Redescription and Phylogenetic Position of Condylostoma arenarium Spiegel, 1926 (Ciliophora, Heterotrichea) from Guanabara Bay, Brazil.

Published by the International Society of Protistologists

The Journal of

Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

Redescription and Phylogenetic Position of Condylostoma arenarium Spiegel, 1926 (Ciliophora, Heterotrichea) from Guanabara Bay, Brazil Noemi M. Fernandesa, Roberto J. P. Diasb, Carlos G. Schragoa & Inacio D. Silva-Netoc rio de Biologia Evolutiva Teo rica e Aplicada, Dept. de Gene tica, Inst. de Biologia, CCS, Universidade Federal do Rio de Janeiro, CEP: a Laborato 21941-617, Ilha do Fund~ao, Rio de Janeiro – RJ, Brazil rio de Protozoologia, Dept. de Zoologia, ICB, Universidade Federal de Juiz de Fora, CEP: 36036-900, Juiz de Fora – MG, Brazil b Laborato rio de Protistologia, Dept. de Zoologia, Inst. de Biologia, CCS, Universidade Federal do Rio de Janeiro – UFRJ, CEP: 21941-590, Ilha do c Laborato Fund~ ao, Rio de Janeiro – RJ, Brazil

Keywords Condylostomatidae; diversity; heterotrichs; infraciliature; marine species; morphology. Correspondence rio de Biologia N. M. Fernandes, Laborato rica e Aplicada, Dept. de GenEvolutiva Teo tica, Inst. de Biologia, CCS, Universidade e Federal do Rio de Janeiro. CEP: 21941-617, Ilha do Fund~ ao, Rio de Janeiro – RJ, Brazil Telephone/FAX number: +55-21-25626363; e-mail: [email protected] Received: 7 December 2014; revised 14 February 2015; accepted March 8, 2015. doi:10.1111/jeu.12228

ABSTRACT Details on Condylostoma arenarium infraciliature have not been described; therefore, it is considered a poorly known species. The lack of detailed description on C. arenarium morphology caused several misidentifications that have accumulated in the literature. In this study, we present the first complete description of C. arenarium infraciliature based on protargol-impregnated organisms and scanning electron microscopy. We also have inferred the phylogenetic position of this species based on 18S rRNA sequences. The main characteristics of C. arenarium population from Guanabara Bay are as follows: in vivo elongated body shape with 350–600 lm length 9 70–220 lm width, they are highly contractile when subjected to disturbances, green-yellowish cortical granules are present, contractile vacuoles absent, V-shaped peristome comprises approximately 1/5 of the total length, adoral zone with 83–145 membranelles, 1–2 small frontal cirrus observed only in impregnated specimens, 10–15 fiber-like stripes arranged transversely on the inner wall of the oral cavity, 30–45 somatic kineties, moniliform macronucleus with 15–20 nodules. Some observations on morphogenesis of C. arenarium were also included. In phylogenetic analyses, C. arenarium clustered with Condylostoma sp. within a clade composed of three C. curva sequences with high support values.

CILIATES of the genus Condylostoma Bory de St. Vincent, 1824 are characterized by elongated body shapes that are often posteriorly tapered. Most species are marine, but certain representatives have been observed in soil and -Fremiet 1958; Foissner 1995; Kahl freshwater (Faure 1932; Penard 1922). The macronucleus is moniliform in most Condylostoma species, and the presence of one to several cirri near the right wall of the oral cavity distinguishes these species from other heterotrichs (Shazib et al. 2014). Condylostoma is a confusing genus for species identification. Most descriptions are sketchy and based solely on in vivo observations, which results in numerous misinterpretations that accumulate in the literature and produce confusion and difficulty in species identification (Agamaliev 1972; Borror 1963, 1972; Bullington

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1940; Burkovsky 1970; Chen et al. 2007; Dragesco 1960; is 1986; Faure -Fremiet Dragesco and Dragesco-Kerne 1958; Kahl 1932; Kim et al. 2012; Petz et al. 1995; Shao et al. 2006; Song and Wilbert 1997; Song et al. 2003; Villeneuve-Brachon 1940; Yagiu 1956). Condylostoma arenarium Spiegel 1926 is a marine species with a broad distribution (Al-Rasheid 1999; Borror 1963; Burkovsky 1970; Dragesco 1960, 1965; Dragesco is 1986; Hartwig 1974; Kattar 1970; and Dragesco-Kerne Raikov 1963; Ricci et al. 1982; Wright 1983). The original description of C. arenarium was solely based on in vivo observations, and the author provided few details on the morphology of this species. Later, other authors described populations identified as C. arenarium (Agamaliev 1972; Al-Rasheid 1999; Borror 1963, 1972; Dragesco

© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 722–732

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is 1986; Kahl 1932; Villeneuve-Braand Dragesco-Kerne chon 1940). However, Song et al. (2003) considered most of these descriptions as misidentifications. Details on C. arenarium morphogenesis and infraciliature have not been described (Kim et al. 2012; Shao et al. 2006). Therefore, Chen et al. (2007) considered C. arenarium a poorly known species. Only two studies report the occurrence of C. arenarium in Brazil (Kattar 1970; Wanick and da Silva-Neto 2004), but do not include the species’ morphology. This study provides the first complete description of C. arenarium infraciliature based on protargol-impregnated organisms and scanning electron microscopy. Moreover, the phylogenetic position of C. arenarium was inferred based on 18S rRNA sequences. MATERIALS AND METHODS Sampling, culture, and morphology The C. arenarium population was obtained from sediment samples collected at Guanabara Bay, Rio de Janeiro, Brazil (approximate geographic coordinates: 22°150 32″N, 43°130 12″E). These samples were set out in cultures using Petri dishes with water from the sample site and mashed rice grains (Foissner 1992). The general morphology and measurements were observed in vivo through differential interference contrast microscopy at a 100–200X magnification and at 1,000X magnification for cortical granule observations. To reveal the total infraciliature and nuclear apparatus, we used the protargol impregnation method proposed by Dieckmann (1995). In accordance with SilvaNeto (1994), scanning electron microscopy was also performed to visualize the oral and somatic ciliature pattern. Morphometric data were obtained from 30 randomly selected protargol-impregnated specimens. The software used for morphometry was Image Pro-plus 5.0â (Media Cybernetics, Atlanta, GA). The adopted terminology primarily follows Lynn (2008). Voucher slides were deposited rio de Protistologia, Inst. de Biin the collection of Laborato ologia, Universidade Federal do Rio de Janeiro.

Redescription of Condylostoma arenarium

Phylogenetic inference To determine the phylogenetic position of C. arenarium, we composed a dataset of 18S rRNA gene sequences from 42 heterotrich species and 4 karyorelictids which were used as outgroup for all analyses (Table 1). The alignments were conducted with the Geneious Software. Primer sequences were removed and the final alignment was cured with Gblocks server for cleaning ambiguously aligned positions (Castresana 2000; http://molevol.cmima.csic.es/castresana/Gblocks_server.html). Bayesian inference (BI) analysis was performed using the MrBayes 3.1.2 program (Ronquist and Huelsenbeck 2003) implemented on the CIPRES portal (https://www.phylo.org) using the GTR+ I+ Γ substitution model. This model was the best fit to our data according to the ModelTest module implemented using the HyPhy program (Pond et al. 2005). Markov Chain Monte Carlo analysis consisted of two independent trials with four chains each. Each chain was run for 10,000,000 generations and sampled every 100th cycle. The first 250,000 generations were discarded as burn-in. BI tree consisted of the 50% majorityrule consensus of the topologies collected after the burnin and clade support was calculated by the frequency of each clade in sampled topologies. Maximum likelihood analysis was performed in PhyML 3.0 (Guindon et al. 2010), as implemented in the ATGC bioinformatics platform (http://www.atgc-montpellier.fr/), using the same substitution model. Clade support was estimated using 1,000 bootstrap replicates. The similarity and absolute difference matrix were exported from Geneious. RESULTS Heterotrichea Stein, 1859 Heterotrichida Stein, 1859 Condylostomatidae Kahl in Doflein & Reichenow, 1929 (syns. Condylostomidae, Condylostentoridae) Condylostoma Bory de St. Vincent, 1824 Condylostoma arenarium Spiegel, 1926 Improved diagnosis

DNA isolation, amplification, and sequencing Genomic DNA extraction, PCR amplification, and sequencing of 18S rRNA gene of C. arenarium were performed according to the method described by Gong et al. (2009). The target segment of the 18S rRNA gene sequence was amplified using the eukaryotic universal primers EukA/ EukB (Medlin et al. 1988), which encompassed the full length of the gene. The PCR products were purified using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) and sequenced directly. Both strands of DNA were sequenced using the same universal Eukarya-specific primers. The sequence obtained was edited using the Geneious Software (Geneious Version 7; Biomatters, Biomatters Ltd, Auckland, New Zealand) and compared with sequences in the NCBI database using the BLASTN algorithm.

Condylostoma arenarium measured 350–600 9 70– 220 lm (n = 30) in vivo with an elliptical body shape dorsoventrally flattened and highly contractile when subjected to disturbances. Green-yellowish cortical granules and colorless cytoplasm. A V-shaped peristome comprises to approximately 1/5 of the total length. Adoral zone with 83–145 membranelles and a conspicuous paroral membrane (PM) at right side. One or two small and inconspicuous frontal cirrus were observed only in impregnated organisms. Approximately 10–15 fiber-like stripes are arranged transversely on the inner wall of the oral cavity. A holotrichous somatic ciliature was composed of 30–45 longitudinally arranged kineties. Near the posterior end, some ventral kineties are shortened and form a V-shaped suture. A moniliform macronucleus with 15–20 nodules is located at the right edge of the body.


