␮m in length. Cells stenopodia; Dyková et al. 2010; Geisen et al. 2014; uninucleate (average: 10.6 ␮m; range: 8.5 ␮m Peglar et al. 2016; Smirnov et al. 2007), and are the to 13 ␮m; oval to round in shape) with a single, smallest thecamoebid cells ranging from 10.1 ␮m to central and spherical to oval-shaped