Zoologica Scripta

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Molecular phylogeny of three ambiguous ciliate genera: Kentrophoros, Trachelolophos and Trachelotractus (Alveolata, Ciliophora)

SHAN GAO,MICHAELA C. STRU¨ DER-KYPKE,KHALED A. S. AL-RASHEID,XIAOFENG LIN &WEIBO SONG

Submitted: 12 May 2009 Gao, S., Stru¨der-Kypke, M.C., Al-Rasheid, K.A.S., Lin, X. & Song, W. (2010). Molecular Accepted: 31 October 2009 phylogeny of three ambiguous ciliate genera: Kentrophoros, Trachelolophos and Trachelotractus doi:10.1111/j.1463-6409.2010.00416.x (Alveolata, Ciliophora).—Zoologica Scripta, 39, 305–313. Very few molecular studies on the phylogeny of the karyorelictean ciliates have been car- ried out because data of this highly ambiguous group are extremely scarce. In the present study, we sequenced the small subunit ribosomal RNA genes of three morphospecies rep- resenting two karyorelictean genera, Kentrophoros, Trachelolophos, and one haptorid, Trache- lotractus, isolated from the South and East China Seas. The phylogenetic trees constructed using Bayesian inference, maximum likelihood, maximum parsimony and neighbor-joining methods yielded essentially similar topologies. The class Karyorelictea is depicted as a monophyletic clade, closely related to the class Heterotrichea. The generic concept of the family Trachelocercidae is confirmed by the clustering of Trachelolophos and Tracheloraphis with high bootstrap support; nevertheless, the order Loxodida is paraphyletic. The transfer of the morphotype Trachelocerca entzi Kahl, 1927 to the class Litostomatea and into the new haptorid genus Trachelotractus, as suggested by previous researchers based on morpho- logical studies, is consistently supported by our molecular analyses. In addition, the poorly known species Parduczia orbis occupies a well-supported position basal to the Geleia clade, justifying the separation of these genera from one another. Corresponding author: Weibo Song, Laboratory of Protozoology, Ocean University of China, Qingdao 266003, China. Tel: +86 532 8203 2283; E-mail: [email protected] Shan Gao & Weibo Song, Laboratory of Protozoology, Ocean University of China, Qingdao 266003, China. E-mail: [email protected], [email protected] Michaela C. Stru¨der-Kypke, Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada. E-mail: [email protected] Khaled A. S. Al-Rasheid, Zoology Department, King Saud University, PO Box 2455, Riyadh 11451, Saudi Arabia. E-mail: [email protected] Xiaofeng Lin, Laboratory of Protozoology, College of Life Science, South China Normal University, Guangzhou, 510631, China. E-mail: [email protected]

Introduction Dragesco (1996a,b) and Foissner & AL-Rasheid (1999a,b) Karyorelictean ciliates play an important role in under- obtained excellent preparations from all main groups of standing nuclear dimorphism and phylogeny of ciliates karyorelicteans and revealed a world of new details using a because their diploid macronuclei are non-dividing and ‘strong’ fixative and Wilbert’s protargol technique (Wil- originate from micronuclei during and after each cell divi- bert 1975). sion (Corliss 1974; Lynn & Small 1997). However, mor- Until now, numerous conflicting classifications based on phological analyses of the karyorelicteans were limited for morphological, developmental or ultrastructural features a long time by their extreme fragility, although many basic have been proposed for karyorelicteans (Corliss 1979; features (e.g. body shape, nuclear structure and somatic Lynn & Corliss 1991; de Puytorac et al. 1993; Foissner cortical ultrastructure) were explored in the pioneering 1998). Emphasizing oral structures, the classification studies by Dragesco (1960), Dragesco & Dragesco-Kerne´is scheme of Corliss (1979) united the orders Karyorelictida, (1986), Raikov et al. (1975) and Wilbert (1986). More Primociliatida, Prostomatida, Haptorida and Pleurostom- recently, Foissner (1995a,b,c, 1996, 1997a,b), Foissner & atida into the subclass Gymnostomata and deemed

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Molecular phylogeny of three ciliate genera d S. Gao et al.