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Table 1. Classification of species used in phylogenetic analyses, accession number, and reference Class Heterotrichea

Family Blepharismidae

Chattonidiidae Climacostomidae Condylostomatidae

Fabreidae Folliculinidae Gruberiidae Maristentoridae Peritromidae

Spirostomidae

Stentoridae

Karyorelictea

Loxodiidae

Species

Accession number

Reference

Blepharisma sinuosum Blepharisma americanum Blepharisma undulans Blepharisma japonicum Blepharisma musculus Blepharisma elongatum Blepharisma hyalinum Blepharisma steini Chattonidium setense Climacostomum virens Climacostomum virens Condylostentor auriculatus Condylostoma arenarium Condylostoma cf. arenarium Condylostoma curva Condylostoma curva Condylostoma curva Condylostoma minutum Condylostoma minutum Condylostoma spatiosum Condylostoma spatiosum

JN627438 AM713182 AM713183 AM713185 KJ651813 AM713186 AM713184 AM713187 AM295495 EU583990 KJ651814 DQ445605 JQ282895 FJ998021 FJ868179 KJ651827 EU379939 DQ822482 KJ651815 DQ822483 HM140390

Condylostoma spatiosum Condylostoma sp. Condylostoma tropicum Condylostomides sp. Fabrea salina

KJ651816 AM295496 FJ868178 AM713188 DQ168805

Folliculina sp. Eufolliculina uhligi Gruberia sp. Maristentor dinoferus Peritromus faurei Peritromus kahli Peritromus sp. Spirostomum minus Spirostomum ambiguum

EU583992 EUU47620 KJ651818 AY630405 EU583993 AJ537427 KJ651830 JQ282896 JQ723981

Spirostomum caudatum Anigsteinia clarissima

KJ651820 HM140405

Stentor polymorphus Stentor polymorphus

JQ282898 AF357144

Stentor roeseli

AF357913

Stentor muelleri Stentor coeruleus Stentor amethystinus Loxodes vorax Loxodes vorax Loxodes striatus Tracheloraphis huangi

KJ651824 AM713189 AM713191 KJ651829 KJ524909 KJ524910 KJ609039

Fernandes et al. (2013) Schmidt et al. (2007) Schmidt et al. (2007) Schmidt et al. (2007) Shazib et al. (2014) Schmidt et al. (2007) Schmidt et al. (2007) Schmidt et al. (2007) Modeo et al. (2006) Miao et al. (2009) Shazib et al. (2014) M. Miao and W. Song (unpubl. observ.) Present study Zhou et al. (2010) Yan et al. (2014a) Shazib et al. (2014) W. Guo and W. Song (unpubl. observ.) W. Guo and W. Song (unpubl. observ.) Shazib et al. (2014) W. Guo and W. Song (unpubl. observ.) S.N. Khan and M.K. Shin (unpubl. observ.) Shazib et al. (2014) Modeo et al. (2006) Yan et al. (2014a,b) Schmidt et al. (2007) G. Di Giuseppe and F. Dini (unpubl. observ.) Miao et al. (2009) Hammerschmidt et al. (1996) Shazib et al. (2014) Miao et al. (2005) Miao et al. (2009) Rosati et al. (2004) Shazib et al. (2014) Fernandes and da Silva Neto (2013) M. Dunthorn, K. Wolf, H.-W. Breiner, T. Stoeck and W. Foissner (unpubl. observ.) Shazib et al. (2014) S.N. Khan and M.K. Shin (unpubl. observ.) Fernandes et al. (2014) F. Zhu, Y. Yu and Y. Shen (unpubl. observ.) F. Zhu, Y. Yu and Y. Shen (unpubl. observ.) Shazib et al. (2014) Schmidt et al. (2007) Schmidt et al. (2007) Shazib et al. (2014) Xu et al. (2014) Xu et al. (2014) Yan et al. (2014b)

Karyorelictea species were used as outgroup in all analyses. In bold, the new obtained sequence.

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Table 1. Classification of species used in phylogenetic analyses, accession number, and reference Class Heterotrichea

Family Blepharismidae

Chattonidiidae Climacostomidae Condylostomatidae

Fabreidae Folliculinidae Gruberiidae Maristentoridae Peritromidae

Spirostomidae

Stentoridae

Karyorelictea

Loxodiidae

Species

Accession number

Reference

Blepharisma sinuosum Blepharisma americanum Blepharisma undulans Blepharisma japonicum Blepharisma musculus Blepharisma elongatum Blepharisma hyalinum Blepharisma steini Chattonidium setense Climacostomum virens Climacostomum virens Condylostentor auriculatus Condylostoma arenarium Condylostoma cf. arenarium Condylostoma curva Condylostoma curva Condylostoma curva Condylostoma minutum Condylostoma minutum Condylostoma spatiosum Condylostoma spatiosum

JN627438 AM713182 AM713183 AM713185 KJ651813 AM713186 AM713184 AM713187 AM295495 EU583990 KJ651814 DQ445605 JQ282895 FJ998021 FJ868179 KJ651827 EU379939 DQ822482 KJ651815 DQ822483 HM140390

Condylostoma spatiosum Condylostoma sp. Condylostoma tropicum Condylostomides sp. Fabrea salina

