(Ciliophora, Hypotricha): Ontogenetic, Morphologic, and Molecular Data Suggest the Establishment of a New Genus Apourostylopsis N

The Journal of Published by 中国科技论文在线 http://www.paper.edu.cnthe International Society of Eukaryotic Microbiology Protistologists

J. Eukaryot. Microbiol., 58(1), 2011 pp. 11–21 r 2010 The Author(s) Journal of Eukaryotic Microbiology r 2010 International Society of Protistologists DOI: 10.1111/j.1550-7408.2010.00518.x Re-Evaluation on the Diversity of the Polyphyletic Genus Metaurostylopsis (Ciliophora, Hypotricha): Ontogenetic, Morphologic, and Molecular Data Suggest the Establishment of a New Genus Apourostylopsis n. g.

WEIBO SONG,a NORBERT WILBERT,b LIQIONG LIa and QIANQIAN ZHANGa aLaboratory of Protozoology, Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China, and bZoologisches Institut, Universita¨t Bonn, 53115 Bonn, Germany

ABSTRACT. The urostylid genus Metaurostylopsis Song et al., 2001 was considered to be a well-outlined taxon. Nevertheless, recent evidence, including morphological, ontogenetic, and molecular information, have consistently revealed conflicts among congeners, regarding their systematic relationships, ciliature patterns, and origins of ciliary organelles. In the present work, the morphogenetic and morphogenetic features were re-checked and compared, and the phylogeny of nominal species was analysed based on information inferred from the small subunit ribosomal RNA (SS rRNA) gene sequence. In addition, the binary divisional process in a new isolate of Meta- urostylopsis struederkypkeae Shao et al., 2008 is described. All results obtained reveal that the genus is a polyphyletic assemblage whose nominal congeners fall into three clades within the core Urostylida, based on SS rRNA gene sequences. These three clades not match the groups inferred from morphological/morphogenetical evidences. Some conflicting data from molecular and ontogenetic studies also indicate that single-gene information might not be consistently reliable in detecting the phylogenetic relationships among closely related groups and comprehensive multi-gene analyses are necessary to give a more exact evaluation for this divergent assemblage. According to our new understandings, five forms are confirmed to be true Metaurostylopsis. The morphotype Metaurostylopsis sinica Shao et al., 2008 should be excluded from the genus and represents a distinct type, and, thus, a new genus Apourostylopsis n. g. with it as the type specie, i.e. Apourostylopsis sinica (Shao et al., 2008) n. comb. Key Words. Hypotricha, Metaurostylopsis and Apourostylopsis, molecular phylogeny, new genus, ontogenesis.