karyorelictids to be the most primitive ciliates. However, morphological characters were combined to achieve a new this hypothesis was challenged by ultrastructural data that evaluation of the phylogenetic relationships of the karyor- suggested a close relationship between karyorelictids and elictean ciliates. heterotrichs, which both possess overlapping postciliary microtubular ribbons forming the conspicuous postcili- Materials and methods odesmata to the right side of the kinety (Raikov et al. Ciliate collection and identification 1975). The schemes proposed by later authors (Foissner Kentrophoros fasciolatum and Kentrophoros sp.-QD-06112601 1998; Lynn & Small 2002; Lynn 2008) generally sup- were collected from the coast of Qingdao, northern China ported this relationship but they differ with respect to the (3608¢N 12043¢E). Trachelotractus entzi (Kahl, 1927) taxonomic structure within the class Karyorelictea Foissner 1997a,b and T. gigas were sampled from Daya (Table 1). Bay (2242¢N 11432¢E), southern China. Isolation and Compared with the large number of morphospecies morphological investigations of species carried out accord- described and new taxa recognized to date, the number of ing to Foissner (1991) and Foissner & Dragesco (1996a). karyorelictean species with SS rRNA gene sequence infor- mation available from GenBank is low (over 130 described DNA extraction and polymerase chain reaction (PCR) morphospecies and just eight sequenced). Some key taxa amplification are still needed in analyses to achieve better understanding Isolation of cells and extraction of genomic DNA were and interpretation of the phylogenetic relationships of performed according to Yi et al. (2008b). In brief, cells these organisms. We have identified three karyorelictean were starved in sterilized seawater at room temperature species, i.e. two Kentrophoros species and Trachelolophos overnight to minimize contents of food vacuoles and con- gigas, as well as Trachelotractus entzi, which was formerly taminants, and then DNA was extracted using an REDEx- classified as Trachelocerca within the class Karyorelictea, tract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO, but has been transferred to the class Litostomatea by USA) according to the manufacturer’s protocol, with the Foissner (1997a). None of these sequences has so far been slight modification that only 1 ⁄ 10 of the volume suggested analysed in phylogenetic studies. Gene sequences and for each reagent solution was used (Gong et al. 2007). DNA samples were stored at )20 C. The universal eukaryotic forward primer Euk A (5¢- AACCTGGTTGATCCTGCCAGT-3¢) and reverse Euk Table 1 Four taxonomic schemes for the classification of B(5¢-TGATCCTTCTGCAGGTTCACCTAC-3¢) were karyorelictean ciliates. used to amplify the SS rRNA gene (Medlin et al. 1988) using the PCR protocol of Gao (2008). Corliss (1979) Foissner (1998) Lynn & Small (2002) Lynn (2008) et al.

C. Kinetofragminophora C. Karyorelictea C. Karyorelictea C. Karyorelictea Cloning and sequencing of SS rRNA gene S.C. Gymnostomata S.C. Trachelocercia O. Karyorelictida O. Loxodida O. Protostomatida O. Protostomatida Polymerase chain reaction products were purified using an F. Trachelocercidae F. Kentrophoridae F. Kentrophoridae F. Kentrophoridae H.Q & Q Gel Extraction Kit II (U-gene, Jixi, Anhui, Trachelocerca Kentrophoros Kentrophoros Kentrophoros China) and then inserted into pUCm-T vectors (Sangon, Trachelonema F. Cryptopharyngidae Prototrachelocerca F. Trachelocercidae Tracheloraphis Apocrypharynx Trachelolophos Kovalevaia Toronto, ON, Canada). Plasmids were harvested using the F. Loxodidae Cryptopharynx Trachelonema Prototrachelocerca Mini-prep Spin Column kit (Sangon) and were sequenced Kentrophoros F. Loxodidae Tracheloraphis Sultanophrys by the Invitrogen sequencing facilities in Shanghai and Loxodes Loxodes F. Trachelocercidae Trachelocerca Remanella Remanella Trachelocerca Trachelolophos Guangzhou, China. Subsequent sequencing was performed F. Geleiidae O. Trachelocercida O. Loxodida Tracheloraphis in both directions by primer walking (Yi et al. 2008a). Avelia F. Trachelocercidae F. Cryptopharyngidae O. Loxodida Geleia Trachelocerca Apocrypharynx F. Cryptopharyngidae O. Primociliatida Trachelophos Cryptopharynx Apocrypharynx Phylogenetic analyses O. Prostomatida Tracheloraphis F. Loxodidae Cryptopharynx Other sequences used in this study were obtained from O. Haptorida F. Prototrachelocercidae Loxodes F. Loxodidae the NCBI ⁄ GenBank database (Table 2). Sequences were O. Pleurostomatida Prototrachelocerca Remanella Loxodes S.C. Protoheterotrichia O. Protoheterotrichida Remanella aligned using CLUSTAL W 1.83 (Thompson et al. 1994). O. Protoheterotrichida F. Geleiidae O. Protoheterotrichida Ends were trimmed and the ambiguously aligned sites F. Geleiidae Avelia F. Geleiidae were Gblocks v.0.91b (Castresana, 2000), yielding an Avelia Geleia Avelia Geleia Gellertia Geleia alignment of 1387 characters. Gellertia The program MRMODELTEST v.2 (Nylander 2004) Parduczia selected GTR + I (= 0.3474) + G (= 0.6060) under AIC Our new sequences are underlined. criterion as the best model, which was then used in the