KJ651816 AM295496 FJ868178 AM713188 DQ168805

Folliculina sp. Eufolliculina uhligi Gruberia sp. Maristentor dinoferus Peritromus faurei Peritromus kahli Peritromus sp. Spirostomum minus Spirostomum ambiguum

EU583992 EUU47620 KJ651818 AY630405 EU583993 AJ537427 KJ651830 JQ282896 JQ723981

Spirostomum caudatum Anigsteinia clarissima

KJ651820 HM140405

Stentor polymorphus Stentor polymorphus

JQ282898 AF357144

Stentor roeseli

AF357913

Stentor muelleri Stentor coeruleus Stentor amethystinus Loxodes vorax Loxodes vorax Loxodes striatus Tracheloraphis huangi

KJ651824 AM713189 AM713191 KJ651829 KJ524909 KJ524910 KJ609039

Fernandes et al. (2013) Schmidt et al. (2007) Schmidt et al. (2007) Schmidt et al. (2007) Shazib et al. (2014) Schmidt et al. (2007) Schmidt et al. (2007) Schmidt et al. (2007) Modeo et al. (2006) Miao et al. (2009) Shazib et al. (2014) M. Miao and W. Song (unpubl. observ.) Present study Zhou et al. (2010) Yan et al. (2014a) Shazib et al. (2014) W. Guo and W. Song (unpubl. observ.) W. Guo and W. Song (unpubl. observ.) Shazib et al. (2014) W. Guo and W. Song (unpubl. observ.) S.N. Khan and M.K. Shin (unpubl. observ.) Shazib et al. (2014) Modeo et al. (2006) Yan et al. (2014a,b) Schmidt et al. (2007) G. Di Giuseppe and F. Dini (unpubl. observ.) Miao et al. (2009) Hammerschmidt et al. (1996) Shazib et al. (2014) Miao et al. (2005) Miao et al. (2009) Rosati et al. (2004) Shazib et al. (2014) Fernandes and da Silva Neto (2013) M. Dunthorn, K. Wolf, H.-W. Breiner, T. Stoeck and W. Foissner (unpubl. observ.) Shazib et al. (2014) S.N. Khan and M.K. Shin (unpubl. observ.) Fernandes et al. (2014) F. Zhu, Y. Yu and Y. Shen (unpubl. observ.) F. Zhu, Y. Yu and Y. Shen (unpubl. observ.) Shazib et al. (2014) Schmidt et al. (2007) Schmidt et al. (2007) Shazib et al. (2014) Xu et al. (2014) Xu et al. (2014) Yan et al. (2014b)

Karyorelictea species were used as outgroup in all analyses. In bold, the new obtained sequence.

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Figure 2 Condylostoma arenarium after protargol impregnation. A. Ventral view of a typical individual showing the macronucleus and peristome. B, C. Details on the peristome to show the paroral membrane located deeply on right side of the buccal cavity, and the AZM forming a spiral that ends at the cytostome. The black arrow indicates fiber-like stripes on the inner wall of the peristome. D. Details of oral apparatus to show frontal cirri and paroral membrane. E. Ventral view of the anterior end to show one small frontal cirri located right of the paroral membrane. F. Ventral sutures (arrows) located near the posterior end. G. Details on the ventral suture. AZM = adoral zone of membranelles; FC = frontal cirri; Ma = macronucleus; P = peristome; PM = paroral membrane. Scale bars: (A) 100 lm; (B, C, F) 50 lm; (D, E, G) 20 lm.

the oral region (Fig. 1A, B, 2A, 3A, B, 4A, B). The somatic cilia are approximately 8.5 lm (Fig. 3A, D; Table 2). Some ventral kineties were shorter and formed longitudinal V-shaped sutures near the posterior end (Fig. 2F, G, 4A, B). A moniliform macronuclei with 15–20 nodules is located along the right side of the body (Fig. 2A, 4A, B). Each node is 20 9 11 lm on average and was joined by nuclear bridges (Fig. 3F; Table 2). Numerous micronuclei are located near the macronucleus (Fig. 4A). The ciliates move slowly, crawling on the bottom of the Petri dishes or swimming around the main axis of the body. Further, they are highly contractile when subjected to disturbances and are voracious omnivorous predators that feed on small diatoms, dinoflagellates, and filamentous algae.

Figure 3 Scanning electron microscopy images of Condylostoma arenarium. A, B. Ventral and dorsal general views, respectively. C. Individual in final stages of division. D. Details on the peristome showing the AZM (note that the inner cilia are shorter) and paroral membrane. The black arrow indicates probably two small frontal cirrus (?), and the white arrows shown the fiber-like stripes on the inner wall of the buccal cavity. E. Details on the AZM and paroral membrane. Note that the posterior and inner rows of the membranelle cilia are shorter. F. Internal view showing part of the macronucleus with three nodules connected by nuclear threads. AZM = adoral zone of membranelles; Ma = macronucleus; PM = paroral membrane. Scale bars: (A–C) 200 lm; (D, E) 50 lm; (F) 5 lm.

details of C. arenarium divisional process because, unfortunately, we could not collect good pictures of impregnated ciliates in division. During C. arenarium stomatogenesis, a new oral region (opisthe) is formed, with the fully developed adoral zone of membranelles and PM. (Fig. 5A, B). We observed macronuclear nodules compaction or assembled together (Fig. 5C), which formed a string-shaped structure that stretches and divides upon cytokinesis (Fig. 5D). The macronuclear nodules shape and number were re-established after division.