HE ciliate group Hypotricha (s.l.) contains approximately positions of taxa (Chen et al. 2010b; Wang et al. 2011; Yi et al. T 1,000 nominal species and all of them exhibit complicated 2008). morphogenesis whose features of physiological reorganization In the present work, we have compared the ciliature patterns and ontogenesis play important roles in defining the relationships and modes of cortical development of all known members so far among them (Berger 1999, 2006, 2008; Chen et al. 2010b; assigned to this genus, and observed the morphology and mor- Foissner 1996; Foissner et al. 2010; Hemberger 1982; Ku¨ppers phogenesis of a new isolate of Metaurostylopsis struederkypkeae and Claps 2010; Liu et al. 2010; Paiva and Silva-Neto 2009; Shao Shao et al., 2008, and we have analysed the systematic relation- et al. 2007a). The monophyletic origin of this assemblage is ba- ship of this species complex based on sequences of the SS rRNA sically supported by both small subunit ribosomal RNA (SS gene. We had three main aims: (1) to re-check the systematic rRNA) gene phylogenies (Bernhard, Stechmann, and Foissner arrangement of this assemblage, (2) to get further insight into the 2001; Huang et al. 2010; Jiang et al. 2010a, b; Paiva et al. 2009; diversity of their ontogenetic processes, and (3) to confirm the Schmidt et al. 2007), by the conservative ciliature patterns, and by distinction of congeners using more comprehensive data. features of stomatogenesis (Berger 1999, 2006, 2008; Berger and Foissner 1997; Borror 1979; Chen et al. 2010c; Li et al. 2010). On the other hand, hypotrichs display highly diverse somatic struc- MATERIALS AND METHODS tures and the origin of the ciliature as well as the modes of cortical Sample collection, morphological and morphogenetic stud- development divide them into many polyphyletic subgroups (Ber- ies. All isolates used in the present studies were either from the ger 2006; Foissner and Stoeck 2008; Foissner et al. 2004; Jiang cell bank of the Laboratory of Protozoology, Ocean University of and Song 2010; Song et al. 2009). Nevertheless, recent studies China, which were collected either from costal areas near Qingdao using molecular information have consistently questioned the (Tsingtao, 1201180E; 361040N) or from the Daya Bay, Southern current systematic arrangement regarding the relationships and China (1141320E; 221420N). Material identification and cultiva- assignments of many families and orders which were traditionally tion were carried out according to Song et al. (2001). Morpho- believed to be monophyletic, such as many taxa in the orders logical and morphogenetic studies on M. struederkypkeae were Urostylida, Sporadotrichida, and Stichotrichida (Hu et al. 2009b; performed after Wilbert (1975) and Li et al. (2009b). To illustrate Lynn 2008; Yi et al. 2009a). This bewildering situation makes it the changes occurring during morphogenesis, old (i.e. parental) difficult to describe their evolutionary relationships using only a cirri are depicted with contour whereas new cirri are shaded black. single line of evidence (i.e. morphological/morphogenetical or Terminology and systematics are mainly according to Song et al. molecular) (Hu et al. 2009a). (2001) and Berger (2006). The urostylid genus Metaurostylopsis currently contains eight DNA extraction, polymerase chain reaction (PCR) amplifi- morphotypes (Table 1) (Chen, Huang, and Song 2010a; Lei et al. cation, and sequencing. Two populations of Metaurostylopsis 2005; Shao et al. 2008a, c; Song and Wilbert 2002; Song, Petz, cheni and one population of Metaurostylopsis flavicans were and Warren 2001; Wang et al. 2011), and morphogenesis has been treated in the present work. Genomic DNA extraction, PCR am- reported for four of them (Chen et al. 2010a; Shao et al. 2008c; plification, and sequencing of the SS rRNA gene were performed Song et al. 2001). Recent reports indicate that there is greater according to Gong et al. (2009). The SS rRNA gene was amplified diversity within the genus, leading to confusion regarding the by PCR with primers Euk A (50-AACCTGGTTGATCCTGC identification of species, genus definition, and the systematic CAGT-30) and Euk B (50-TGATCCTTCTGCAGGTTCACC TAC-30). Polymerase chain reaction amplification and sequenc- Corresponding Author: W. Song, Laboratory of Protozoology, Insti- ing were according to Miao et al. (2009). tute of Evolution & Marine Biodiversity, Ocean University of China, Phylogenetic analyses. The new SS rRNA gene sequences, Minxing Building B, Qingdao 266003, China—e-mail: wsong@ouc. together with sequences of 52 hypotrichous taxa (s.l.) downloaded edu.cn from GenBank database (see Fig. 37 for GenBank accession 11 转载中国科技论文在线 http://www.paper.edu.cn

12 J. EUKARYOT. MICROBIOL., 58, NO. 1, JANUARY– FEBRUARY 2011

numbers), were aligned using CLUSTAL W implemented in Bio- buccal cavity. Subsequently, seven morphotypes have been edit 7.0 (Hall 1999). Phacodinium metchnikoffi was selected as the assigned to this genus (Chen et al. 2010a; Lei et al. 2005; Shao out-group species. Ambiguously aligned regions and gaps were et al. 2008a, c; Song and Wilbert 2002; Wang et al. 2011) (Table 1). excluded before phylogenetic analyses. Bayesian inference (BI), Characteristically, all known forms are found in marine or maximum likelihood (ML), and maximum parsimony (MP) were brackish waters and all of them seem to have remarkable cortical used to construct phylogenetic trees. Model selection and phylo- granules, which often render the cell coloured (e.g. yellow-brown- genetic analyses were according to Yi et al. (2009c). ish, reddish, or yellow-greenish) (Fig. 4–6). Three morphotypes The AU tests were used to test for competing phylogenetic even have two types of granules, that is, larger and smaller ones hypotheses of monophyly of the genus (Shimodaira 2002), with (Table 1). The larger granules are mostly grouped and located the constraint tree generated by PAUP. The MP analysis was per- along/near the ciliary organelles while the smaller ones are usu- formed with the software package PAUPÃ 4.0b10, and support for ally sparsely distributed on both sides of the cell. the internal branches was estimated using the bootstrap method Three morphological groups can be recognized according to the with 1,000 replicates (Yi et al. 2010). TREEVIEW v1.6.6 and ciliature patterns. Group I consists of five members, making the MEGA 4.0 were used to visualize tree topologies (Tamura et al. ‘‘core group,’’ with a pattern typical of the type species (Fig. 3) 2007). (Chen et al. 2010a; Lei et al. 2005; Shao et al. 2008a, c; Song et al. 2001; Song and Wilbert 2002). In contrast, groups II and III have only one or two species: Metaurostylopsis sinica (group II; Fig. 1), RESULTS Metaurostylopsis songi and M. flavicans (group III; Fig. 2). Morphology and infraciliature of nominal species in Meta- Among them, M. sinica differs from the group I in (1) having urostylopsis. The genus Metaurostylopsis was established by only two frontoterminal cirri that are generated from splitting of Song et al. (2001) based on the following morphological and mor- the last FVT-cirral anlage (vs. four or more in group I, see below); phogenetical characters: having a long row of frontoterminal cirri (2) lacking the genus-diagnostic ventral row of single cirri that come from the whole rightmost or last frontoventral (FVT)- whereas the midventral, staggered row extends caudally almost cirral anlage; frontal cirri clearly differentiated; typical urostylid to the transverse cirri (vs. typically present and posterior to the midventral rows or a midventral complex, short and terminating at equatorial termination of the very short midventral row in group about the cell equator, and then continued as a single-rowed ven- I); and (3) possessing several pretransverse cirri (vs. absent in tral row; buccal and transverse cirri present; on both cell sides, group I) (Fig. 1). Group III, represented by M. songi and M. flavi- more than one row of marginal cirri that derive intrakinetally from cans, is an intermediate type, also has two frontoterminal cirri but individual anlagen within each parental row; oral primordium for lacks the pretransverse cirri, and its midventral complex extends the proter developing from a ‘‘pocket’’ beneath the pellicle of posteriorly only to about two-thirds of cell length with several