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S. Gao et al. d Molecular phylogeny of three ciliate genera

Table 2 Small subunit rRNA gene sequences from GenBank used A maximum parsimony (MP) tree was computed based in this study. on 464 parsimony informative sites with PAUP* 4.0b 10 (Swofford 2003). The reliability of internal branches was GenBank GenBank estimated by bootstrap re-sampling with 1000 replicates. Species name acc. No Species name acc. No A Bayesian inference analysis was performed with Blepharisma M97909 Loxodes magnus L31519 MRBAYES 3.1.2 (Ronquist & Huelsenbeck 2003) using the americanum GTR + G + I evolutionary model indicated by MRMODEL- Bresslaua vorax AF060453 Loxodes striatus U24248 TEST v.2 (Nylander 2004). The program was run for Colpoda inflata M97908 Loxophyllum jini EF123708 Condylostentor DQ445605 Metopus palaeformis M86385 1 000 000 generations with a sampling frequency of every auriculatus 100th tree and a burn-in of 2500. The posterior probabi- Dysteria procera DQ057347 Nyctotheroides AF145353 lites were calculated from the remaining 1304 trees and a deslierrae majority rule consensus tree was constructed. Frontonia lynni DQ190463 Obertrumia Georgiana X65149 Neighbor-joining (NJ) analysis was performed with Frontonia tchibisovae DQ883820 Parduczia orbis AY187924 Furgasonia blochmanni X65150 Phialina salinarum EU242508 MEGA 4 (Tamura et al. 2007) using the Tamura-Nei model Geleia fossata AY187925 Plagiopyla frontata Z29440 (Tamura & Nei 1993), and the support for the internal Geleia simplex AY187927 Prorocentrum micans EF492511 branches was estimated using the bootstrap method with strain 00Nah 1000 replicates. Geleia simplex AY187932 Prorodon teres X71140 strain 2000Nah Geleia sp. AY187926 Prorodon viridis U97111 Results Geleia swedmarkii AY187933 Trachelolophos gigas FJ463746 The newly obtained SS rRNA gene sequences have been Harmannula derouxi AY378113 Tracheloraphis sp. L31520 deposited in the GenBank with the following accession Kentrophoros FJ467505 Trachelotractus entzi FJ463745 numbers: Kentrophoros fasciolatum-FJ467505, Kentrophoros fasciolatum sp.-QD-06112601-FJ476506, -FJ463746 and Kentrophoros FJ476506 Trimyema compressum Z29438 T. gigas T. sp.-QD-06112601 entzi-FJ463745 (Table 2). The two trachelocercids, T. Lacrymaria marina DQ777746 Uronychia setigera AF260120 gigas and Tracheloraphis sp., share a sequence similarity of Litonotus paracygnus EU242509 Uronychia transfuga EF198669 95.2%, and the two species of Kentrophoros are 99.6% sim-