Morphogenesis observations Condylostoma morphogenesis is relatively unknown, and for many species, including C. arenarium, it has not been fully described (Foissner 1996). We observed only a few

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Distribution Condylostoma arenarium has been reported in marine environments from Italy, France, Africa, North and Central


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Figure 4 Morphology and infraciliature of Condylostoma arenarium from live specimens (A, D) and after protargol impregnation (B, C). A. Ventral view of a typical individual. The white arrows shown micronuclei and the black ones shown shortened somatic kineties; B. Ventral view of impregnated specimen. The black arrows shown shortened somatic kineties; C. Ventral view of the anterior end, to show the oral apparatus. Note the small frontal cirri positioned at right of the paroral membrane. D. Part of the pellicle, showing the shape and distribution of cortical granules. Scale bars: (A, B) 100 lm; (C, D) 10 lm.

America, Japan, and Mideast (Al-Rasheid 1999). In this study, the author presents an extensive list of sites where C. arenarium has been observed, but most of the reports are likely misidentifications (Song et al. 2003). Only one study has reported C. arenarium from freshwater in Thailand (Charubhun and Charubhun 2000); however, the authors did not describe the morphology of the ciliate, and thus, the identification of this population is also questionable. 18S rRNA gene sequence and phylogenetic position The 18S rRNA gene sequence of C. arenarium was deposited in the NCBI/GenBank database under accession number JQ282895. The nearly complete 18S rRNA gene sequence is composed of 1,655 bp, of which 27.1% are adenines, 20.9% cytosines, 26.9% guanines, and 25.1% thymines. The BLASTN algorithm indicates that it is 98% identical with C. curva (accession numbers EU379939) and has the highest level of similarity (98.7%) with Condylostoma sp. from Modeo et al. (2006) (accession number: AM295496), differing from this sequence at 21 nucleotide sites (Table 3). In the inferred phylogenies, C. arenarium was positioned as sister taxon of Condylostoma sp. with high support values (Fig. 6). These two sequences, in turn, were positioned within the cluster composed of 3 sequences of C. curva with high support values both in BI (100%) and ML (95%) trees. The topologies obtained from BI and ML analyses were in agreement, therefore, only BI topology is presented (Fig. 6). For both analyses, Condylostoma was recovered as a paraphyletic group.


Figure 5 Condylostoma arenarium morphogenesis, protargol impregnations. A, B. Ventral view showing the oral primordium formation. C. The beginning of macronuclear compaction. D. The final stages of morphogenesis; the macronucleus is elongated, similar to a string, before daughter cell separation. Ma = macronucleus; OP = oral primordium. Scale bars: (A–D) 100 lm.

DISCUSSION Condylostoma is one of the most confusing Ciliophora genera for species identification and separation (Chen et al. 2007; Kim et al. 2012; Shao et al. 2006; Song et al. 2003). This genus is confusing because few characters are used for the species description, and most of the putative species are extremely variable in many morphological aspects, such as body shape and size, number of macronuclear nodules, and number of somatic kineties. The character states also overlap among many species (Tables 4, 5). Therefore, the literature includes several misidentifications or inadequate descriptions (Borror 1963; Kahl 1932; Petz et al. 1995; Song and Wilbert 1997; Song et al. 2003; Villeneuve-Brachon 1940). Thus, studies involving Condylostoma spp. identification should be accompanied by meticulous details on morphology using techniques that facilitate the visualization of the infraciliature pattern, nuclear apparatus, and cortical granulation. Condylostoma arenarium was first characterized by Spiegel (1926); the unique characteristics presented were as follows: live organisms with a slightly tapered ellipsoid shape at the posterior end, total length range from 400 to 600 lm, 28–32 longitudinally arranged somatic kineties, oral region corresponds to 1/5 to 1/4 of the total length, moniliform macronucleus with 14–16 nodules, organisms with little pigmentation exhibit a yellow color when viewed under low magnification, frontal cirri and contractile vacuole not been observed (Fig. 7A). Subsequently, Kahl (1932), Villeneuve-Brachon (1940), and Borror (1963) described C. arenarium populations, but these three authors reported the presence of 4–5 frontal cirri (Table 4 is and Fig. 7B–E, G, H). Dragesco and Dragesco-Kerne (1986) presented a more detailed description for a population identified as C. arenarium with 6–7 frontal cirri (Table 4 and Fig. 7J). However, in a detailed study on C. curva morphology and infraciliature, Song et al. (2003) synonymized the C. arenarium population described by Kahl


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Table 3. Absolute distance (above the diagonal) and similarity (in %, below the diagonal) between the sequences of the clade (C. curva + C. cf. arenarium + C. arenarium)

Species C. curva EU379939 C. curva FJ868179 C. cf. arenarium FJ998021 C. curva KJ651827 Condylostoma sp. AM295496 C. arenarium present study

C. curva EU379939

C. curva FJ868179 19

98.8 97.1 98.9 99.2 98.5

96.8 99.7 98.6 98.1

C. cf. arenarium FJ998021 46 50 96.9 97.1 96.9

C. curva KJ651827 17 4 48 98.7 98.2

Condylostoma sp. AM295496 14 23 46 21

C. arenarium present study 23 30 49 28 21

98.7

The smallest absolute distance and the highest similarity are in bold.