Fig. 1–6. Infraciliature (1–3) and living morphology (4–6) of species in the Metaurostylopsis complex (1, 5, Apourostylopsis sinica n. g., n. comb.; 2, 4, Metaurostylopsis ﬂavicans (incertae sedis); 3, 6, Metaurostylopsis struederkypkeae) (Fig. 1, 5, from Shao et al. 2008c, 2, 4, from Wang et al. 2011; with permission; Fig. 3, 6, original). 1–3. Ventral views, to show the infraciliature on ventral side (arrow in two marks some non-zigzagged cirri, while the arrowhead points to the frontoterminal cirri). 4. Ventral view of a typical specimen. 5. Ventral view, to show the larger cortical granules (arrows). 6. Dorsal view, to show the cortical granules; black arrowheads mark the larger cortical granules, while small white arrows indicate the tiny ones. AZM, adoral zone of membranelles; BC, buccal cirrus; FC, frontal cirri; FTC, frontoterminal cirri; LMR, left marginal rows; MVR, midventral rows (midventral complex); PTC, pretransverse cirri; RMR, right marginal rows; TC, transverse cirri; VR, ventral row. Scale bars in Fig. 1, 2, 4, 5 5 50 mm, in Fig. 3 5 20 mm. 中国科技论文在线 http://www.paper.edu.cn

SONG ET AL.—DIVERSITY AND PHYLOGENY OF METAUROSTYLOPSIS 13

irregularly arranged cirri (ventral cirri ?), which are positioned far a away from the transverse cirri (Fig. 2; Lei et al. 2005; Wang et al. 30–60 2011). Morphogenesis of Metaurostylopsis struederkypkeae Shao (2011) et al., 2008. Based on a new isolate collected from the beach Wang et al. near Qingdao, north China, we observed its morphogenetic processes during the binary ﬁssion. Because it exhibits, nevertheless, exactly the same mode of cortical development as reported for the type species, Metaurostylopsis marina (see Song et al. 2001), we only highlight the critical points and some minor differences here 25–35 130–200

(Fig. 7–35): Shao (1) The entire parental ciliature, including the old adoral zone

et al. (2008c) of membranelles (AZM), is completely renewed with a typical M. marina mode (Fig. 11–21). (2) The new AZM and undulating membranes in the proter originate from a newly formed oral primordium located beneath the surface of the buccal cavity (thus genus

20–30 100–120 typical), that is, in a de novo and subapokinetal mode

(Fig. 7–11). (3) The new frontoterminal cirri, as a short row, come from the

present work non-splitting, rightmost FVT-cirral anlage in both dividers

Shao et al. (2008a); (Fig. 16, 18, 20). Thus, they are formed in an apomorphic way. (4) In at least in some individuals, the generation of dorsal kineties in the opisthe seems to be ‘‘abnormal,’’ possible 40–60 90–120 one more anlage appears near the middle one (Fig. 12, double arrowheads; Fig. 17, arrows); but later, such extra

(2010a) structure might be absorbed and thus the ﬁnal number in Chen et al. the non-dividing stage will be invariably three (Fig. 15, 17,

Type II: colourless Type II: dark-reddish Type II: colourless 19, 21). (5) Fusion of the macronuclear nodules results in an irregular mass with only a few branches (Fig. 12, 30).

assemblage. (6) Usually two to three pairs of dorsal cilia (dikinetids; Fig. 15–30 100–140 17, arrowheads; 18, arrows) are positioned anterior to the rightmost marginal row (RMR) (Fig. 19, arrows). Their

M. salina M. cheni M. struederkypkeae Apourostylopsis sinica M. flavicans origin is undetermined, yet very likely is from the anterior