Species sequenced by the present authors are marked in bold. New published ilar. The sequence of T. entzi shows only low similarities sequences in the present study are underlined. to the other karyorelicteans, ranging from 72.10% to 75.92% (Table 3) but shares 90% sequence similarity with maximum likelihood (ML) analysis. An ML tree was con- haptorid species (data not shown). structed using the PHYML v2.4.4 program (Guindon & Trees constructed using different algorithms have com- Gascuel 2003). The reliability of internal branches was parable topologies (Figs. 1, 2). The monophyletic class assessed using the non-parametric bootstrap method with Karyorelictea is fully supported by all methods 1000 replicates. (BI ⁄ ML ⁄ MP ⁄ NJ, 1.00 ⁄ 100 ⁄ 100 ⁄ 100) and forms a sister

Table 3 Sequence similarities of karyorelictean ciliates included in the present study.

G. swedmarkii G. simplexa G. simplexb G.sp. G. fossata P. orbis T. sp. L. striatus L. magnus T. gigas K. fasciolatum K. sp. T. entzi

G. swedmarkii G. simplexa 98.96 G. simplexb 98.89 99.93 G. sp. 97.56 97.77 97.70 G. fossata 98.19 98.33 98.26 97.35 P. orbis 89.48 89.69 89.62 89.41 89.34 T. sp. 82.56 82.56 82.49 81.97 82.16 81.90 L. striatus 82.56 82.75 82.69 82.18 82.69 81.79 86.55 L. magnus 82.45 82.65 82.58 82.06 82.58 81.61 86.58 99.35 T. gigas 82.72 82.72 82.65 82.46 82.26 82.26 95.20 89.03 88.70 K. fasciolatum 82.46 82.65 82.65 81.81 82.33 81.49 88.43 89.52 89.20 89.20 K. sp. 82.70 82.77 82.77 82.06 82.57 81.61 88.68 89.77 89.45 89.07 99.55 T. entzi 72.82 72.75 72.69 72.10 72.75 72.43 75.34 75.86 75.40 75.92 75.84 75.88

aGeleia simplex strain 2000Nah. bGeleia simplex strain 00Nah. Species sequenced by the present study are marked in bold.

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Molecular phylogeny of three ciliate genera d S. Gao et al. Intramacronucleata desmatophora Postcilio-

Fig. 1 Phylogenetic tree inferred from SS rRNA gene sequences of selected ciliate taxa. The numbers at the internal nodes are Bayesian posterior probability values, followed by the bootstrap values for the maximum likelihood (ML) analysis. Solid circles represent full bootstrap support in both algorithms. Species sequenced in the present study are shown in bold type. ‘–’ reflects minor differences between Bayesian and ML methods.

group to the class Heterotrichea. Within the class Karyor- kinety) and Trachelocerca (without brosse and uninterrupted elictea, four major clades are well resolved by both BI and simple circumoral kinety). Lynn (2008) also included the ML methods and also by MP and NJ in most cases. The newer genera Kovalevaia (Foissner 1997b) and Sultanophrys genus Geleia appears monophyletic (BI ⁄ ML ⁄ MP ⁄ NJ, (Foissner & AL-Rasheid 1999a,b) in this family. In the 1.00 ⁄ 100 ⁄ 100 ⁄ 100), with Parduczia branching off basally, present study, Trachelolophos and Tracheloraphis formed a and species of Loxodes and isolates of Kentrophoros form distinct clade, corresponding to the concept of the family two well-supported clades that associate with weak to Trachelocercidae as postulated by Foissner & Dragesco moderate support. Trachelolophos and Tracheloraphis form a (1996b) that was defined by two strong synapomorphies, separate clade, corresponding to the family Trachelocerci- i.e. the apicalization of the oral apparatus and the brosse. dae (BI ⁄ ML ⁄ MP ⁄ NJ, 1.00 ⁄ 100 ⁄ 99 ⁄ 100). Trachelotractus This concept is also embraced by Lynn’s current classifica- entzi clusters basally within the class Litostomatea with tion scheme (Lynn 2008), which places Tracheloraphis full support. within the family Trachelocercidae instead of within the family Kentrophoridae (Lynn & Small 2002) (Table 1). Discussion Recently, Mazei et al. (2009) sequenced the trachelocer- Relationships among trachelocercid karyorelictids cid species Trachelocerca ditis and inferred the phylogenetic Based entirely on infraciliary features, particularly the oral relationships among the trachelocercids in more detail. structures, Foissner & Dragesco (1996a) recognized four The genera Trachelocerca and Trachelolophos cluster trachelocercid genera (Table 1, Foissner 1998): Prototrache- together, with Tracheloraphis branching basally. This find- locerca (with brosse interrupting compound circumoral cil- ing is consistent with the similar circumoral kinety pat- iature), Tracheloraphis (with brosse interrupting simple terns (simple and uninterrupted) shared by Trachelocerca circumoral kinety), Trachelolophos (with brosse near the and Trachelolophos and does not support a closer relation- centre of oral bulge and uninterrupted simple circumoral ship of Trachelolophos and Tracheloraphis, which could be