Figure 6 BI tree based on 18S rRNA sequences, showing the position of Condylostoma arenarium from Guanabara Bay (bold). The support values for nodes are for BI and ML trees, respectively (BI/ML). The asterisks denote full support in both analyses. The accession number in the GenBank database is located after the taxon name. The scale bar represents two substitutions per 100 nucleotide positions.

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– – Al-Rasheid (1999) Brownish Dark-brown Dragesco and is (1986) Dragesco-Kerne – – Borror (1972) – – Agamaliev (1972) Colorless – Borror (1963)

a

Recognized from the in vivo drawings (lack of infraciliature data).

– – Villeneuve-Brachon (1940) Yellowish – Kahl (1932) – – Spiegel (1926)

ca 1/3 ca 1/3 ca 1/4–1/3 –

ca 1/3 –

– –

ca 1/5

– –

60–70 –

4–5a 50–70 19–30 4–5a Slightly tapered 28–32 14–16

4–5 26–34 11

26–32 8–15

ca 1/4

–

–

70–100

70–80

– 40 10–17 – 40–64 13–16 –

6–7 32–42 7–12

300–400 Rounded 187–367 – 170–480 Slightly tapered 500–700 – 400–600 –

213–365 Rounded

350–600 Slightly tapered

300–600 Rounded


Body length in vivo (lm) Shape of the posterior end Frontal cirri, number Somatic kineties, number Macronuclear nodules, number Adoral membranelles, number Buccal field ratio in vivo (lm) Cytoplasm color Cortical granules color Reference

C. arenarium C. arenarium C. arenarium C. arenarium Characters

Table 4. Comparison between populations previously described as Condylostoma arenarium

C. arenarium

C. arenarium

C. arenarium

C. arenarium

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(1932), Borror (1963, 1972) and Dragesco and Dragescois (1986) as C. curva. The population described by Kerne Villeneuve-Brachon (1940) as C. arenarium was also probably misidentified because, compared with the original description, the organisms are smaller, with more macronuclear nodules and 4–5 frontal cirri (Table 4). Subsequent authors might have misidentified certain species likely because their identifications were based on Kahl’s (1932) descriptions. The C. arenarium population described by Agamaliev (1972) is the only population that is fully consistent with the original description (Table 4), but this author did not detail the infraciliature. In fact, C. arenarium infraciliature has not been described using modern methods and, thus, was considered a poorly known species by Shao et al. (2006) and Chen et al. (2007). Due to the many confusing aspects of this genus, the identification of the population from Guanabara Bay, Brazil, was based on the original description by Spiegel (1926); we mainly considered the size, body shape, peristomial field ratio, color and number of macronuclear nodules. Originally, Spiegel did not observe frontal cirri likely because his description was only based on in vivo observations. The C. arenarium population from Guanabara Bay has slightly more somatic kineties and macronuclear nodules compared with the original population. However, such features may vary among C. arenarium populations due to environmental conditions (Kiesselbach 1935). The genus Condylostoma includes five marine morphotypes that should be compared with C. arenarium: C. curva Burkovsky 1970; C. granulosum Bullington 1940; C. magnum Spiegel 1926; C. minutum Bullington 1940 and Condylostoma spatiosum Ozaki and Yagiu, 1944. Condylostoma curva was repeatedly misidentified as C. arenarium (see the discussion above and Song et al. 2003). However, this species is easily distinguished from C. arenarium due to the highest number of frontal cirri (4–8 vs. 1–2 in C. arenarium), a substantially less slender body shape; further, it is smaller (primarily < 250 lm vs. usually > 250 lm). Moreover, C. curva have dark gray cortical granules (vs. green-yellowish in C. arenarium) and a greater peristomial field (Song et al. 2003; Table 5). Condylostoma curva granulosum have a less slender body shape, more prominent oral cavity and colorless cortical granules that are thickly arranged between the kineties (vs. green-yellowish cortical granules that are sparsely arranged in C. arenarium) (Petz et al. 1995; Table 5). The C. magnum infraciliature was fully described by Song and Wilbert (1997). This species differs from C. arenarium in its longer body, which can is 1986; reach 800 lm (Dragesco and Dragesco-Kerne Song and Wilbert 1997), while the length of C. arenarium does not exceed 600 lm (Tables 4, 5). Besides the difference in length, C. magnum has a more tapered posterior end. This feature is even more evident in impregnated specimens (see pictures from Dragesco and is 1986; Kahl 1932; Song and Wilbert Dragesco-Kerne 1997). Moreover, C. magnum has more membranelles and a grayish cytoplasm (Table 5). Condylostoma minu-