Lei et al. (2005); part of the right marginal row anlage as revealed in other Shao et al. (2008c) congeners. Morphogenesis among reported nominal species in Meta- a

20–35 40–120 urostylopsis. Cortical development and/or morphogenesis during

cell division has been described previously in four morphotypes,

(2005) M. marina, M. sinica, M. cheni, and M. struederkypkeae (Chen Lei et al. et al. 2010a; Shao et al. 2008c; Song et al. 2001; current work). Among those, two basic modes, corresponding to the morpholog-

Metaurostylopsis–Apourostylopsis ical groups I and II can be recognized: M. sinica represents a type of its own while the other one is shared by the remaining three, 50–90 90–150 including the type species M. marina. The main processes can be summarized as follows. (2002) Oral primordia (OP). All forms share the same processes in the origin and development of the oral structures: two sets of OP Song and Wilbert are formed for both proter and opisthe. In the proter, the OP is formed de novo and subcortically in the buccal ﬁeld, and the new UM anlage is also generated near the OP beneath the cell surface; 50–80 150–300 the old oral structure will be completely replaced later by the new

3–63–53–5 5–8 6–9 6–7 2–3 3 3–5 3ones. In 2–4the opisthe, 3 4 as usual 3–4 in other 3 4–6 urostylids, 4–5 both 2 3 OP and 3 UM 2 2 4 3 (2001) M. marina M. rubra M. songi Colourless Dark-reddish Colourless Colourless Type I: yellow-green Type I: yellow-green Type I: yellow Bright yellow anlage are developed apokinetally on the surface, and no parental

80–120 structures are involved in forming the OP and UM primordia (see also Fig. 7–11, 13, 14, 16, 18).

, for their taxonomic deﬁnition, see text and Table 2. Frontoventral transverse cirral anlagen (FVTCA). These cirral anlagen are formed apokinetally and none of the old cilia-

Morphological comparison of morphotypes in ture seems to join the development of these primordia. However,

1. two stable and dissimilar types/modes can be recognized among all nominal species. Incertae sedis a Table In the marina–cheni–struederkypkeae type, the core group, the m) m Body size in vivo ( Body colourCortical granules, colour Colourless Brick-reddish Colourless Reddish Colourless Rose-reddish Yellow-brownish Yellow-brownish Characters No., frontoterminal cirri No., midventral pairsVentral rowNo., left marginal rows 7–11No., right marginal rows No., transverse cirriData source Present 8–11 5–9 Present Song et al. 9–12 4–6 Absentfollowing 4–7 Present structures 6–7 are Present 5–9 formed 2–5 from Present the 4–7 FVTCA: 5–8 (1) Absent four 11–15 clearly 2–5 Absent 13–17 5–8 7–10 中国科技论文在线 http://www.paper.edu.cn

14 J. EUKARYOT. MICROBIOL., 58, NO. 1, JANUARY– FEBRUARY 2011

Fig. 7–13. Morphogenesis of Metaurostylopsis struederkypkeae at early to middle stages. 7. Ventral view, to show the oral primordium in the opisthe (double arrowheads) and proter (arrow) respectively. 8, 9. Ventral view, arrowheads indicate the frontoventral (FVT)-cirral anlagen, which are formed de novo, arrows indicate the undulating membrane anlagen, while double arrowheads mark the newly formed anlagen for the marginal rows. 10. A divider in middle stage, arrowheads show the FVT-anlagen, arrows mark the anlagen of marginal rows. 11, 12. Ventral and dorsal views of the same divider, arrowheads mark the dorsal kinety anlagen, while the double arrowheads indicate the extra and irregularly formed anlage(n) (abnormal ? Only on stage was obtained). Note that the macronucleus (Ma, arrow) is in a mass with four branches. 13. Ventral view, arrows mark the ﬁrst frontal cirrus in both dividers arising from the undulating membranes anlagen. Scale bars 5 25 mm.

differentiated frontal cirri; (2) usually more than four frontoter- form the single ventral row posterior to and continuous with the minal cirri, which come from the non-splitting rightmost cirral midventral rows in the core group). anlage; (3) a single buccal cirrus; (4) a relatively short midventral Marginal row anlagen. Both groups show a process similar complex, which terminates equatorially; (5) a short, single-rowed to other related urostylids: in both proter and opisthe they are ventral row; and (6) several transverse cirri (see also Fig. 8–11, formed intrakinetally in an early divisional stage. These anlagen 13, 14, 16, 18, 20). then stretch and gradually replace the absorbed parental rows (see In the contrast, signiﬁcant differences can be seen in the also Fig. 10, 11, 13, 14, 16, 18, 20). FVTCA of the sinica type including: (1) the rightmost anlage Dorsal kinety anlagen. The dorsal kinety development splits into two parts, of which the anterior one moves anteriad exhibits the same mode in both groups, and it is also characteristic becoming the frontoterminal cirri while the posterior part gener- of most urostylids: three anlagen are developed intrakinetally in ates one transverse and likely also one pretransverse cirrus (vs. both dividers. They subsequently elongate and replace the three pa- migrating as a whole structure towards anteriorly and becoming rental dorsal kineties. During the division process, neither fragments the frontoterminal row in the core group, above); (2) the anlage in anlagen nor caudal cirri are formed (see also Fig. 12, 15, 17, 19). n-1 (i.e. the second one from right) will become the pretransverse In addition, dividers are frequently observed with a few dikin- and transverse cirri, but not the ventral row in M. sinica (vs. will etids anterior to the RMR in both proter and opisthe. These 中国科技论文在线 http://www.paper.edu.cn