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S. Gao et al. d Molecular phylogeny of three ciliate genera

AB

Fig. 2 (A) Distance matrix tree inferred from the SS rRNA gene sequences of selected ciliate taxa using the neighbor-joining algorithm. (B) Maximum parsimony tree of the same data set inferred from the SS rRNA gene sequences. The numbers at the forks indicate the percentage of times that specific branch pattern occurred in 1000 trees. Species sequenced in this work are highlighted in bold type.

hypothesized if the tuft was considered as a highly modi- phoridae within the order Protostomatida and placed the fied brosse (like the brosse possessed by Tracheloraphis) ventrostomous families Loxodidae and Cryptopharyngidae (Foissner & Dragesco 1996a,b; Foissner 1998). into the order Loxodida because they considered the diverse oral structures as the primary distinguishing crite- Relationships among Trachelocercidae, Kentrophoridae and ria in this class (Lynn 2008). Foissner (1998) argued that Loxodidae the order Prostomatida Small and Lynn, 1985, uniting the Based on the currently available karyorelictean sequences, Kentrophoridae and Trachelocercidae but excluding the the three families Kentrophoridae, Trachelocercidae and Loxodidae, is very likely artificial because the families Loxodidae are all depicted as monophyletic. However, Kentrophoridae and Loxodidae share the distinct similari- their phylogenetic relationships are not unambiguously ties mentioned above. resolved by the molecular data. Based on three morpho- In our preliminary analyses, the family Trachelocercidae logical characteristics, Foissner (1998) assigned the fami- seems to be separated distinctly from the clustered Ken- lies Kentrophoridae and Loxodidae to the order Loxodida trophoridae and Loxodidae (Figs. 1, 2), which is support- based on the following evidence (i) the somatic cortical ive of Foissner’s scheme to some degree. However, the ultrastructure is distinctly similar in Loxodes and Kentropho- molecular results reject Foissner’s (1998) suggestion that ros (Raikov 1973); (ii) the somatic infraciliature is very sim- the trachelocercids are more derived with respect to their ilar in Loxodes, Remanella and Kentrophoros; and (iii) oral region. It should be noted that the order Loxodida although no oral structures are visible in Kentrophoros, oral sensu Foissner 1998 (Loxodidae + Kentrophoridae) was vestiges have been shown in the anterior body region, paraphyletic in the BI ⁄ MP ⁄ ML analyses, and only the NJ where loxodids have the oral apparatus. Meanwhile, he analysis showed that the families Loxodidae and Kentro- assigned the family Trachelocercidae to the separate order phoridae clustered together, although with very low sup- Trachelocercida, mainly based on the unique features port. At this point, the molecular data do not shared by trachelocercids, namely the apicalization of the unambiguously support either of the two classification oral apparatus and the brosse. However, other researchers schemes. Morphospecies in the family Kentrophoridae are (Lynn & Small 2002; Lynn 2008) included the prostomous distinct from loxodids (and other karyorelicteans) and family Trachelocercidae and the ‘astomous’ family Kentro- show morphological (strongly reduced and probably