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Table 5. Morphological comparison of Condylostoma arenarium and some closely related species Characters

C. granulosum

C. magnum

C. spatiosum

C. minutum

C. curva

C. arenarium

Body, length in vivo (µm) Body, width in vivo (µm) Frontal cirri, number Somatic kineties, number Shortened somatic kineties, number Macronuclear nodules, number Length of a macronuclear nodule (µm) Adoral membranelles, number Buccal field ratio in vivo ( µm) Cytoplasm color

214–590 65–200 1–3 28–65 –

450–800 80–120 2 47–56 3–6

400–700 120 1 51–63 2–5

200–400 80 1–2 26–33 5–7a

150–350 – 4–8 22–32 7–9a

350–600 70–220 1–2 30–45 5–6

6–13

12–19

13–22

10–15

5 a 13

15–20

17–85

20–44

20–60

12–40

123–210

ca 150–200

113–153

67–103

68–108

83–145

ca 1/3

ca 1/4

ca 1/3

ca 1/4

ca 1/3 to 2/5

ca 1/5

Colorless

Yellowish

Colorless

Colorless Petz et al. (1995)

Colorless to slightly yellow-grayish Dark- gray Chen et al. (2007)

Yellow-brownish

Cortical granules color Reference

Colorless to grayish – Song and Wilbert (1997)

Dark- gray Chen et al. (2007)

Dark gray Song et al.(2003)

Green-yellowish Present study

–

10–34

a

Based on drawings.

tum differs from C. arenarium in its smaller size (< 400 lm vs. 400–600 lm in C. arenarium), more conspicuous peristomial cavity (ca. 25% vs. ca. 20% in C. arenarium) and dark-gray cortical granules (Chen et al. 2007; Table 5). Condylostoma spatiosum was well-characterized by Shao et al. (2006) in a comprehensive study on the morphology and morphogenesis of this species. Condylostoma spatiosum differs from C. arenarium mainly in its dark-gray cortical granule color, greater peri-

Figure 7 Drawing of the populations described as Condylostoma arenarium. A. Original description by Spiegel (1926). B–E. Morphotypes presented by Kahl (1932). F. Villeneuve-Brachon (1940). G–H. Borror (1963, 1972), respectively. I. Agamaliev (1972). J. Dragesco and is (1986). Scale bars: (A–E), (G–J) 200 lm. (F) 50 lm. Dragesco-Kerne

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stomial field, and greater number of somatic kineties (51–63 vs. 30–45 in C. arenarium) (Table 5). Most features that are considered taxonomically important for Condylostoma are highly variable; these features include body size, number of kineties, number of macronuclear nodules, and number of membranelles. In addition to intraspecific variability, certain characteristics may change due to environmental conditions. For example, Kiesselbach (1935) revealed that temperature affects body size and macronuclear organization in C. arenarium. We agree with Song and Wilbert (1997); the number and structure of frontal cirri should be a good characteristic for Condylostoma species distinction. However, the states for this characteristic overlap in several species (see the example in Table 5); thus, this feature cannot be solely used to identify Condylostoma spp. Cortical granule coloration is also a primary characteristics used for species identification and separation in the genus Condylostoma. However, the color of the granules is subjective, and as different hues of the same color may be present, many instances can cause confusion; for example, the colors “yellow-brownish” and “yellow-greenish” may be the same color described by different observers. Moreover, the granule color may vary slightly based on the quantity of light used during microscopy observations. The number of shortened somatic kineties is also a taxonomically valuable characteristic for Condylostoma, but it is rarely used in species descriptions. Most Condylostoma species descriptions are highly superficial, the characteristics used to identify species are highly variable, and numerous misidentifications have accumulated in the literature. Therefore, a current revision of the genus is necessary, with detailed descriptions on the morphology and diagnostic characteristics of the spe-


Fernandes et al.