SONG ET AL.—DIVERSITY AND PHYLOGENY OF METAUROSTYLOPSIS 15

Fig. 14–21. Middle and late morphogenetic stages of Metaurostylopsis struederkypkeae. 14, 15. Ventral and dorsal views of the same divider, arrows in 14 mark the last frontoventral (FVT)-anlage, while in 15 indicate the dorsal kineties anlagen. Note the macronucleus is now highly branched. 16, 17. Ventral and dorsal views of the same divider, note that the last FVT-anlage remains unmoved anteriad (arrows in 16); arrows in 17 mark two new dorsal kineties between new DK 1 and DK 3. 18, 19. Ventral and dorsal views of a late divider, to show the newly formed frontoterminal cirri (arrows in 18), the transverse cirri (arrowheads in 18), and the dorsal kineties (arrowheads in 19). Note the old ‘‘extra’’ dikinetids remain unabsorbed (arrows in 19). 20, 21. Ventral and dorsal views, to show the frontoterminal cirri (arrows) and the single-rowed ventral row in both dividers (arrowheads). Scale bars 5 40 mm.

additional dikinetids are formed in the same mode, as seen at least New SS rRNA gene sequence and phylogenetic analyses in M. marina, M. cheni, and M. sinica, and generated from the Data newly sequenced in the present work. In our current rightmost marginal anlage rather than from the dorsal kinety anl- work, the SS rRNA gene of three isolates had length (bp), GC agen (Chen et al. 2010a; Shao et al. 2008c; Song et al. 2001). content, and GenBank accession numbers as follows: 1,767, Division of the nuclear apparatus. Macronuclear division 44.7%, and FJ775720 for M. cheni population JJM2007050803; demonstrates a unique feature in the marina–cheni–struederkyp- 1,767, 44.6%, and HM623916 for M. cheni population keae type: brieﬂy, all macronuclear nodules fuse to form a CXM09050701; and 1,768, 44.5%, and HM623917 for M. ﬂavi- complicated branch-formed structure (Fig. 15) during the middi- cans population CXM08112803. visional stage (vs. generally an oval to bar-shaped mass in Excluding primers, the sequence of M. struederkypkeae differs M. sinica and all other known urostylids). by 44 bp from Metaurostylopsis salina, 82 bp from M. sinica, 中国科技论文在线 http://www.paper.edu.cn

16 J. EUKARYOT. MICROBIOL., 58, NO. 1, JANUARY– FEBRUARY 2011

Fig. 22–35. Photomicrographs of Metaurostylopsis struederkypkeae during morphogenesis (after protargol impregnation). 22, 23. Ventral views of an early divider, to show the oral primordium in the proter (arrow in 22) and the opisthe (arrow in 23). 24, 25. About the same stage, ventral views, to indicate the undulating membrane anlagen (arrowheads) and the frontoventral (FVT)-cirral anlagen (arrows) in the proter (24) and opisthe (25). 26. To indicate the anlagen of the right marginal rows (arrows) in a middivider. 27, 28. Ventral and dorsal views, arrows indicate the left marginal row (27) and dorsal kinety (28) anlagen. 29. Ventral view to show the streak-like FVT-cirral anlagen (arrows). 30. To show the branched macronucleus (arrow) fused by ‘‘numerous’’ nodules. 31, 32. Ventral views, to show the anlage for frontoterminal cirri (now as the last FVT-cirral anlage, double arrowheads) and the ventral row (arrows). 33. Dorsal view of late divider, to show the newly formed dorsal kineties (arrows). 34. Ventral view, noting the frontoterminal row (arrow) and the single-rowed ventral row (arrowhead). 35. A specimen just before division, ventral view.