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Molecular phylogeny of three ciliate genera d S. Gao et al.

functionless oral apparatus) and ecological (symbiotic Molecular phylogenetic placement of Trachelotractus entzi ‘kitchen garden’ of sulphur bacteria) peculiarities (Foissner (Kahl, 1927) Foissner, 1997 1995c); therefore, it may be reasonable to separate Trachelocerca entzi Kahl, 1927, a conspicuous, highly kentrophorids from loxodids at the ordinal level. However, contractile species, has been traditionally considered to be additional gene sequences are needed to test this a karyorelictean because of its similarity with trachelocerc- suggestion. ids in general appearance and contractility (Kahl 1930; Intramacronucleata desmatophora Postcilio-

Fig. 3 Bayesian tree inferred from SS rRNA gene sequences of selected ciliate taxa, with addition of several litostomes to reach a better resolution of this group. The numbers at the internal nodes are Bayesian posterior probability values. Species sequenced in the present study are shown in bold type.

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S. Gao et al. d Molecular phylogeny of three ciliate genera

Fig. 4 The 44 parsimony-informative positions in the alignment of the nuclear SS rRNA gene sequences of 12 karyorelictean ciliates. Positions of homologous nucleotides in the alignment are given at the top of each column. aGeleia simplex strain 2000Nah. bGeleia simplex strain 00Nah.

Dragesco 1960; Carey 1992). Foissner (1997a) provided a We constructed another Bayesian tree after adding 34 very detailed morphological description of T. entzi, and litostome sequences (Fig. 3) to achieve a better resolution transferred the species to the haptorid family Helicop- of litostomes relative to karyorelicateans. Trachelotractus rorodontidae, erecting a new genus, Trachelotractus, based entzi clustered at the base of a well-defined litostome on the following evidences: (i) T. entzi lacks all main infra- clade, basal to the orders Pleurostomatida and Haptorida ciliary characteristics of karyorelictean trachelocercids in this tree. Because of the unresolved relationships within (somatic monokinetids in T. entzi vs. dikinetids in trache- the subclass Haptoria, Lynn (2008) only recognized two locercids; absence vs. presence of a glabrous stripe sur- orders, the Haptorida and the Pleurostomatida and did rounded by a highly specialized bristle kinety); (ii) the not reclassify the families within the Haptorida further. main classification features of T. entzi (the peribuccal ridge According to the haptorian classification scheme of Foiss- with its typical extrusomes, somatic monokinetids, oralized ner & Foissner (1988), the family Helicoprorodontidae somatic kinetids and specialized ciliary rows curving forms its own suborder, Helicoprorodontina, within the abound the pharyngeal opening) are exactly those found in order Pseudoholophryida, and the placement of T. entzi the haptorid genus Helicoprorodon. In the present study, T. might support this suggestion. However, the phylogenetic entzi clustered basally to the litostome subclasses Haptoria tree (Fig. 3) does not support any of the suggested hapto- and Trichostomatia and was strongly supported as a mem- rian classification schemes and depicts the subclass Hapto- ber of this assemblage. The secondary structure of the ria as paraphyletic (Stru¨der-Kypke et al. 2006). Although SSrRNA gene sequences of this clade showed a typical we can conclude that T. entzi is indeed a haptorian ciliate, deletion in helix E23_1 and a missing helix E23_5 (Stru¨- our data are not sufficient to define its phylogenetic rela- der-Kypke et al. 2006). Furthermore, T. entzi shared con- tionships within this group exactly. siderably lower sequence similarity with karyorelicteans than we found between genera within the class Karyorelic- Is the genus Parduczia valid? tea (Table 3), which strongly supports its transfer to the The order Protoheterotrichida (Geleia + Parduczia) and the class Litostomatea by Foissner (1997a). genus Geleia were monophyletic in our analyses (Figs. 1–