cies using effective impregnation techniques, electronic microscopy, and molecular tools. Phylogenetic position of C. arenarium Condylostoma arenarium clustered with Condylostoma sp. (AM295496) within a clade composed of three C. curva sequences with high support values (Fig. 6). As shown in Table 3, within the (C. curva + C. arenarium + C. cf. arenarium) clade, a high number of nucleotide substitutions is observed: the sequence identified as C. cf. arenarium (FJ998021) differed from C. arenarium sequence by 49 nucleotides. Between the three sequences identified as C. curva, there were differences of 4 (FJ868179, KJ651827), 17 (EU379939, KJ651827) and 19 (FJ868179, EU379939) nucleotides. When comparing with other species of the same genus, we found that the absolute distance between the two sequences of C. spatiosum was 0, i.e., they were identical, and the two sequences of C. minutum differed at only three nucleotides sites. The high number of nucleotide substitutions observed between sequences of C. curva when contrasted to the other two species indicates the possibility of a species complex in C. curva. However, the number of available sequences is still very low to infer the rate of substitution for 18S rRNA gene in Condylostoma species. Descriptions of C. curva populations are quite variable (see Burkovsky 1970; Chen et al. 2007, Kim et al. 2012; Song et al. 2003) due to the plasticity of morphological characters used for Condylostoma spp. identifications. For example, Song et al. (2003) after studying three C. curva populations, has found that one of them was “sufficiently different from the other two to suggest it might not be conspecific.” However, because the populations shared some features as the number and arrangement of the macronuclear nodules, the appearance of cortical granules, cell color, and habitat, the authors assumed that they were conspecific. The morphological plasticity in C. curva also corroborates the possibility of a species complex. The genus Condylostoma failed in forming a monophyletic group in our analysis because Chattonidium Villeneuve, 1937 and Condylostentor Jankowski, 1978 grouped within the clade composed of Condylostoma spp. (Fig. 6). This result is in agreement with previous studies on Heterotrichea phylogeny (Schmidt et al. 2007; Thamm et al. 2010; Yan et al. 2014a,b). Nevertheless, only five of the 30 nominal species of Condylostoma have 18S rRNA sequences deposited in GenBank. Increasing the number of Condylostoma, Chattonidium, and Condylostentor species is necessary to elucidate their phylogenetic relationships. ACKNOWLEDGMENTS The authors are grateful to Venicio da Veiga for his technical support with scanning electron microscopy and to Pedro Henrique Nunes for some in vivo pictures. We


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Morphology and phylogenetic position of an unusual Stentor polymorphus (Ciliophora: Heterotrichea) without symbiotic algae.

Morphology and molecular phylogeny of three marine Condylostoma species from China, including two new ones (Ciliophora, Heterotrichea).

Redescription of Phacodinium metchnikoffi (Ciliophora, Hypotrichida): general morphology and taxonomic position.

Redescription of the Little-known Ciliate Apoamphisiella vernalis (Stokes, 1887) Berger, 2006 (Ciliophora, Hypotricha) from the Atlantic Forest, Brazil.

Environmental and Sanitary Conditions of Guanabara Bay, Rio de Janeiro.

Molecular systematics and ultrastructural characterization of a forgotten species: Chattonidium setense (Ciliophora, Heterotrichea).

Microbiological Quality Assessment by PCR and Its Antibiotic Susceptibility in Mangrove Crabs (Ucides cordatus) from Guanabara Bay, Rio de Janeiro, Brazil.

Redescription of two subterranean amphipods Niphargusmolnari Méhely, 1927 and Niphargusgebhardti Schellenberg, 1934 (Amphipoda, Niphargidae) and their phylogenetic position.

Bozasella gracilis n. sp. (Ciliophora, Entodiniomorphida) from Asian elephant and phylogenetic analysis of entodiniomorphids and vestibuliferids.

Redescription of Robustodorus megadorus with Molecular Characterization and Analysis of Its Phylogenetic Position within the Family Aphelenchoididae.

Morphological reports on two species of Dexiotricha (Ciliophora, Scuticociliatia), with a note on the phylogenetic position of the genus.

Geochronology and heavy metal flux to Guanabara Bay, Rio de Janeiro state: a preliminary study.

Phylogenetic position of the genus Cyrtostrombidium, with a description of Cyrtostrombidium paralongisomum nov. spec. and a redescription of Cyrtostrombidium longisomum Lynn & Gilron, 1993 (Protozoa, Ciliophora) based on live observation, protargol impregnation, and 18S rDNA sequences.

Dermacentor everestianus Hirst, 1926 (Acari: Ixodidae): phylogenetic status inferred from molecular characteristics.

Redescription of Atopospira galeata (Kahl, 1927) nov. comb. and A. violacea (Kahl, 1926) nov. comb. with redefinition of Atopospira Jankowski, 1964 nov. stat. and Brachonella Jankowski, 1964 (Ciliophora, Armophorida).

Phylogenetic relationships of Blepharisma americanum and Colpoda inflata within the phylum ciliophora inferred from complete small subunit rRNA gene sequences.

On Prophoca and Leptophoca (Pinnipedia, Phocidae) from the Miocene of the North Atlantic realm: redescription, phylogenetic affinities and paleobiogeographic implications.

Ciliates from eutrophized water in the northern Brazil and morphology of Cristigera hammeri Wilbert, 1986 (Ciliophora, Scuticociliatia).

Phylogenetic framework of the systematically confused Anteholosticha-Holosticha complex (Ciliophora, Hypotrichia) based on multigene analysis.

Molecular detection and phylogenetic analysis of bovine astrovirus in Brazil.

Phylogenetic position of Guihaiothamnus (Rubiaceae): its evolutionary and ecological implications.

Phylogenetic position of avian nocturnal and diurnal raptors.

Phylogenetic study of Class Armophorea (Alveolata, Ciliophora) based on 18S-rDNA data.

Helichrysum arenarium subsp. arenarium: phenolic composition and antibacterial activity against lower respiratory tract pathogens.