95–96 bp from the M. flavicans populations, and 40–43 bp from ent bootstrap values (Fig. 37). Clade I is a strongly supported four populations of M. cheni (Fig. 36). The genetic distance group with six isolates belonging to three species. The four among species in Metaurostylopsis assemblage was 0.008– populations of M. cheni group together, and associate first with 0.058. The four populations of M. cheni differ by one to three M. salina with maximum support and then cluster with nucleotides, excluding primers while the two populations of M. struederkypkeae forming a well-supported lineage (100% M. flavicans differ by only one nucleotide (Fig. 36). ML, 1.00 BI, 100% MP). This group is then a weakly supported Phylogenetic analyses based on SS rRNA gene data. sister branch (74% ML/0.96 BI/53% MP) to Clade II, which Trees constructed by all three methods have similar topologies has only one former Metaurostylopsis species, now Apourosty- and, thus, are combined into a single consensus tree for clarity of lopsis sinica n. comb. In Clade II, A. sinica is basal to the presentation. In all phylogenetic analyses, all nine populations of Thigmokeronopsis–Apokeronopsis complex with low support five species in the Metaurostylopsis complex, composed of Meta- (54% ML, 0.87 BI). Clade III, which had maximum support in urostylopsis and Apourostylopsis n. g. (for the latter, see below in all the trees, has two populations of M. flavicans and one Ante- discussion part), are divergently positioned in the core Urostylida holosticha species. Clade III is positioned deeply and as an ‘‘out- (95% ML, 1.00 BI, 88% MP) but fall into three clades with differ- group’’ to the large cluster containing the most Metaurostylopsis 中国科技论文在线 http://www.paper.edu.cn

SONG ET AL.—DIVERSITY AND PHYLOGENY OF METAUROSTYLOPSIS 17

Fig. 36. The E23 region of the small subunit ribosomal RNA sequences of nine populations of ﬁve Metaurostylopsis species and Apourostylopsis n. g. The differences in sequence length are compensated by introducing alignment gaps (-) in the sequences. Matched sites are marked with dots. Note that all sites in four populations of Metaurostylopsis cheni are completely matched. Arrow marks the only unmatched site between two populations of Metaurostylopsis ﬂavicans.

(Clade I), the Thigmokeronopsis–Apokeronopsis–Apourostylopsis Hence, the morphogenesis-based groups are consistent with assemblage (Clade II), and an ‘‘inserted’’ clade consisting of four grouping assignment inferred from the morphological information urostylid genera (100% ML, 1.00 BI, 99% MP). though the group III (i.e. M. flavicans and M. songi) is as yet undescribed morphogenetically. Evidences from molecular data. Based on the SS rRNA DISCUSSION gene sequence for five species (i.e. M. salina, M. struederkypkeae, All morphological, morphogenetical, and molecular infor- M. flavicans, M. cheni, and A. sinica n. comb.), they were always in mation indicating the genus Metaurostylopsis to be a divergent three clusters: the main branch (Clade I) corresponds to the mor- assemblage phological/morphogenetic group I, the core Metaurostylopsis, Evidences from morphology or/and morphogenesis. As which indicate that this is a well-outlined group. As to the two mentioned in the morphological part, three clearly separated types morphologically similar species (i.e. M. flavicans and A. sinica), can be recognized in all eight nominal species: the main group the fact that M. flavicans and A. sinica branch to two different (or the core Metaurostylopsis) including the type species contains clades outside Clade I shows that they cannot be considered as core five morphotypes, while the remaining three forms (i.e. M. sinica, forms (Clade I) assigned to the genus Metaurostylopsis.InAU M. songi and M. flavicans) represent other two groups. Further- tests, the constraint tree with all nominal species grouping together more, at least two groups can be defined based on morphogenetic was obviously rejected (AUo0.005), while the association with features among the four species that have been morphogenetically M. flavicans individually to the Clade I was also clearly rejected investigated (Chen et al. 2010a; Shao et al. 2008b; Song et al. (AUo0.001). On the other hand, the AU test in this database 2001; present work). Similar to the morphological studies, shows that a monophyletic clade containing A. sinica and Clade I M. sinica shows a different mode to that of the core Metaurosty- cannot be rejected by (AU40.1), nevertheless, we prefer to lopsis. The main differences include (1) the macronuclear nodules acknowledge the divergent topologies due to the rather low node fuse into a branched structure before cell division in the core supports (54% ML, 0.87 BI). Hence, the molecular data strongly Metaurostylopsis (vs. to form a non-branched, sausage-like mass question the morphology-based relationship between them and in M. sinica); (2) FVT-cirral anlage n-1 forms a short ventral row indicate that two forms should not belong to the same genus. posterior to the zigzagged midventral rows (i.e. the midventral In summary, the morphological, morphogenetical, and molec- complex) in the core Metaurostylopsis (vs. generates an extra cir- ular data consistently demonstrate that the genus Metaurostylopsis rus, the pretransverse one and one transverse cirrus in M. sinica); contain five species: M. marina, M. rubra, M. salina, M. cheni, and (3) all the cirri formed from the rightmost FVT-anlage (i.e. and M. struederkypkeae. Whereas the morphospecies, M. sinica streak n) migrate anteriad and become the frontoterminal cirri in Shao et al., 2008, should be excluded from this genus and be the core Metaurostylopsis (vs. the streak splits into two parts assigned to a new genus Apourostylopsis n. g. (see below in text; and forms two frontoterminal cirri in the anterior part, while the Table 2). posterior one develops likely to a pretransverse and a transversal With references to two morphologically related forms, M. flavi- cirrus in M. sinica). cans and M. songi, discrepancies in the molecular (only for the 中国科技论文在线 http://www.paper.edu.cn