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3). This is consistent with the characteristic features References shared by the geleiids, i.e. holotrichous somatic ciliation Carey, P. G. (1992). Marine Interstitial Ciliates. London, New and paracytostomal monokinetids (Dragesco & Dragesco- York, Tokyo, Melbourne, Madras: Chapman and Hall. Kerne´is 1986). Foissner (1998) postulated that the order Castresana, J. (2000). Selection of conserved blocks from multiple Protoheterotrichida is the most ancient group of the Kar- alignments for their use in phylogenetic analysis. Molecular Biology and Evolution, 17, 540–552. yorelictea, based on the hypothesis that the common Corliss, J. O. (1974). Remarks on the composition of the large ancestor of the karyorelictids and heterotrichs probably ciliate class Kinetofragmophora de Puytorac et al., 1974, and had the holotrichous ciliation seen in present-day proto- recognition of several new taxa therein, with emphasis on the heterotrichids. 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European Journal the species of Geleia but not P. orbis shared unique nucleo- of Protistology, 32, 234–250. tides in 44 sites (including 10 gaps) in semiconserved, par- Foissner, W. (1995b). The infraciliature of Cryptopharynx setigerus Kahl, 1928 and Apocryptopharynx hippocampoides nov. simony-informative regions of our alignment (Fig. 4). In gen., nov. spec. (Ciliophora, Karyorelictea), with an account addition, the sequence similarities between P. orbis and on evolution in loxodid ciliates. Archiv fu¨r Protistenkunde, 146, Geleia spp. are lower than those within the genus Geleia 309–327. (Table 3). Thus, our results support the separation of Par- Foissner, W. (1995c). Kentrophoros (Ciliophora, Karyorelictea) has duczia from Geleia as suggested by Dragesco (1999). oral vestiges: a reinvestigation of K. fistulosus (Faure´-Fremiet, Dragesco (1999) erected a new family Avellidae to 1950) using protargol impregnation. Archiv fu¨r Protistenkunde, embrace large, vermiform species, such as Avelia and Par- 146, 165–179. Foissner, W. (1996). Updating the trachelocercids (Ciliophora, duczia that possess two invariable paroral stuctures (an in- Karyorelictea). II. Prototrachelocerca nov. gen. trabuccal kinety and a series of polykineties bordering the (Prototrachelocercidae nov. fam.), with a redescription of P. buccal opening vs. the monokinetid rows in the Geliidae). fasciolata (Sauerbrey, 1928) nov. comb. and P. caudata (Dragesco However, this was not accepted in newer classification & Raikov, 1966) nov. comb. European Journal of Protistology, 32, schemes (Lynn & Small 2002; Lynn 2008), which included 336–355. all known protoheterotrichids in the family Gellidae. Foissner, W. (1997a). Updating the Trachelocercids (Ciliophora, Therefore, the validity of the family Aveliidae sensu Karyorelictea). IV. Transfer of Trachelocerca entzi Kahl, 1927 to the Gymnostomatea as a new genus, Trachelotractus gen. n. Dragesco 1999 still needs to be confirmed by testing the (Helicoprorodontidae). Acta Protozoologica, 36, 63–74. phylogenetic relationships among karyorelicteans including Foissner, W. (1997b). Updating the trachelocercids (Ciliophora, sequences of more protoheterotrichid species. Karyorelictea). V. Redescription of Kovalevaia sulcata (Kovaleva, 1966) gen. n., comb. n. and Trachelocerca incaudata Kahl, 1933. Acknowledgements Acta Protozoologica, 36, 197–219. This work was supported by the National Natural Science Foissner, W. (1998). The karyorelictids (Protozoa: Ciliophora), a Foundation of China (Project No. 30870280), the Center unique and enigmatic assemblage of marine, interstitial ciliates: a review emphasizing ciliary patterns and evolution. In G. H. of Excellence in Biodiversity, King Saud University and Coombs, K. Vickerman, M. A. Sleigh & A. Warren (Eds) the 111 Project (No. B08049). Many thanks are also due Evolutionary Relationships Among Protozoa. pp. 305–325. London: to Ms. Zhenzhen Yi, Mr. Weiwei Liu, Yangang Wang Chapman and Hall. and Xinpeng Fan, graduates of our laboratory, OUC, for Foissner, W. & AL-Rasheid, K. A. S. (1999a). Updating the technical help. trachelocercids (Ciliophora, Karyorelictea). VI. A detailed

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