18 J. EUKARYOT. MICROBIOL., 58, NO. 1, JANUARY– FEBRUARY 2011

Fig. 37. Phylogenetic tree based on small subunit ribosomal RNA gene sequences, showing the position of Metaurostylopsis species and Apouro- stylopsis n. g. by maximum likelihood (ML) applying the GTR1G1I model. All nominal forms in the Metaurostylopsis–Apourostylopsis assemblage are shaded in grey. The newly sequenced forms are indicated with asterisks (Ã) and in bold type. Numbers near branches denote ML bootstrap value/Bayesian inference (BI) posterior probability value/maximum parsimony (MP) bootstrap vales. Dashes indicate topologies that differ in the ML, BI, and MP phylogenies while nodes that were maximally supported (498% ML, 1.00 BI, 498% MP) are indicated by solid circles. The scale bar corresponds to ten substitutions per 100 nucleotide positions. Inset ﬁgures from top to bottom: from Wang et al. (2011), Chen et al. (2010a), and Shao et al. (2008c) with permission, to show the morphology and infraciliature of representatives of related groups. Scale bars 5 50 mm.

former) and morphological data suggest neither assignment to any Wang et al. 2011). Unfortunately, ontogenetic data are not yet known genera nor a close relationship with them (Lei et al. 2005; available for both forms, and these might provide some critical information about the origin of ciliature. Thus, the systematic Table 2. List of eight species in Metaurostylopsis complex. positions of M. flavicans and M. songi remain, in our opinion, unresolved at the moment. Hence, they should be considered Basionym or misidentified Current name incertae sedis. Phylogenetic positions of the Metaurostylopsis–Apourosty- Urostyla marina Kahl, 1932 M. marina lopsis complex. All molecular data support the placement of Paraurostyla marina (Kahl, 1932) As above Metaurostylopsis–Apourostylopsis taxa within the order Urostyl- Borror, 1972 Urostyla thompsoni Jankowski, 1979 sensu As above ida. Concerning the relationships of Metaurostylopsis with other Wiackowski (1991; par lapsus) related urostylids, however, no clear solution is provided by the M. rubra Song and Wilbert, 2002 M. rubra SS rRNA gene information because of the low bootstrap values in M. songi Lei et al., 2005 Incertae sedis our trees. As revealed in the topologies, Metaurostylopsis is (genus not confirmed) always the sister group to the Thigmokeronopsis–Apokeronopsis M. salina Lei et al., 2005 M. salina lineage while the latter is clearly separated from the morpholog- M. struederkypkeae Shao et al., 2008 M. struederkypkeae ically similar genus Pseudokeronopsis, which is located as a a Metaurostylopsis sinica Shao et al., 2008 Apourostylopsis sinica peripheral branch to other core urostylids in all the trees. Never- M. flavicansWang et al., 2011 Incertae sedis theless, based on both morphological and morphogenetic features, (genus not confirmed) M. cheni Chen et al., 2010 M. cheni Thigmokeronopsis and Apokeronopsis appear to be more closely related to Pseudokeronopsis than to Metaurostylopsis (Li et al. aNew combination suggested in the present work. Italics signify con- 2009a; Shao et al. 2007b, 2008a, b). For example, all three genera firmation proved in the current paper. have a Pseudokeronopsis-like ciliature pattern, including a 中国科技论文在线 http://www.paper.edu.cn

SONG ET AL.—DIVERSITY AND PHYLOGENY OF METAUROSTYLOPSIS 19

bicorona of frontal cirri and single marginal row on each side (vs. Table 3. Morphological and morphogenetical comparison of Metauro- distinctly differentiated frontal cirri and several marginal rows on stylopsis and Apourostylopsis n. g. each side in Metaurostylopsis). Furthermore, during morphogenesis, no clearly differentiated frontal cirri are developed from the Characters Metaurostylopsis Apourostylopsis n. g. FVT-anlagen in Apokeronopsis, Thigmokeronopsis, and Pseudo- Ventral row (VR)a Present Absent keronopsis (Berger 2006; Li et al. 2009a; Shao et al. 2008b, c). On Pretransverse cirri Absent Present the other hand, Metaurostylopsis and Pseudokeronopsis share (PTC) some features, which are absent in Thigmokeronopsis and Formation of In the apomorphic In the plesiomorphic Apokeronopsis: the cirri of the midventral rows are arranged in frontoterminal cirri wayb way a typical urostylid zigzag pattern in the former two whereas they Midventral complex Short, terminating at Long, extending to are arranged in two distinct midventral rows in Thigmokeronopsis (MVR) about cytostome subcaudal level and Apokeronopsis. Additionally, the marginal and dorsal kineties level in Metaurostylopsis and Pseudokeronopsis develop intrakinetally Fate of FVT-cirral Forming completely Forming PTC, TC anlage n-1c VR and possibly the last (vs. apokinetally in Apokeronopsis and Thigmokeronopsis)(Hu, cirral pair in MVR Warren, and Song 2004; Petz 1995; Song et al. 2001). A similar Macronuclear Fusing to form a To form a single, situation can be also seen in A. sinica and in the morphospecies, nodules in branching mass non-branching mass M. flavicans, which are separated by four divergent urostylid gen- middivisional stage era in the gene trees while morphology indicates that they could be closely related though no morphogenetical data are available in aSingle cirral row posterior to the zigzagged midventral complex. b the latter (Shao et al. 2008c; Wang et al. 2011). This refers to the origin of the frontoterminal cirri: the whole rightmost Thus, the conflict between morphological/morphogenetical and FVT-cirral anlage moves anteriad and becomes the FTC (vs. plesiomorphic: splits into two parts, while only the anteriormost cirri migrate molecular data rejects a consistent assignment for the members in anteriorly. the Metaurostylopsis–Apourostylopsis assemblage. Extreme cThe second FVT-cirral anlage from the right. confusion comes from the SS rRNA gene information, which FVT, frontoventral transverse; FTC, frontoterminal cirri; TC, transverse challenge almost all the ‘‘traditional’’ arrangement based on mor- cirri. phology and morphogenesis. This contradiction might indicate that the SS rRNA gene is not a good marker to clarify the exact relationship among these highly divergent species and genera, as of the following characters: (1) lacking the single-rowed ventral has been noted for the oxytrichid stichotrichs (Foissner et al. 2004; row which is characterized seen in the latter; (2) present one or Yi et al. 2009a). Yet another reason might be the low sampling several pretransverse cirri (vs. absent in Metaurostylopsis); (3) size of taxa analysed, as noted by others (Foissner et al. 2004; frontoterminal cirri generating from the anterior part of the right- Hu et al. 2009a; Paiva et al. 2009; Stru¨der-Kypke and Lynn 2010; most FVT-cirral anlage (vs. from whole anlage in Metaurosty- Yi et al. 2009a, b; Zhang et al. 2010). lopsis); and (4) midventral rows long and extending to the Establishment of a new urostylid genus Apourostylopsis. As posterior cell end (vs. conspicuously shortened, typically termi- argued above, a new genus Apourostylopsis is suggested according nated at about cytostome level). In addition, the macronuclear to both morphological/morphogenetical and molecular information: nodules fusing into a branching mass (vs. a normal single mass in Order Urostylida Metaurostylopsis) is likely also one of criteria to separate both Family Urostylidae genera (Chen et al. 2010a; Shao et al. 2008c) (Table 3). Apourostylopsis n. g. Diagnosis. Marine Urostylidae with several marginal rows on ACKNOWLEDGMENTS both sides; frontoterminal and transverse cirri clearly differentiated; frontoterminal cirri present which derived from the anterior This work was supported by the ‘‘the Natural Science Foun- part of the rightmost FVT-cirral anlage; single buccal and pre- dation of China’’ (Project No. 31030059), and partly supported by transverse cirri present; midventral rows arranged in Holosticha- a visiting grant by DAAD (Deutscher Akademischer Austausch mode without continued ventral row; caudal cirri lacking. Dienst), Germany awarded to W.S. Type species. Apourostylopsis sinica (Shao et al., 2008) n. comb. (basionym: M. sinica Shao et al., 2008). LITERATURE CITED Derivation. Composite of the Greek prefix apo- (from, off, away, after, without, separate) and the posterior part (-urostylop- Berger, H. 1999. Monograph of the Oxytrichidae (Ciliophora, Hypot- sis) of the genus Metaurostylopsis. It alludes to the fact that it richa). Monograph. Biol., 78:1–1080. differs from the ‘‘stem genus’’ Metaurostylopsis. Like the latter, Berger, H. 2006. Monograph of the Urostyloidea (Ciliophora, Hypotricha). feminine gender. Monograph. Biol., 85:1–1304. Berger, H. 2008. Monograph of the Amphisiellidae and Trachelostylidae Species assignable. So far, the new genus contains only the (Ciliophora, Hypotricha). Monograph. Biol., 88:1–737. type, A. sinica. Two other morphotypes, M. flavicans Wang et al., Berger, H. & Foissner, W. 1997. 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