Morphology, morphogenesis, and molecular phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a "Stylonychine Oxytrichidae" (Ciliophora, Hypotrichida) without transverse cirri.

Published by the International Society of Protistologists

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Eukaryotic Microbiology

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE

Morphology, Morphogenesis, and Molecular Phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a “Stylonychine Oxytrichidae” (Ciliophora, Hypotrichida) Without Transverse Cirri Santosh Kumara,b, Daizy Bhartia, Silvia Marinsaltia, Emilio Insoma & Antonietta La Terzaa a School of Environmental Science, University of Camerino, Via Gentile III da Varano, 62032, Camerino (MC), Italy b Ciliate Biology Laboratory, Sri Guru Tegh Bahadur Khalsa College, University of Delhi, Delhi, 110007, India

Keywords Beech forest; flexible/rigid body; Italian fauna; Sibillini Park; soil ciliates. Correspondence S. Kumar and A. La Terza, School of Environmental Science, University of Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy Telephone number: +39 0737 403272; FAX number: +39 0737 403290; e-mail: [email protected] (S. Kumar); [email protected] (A. La Terza) Received: 1 August 2013; revised 25 November 2013; accepted December 12, 2013. doi:10.1111/jeu.12103

ABSTRACT A terrestrial oxytrichid ciliate Paraparentocirrus sibillinensis n. gen., n. sp., which was found in soil samples of a beech forest stand within the National Park of Sibillini Mountains, Italy, was investigated using live observation and protargol impregnation. The morphology of interphase, morphogenesis, and molecular phylogeny inferred from SSU rDNA sequences of this ciliate were studied. Paraparentocirrus n. gen., is mainly characterized by a semirigid body, an undulating membrane in the Oxytricha pattern, six fronto-ventral (FV) rows, the absence of transverse cirri, one right and one left row of marginal cirri, four dorsal kineties, two dorsomarginal rows, and caudal cirri at the end of dorsal kinety 4. During morphogenesis, oral primordia develop through the proliferation of basal bodies from some cirri of FV rows 4 and 5, and FV row 6 takes part in the anlagen formation of the proter. The dorsal morphogenesis was typical of oxytrichids, with simple fragmentation of dorsal kinety 3, and the dorsomarginal rows developed from the right marginal row. Phylogenetic analyses based on the SSU rDNA sequences support the classification of this new genus in the stylonychines.

USING the 18 frontal-ventral-transverse (FVT) cirri pattern as an apomorphy, the family Oxytrichidae Ehrenberg 1838 was divided into the subfamilies Stylonychinae (rigid body, lack of cortical granules) and Oxytrichinae (participation of postoral ventral cirrus V/3 in primordia formation) (Berger 1999; Berger and Foissner 1997). However, some taxa with cirral patterns in fronto-ventral (FV) rows (more than 18 FVT cirri) were placed as taxa of unknown position within the Oxytrichidae (Berger 1999): Ancystropodium, Apoamphisiella, Gastrostyla, Kerona, Paraurostyla, Parentocirrus, Territricha, and Pseudouroleptus. Later, Berger (2008) eliminated the paraphyletic group Oxytrichinae by assuming the 18-cirri pattern was a plesiomorphic feature and, hence, could not be used to define the oxytrichids. Thus, a new hypothesis was proposed to include all those genera in the family Oxytrichidae, which possess as apomorphy a fragmentation of kinety 3. Therefore, regardless of their ciliature (18 FVT cirri or FV rows), the family Oxytrichidae is divided into two groups, namely, the Stylonychinae (body rigid, adoral zone of membranelles

(AZM) ≥ 40% of body length, cortical granules absent) and nonstylonychine oxytrichids (body flexible, AZM ≤ 40% of body length, cortical granules present/absent) (Berger 2006, 2008). The following genera are now included in the Stylonychinae: Coniculostomum, Gastrostyla, Histriculus, Laurentiella, Onychodromous, Pattersoniella, Pleurotricha, Rigidocortex, Steinia, Sterkiella, Stylonychia, Styxophrya, and Tetmemena; and in nonstylonychinae Oxytrichidae: Amphisiellides, Apoamphisiella, Architricha, Australocirrus, Cyrtohymena, Neokeronopsis, Notohymena, Oxytricha, Paraurostyla, Ponturostyla, Pseudouroleptus, Rubrioxytricha, and Territricha. The ventral ciliature of some oxytrichid genera (e.g., Apoamphisiella, Paraurostyla, Parentocirrus) with FV rows are similar to genera of the family Kahliellidae (e.g., Parakahliella, Fragmocirrus) (Berger 2011; Foissner 2000); therefore, when based on morphology, it becomes difficult to assign species to their respective genera. However, it has been reported that ontogenetic details are less variable and, thus, when combined with morphology,

© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2014, 61, 247–259

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offer important information on the identification and construction of evolutionary lineages (Blatterer and Foissner 2003). This present study adds a new species, with an increased number of FV rows, to stylonychines (Berger 2006, 2008). Similarity in the ciliature of the new species on the ventral (except for the absence of transverse cirri) and dorsal surfaces with Parentocirrus hortualis compelled us to place the species in the genus Parentocirrus by assuming the species had lost transverse cirri. However, ontogenetic studies showed differences that, in combination with morphology, demanded the separation of the species into a new genus. Currently, all known genera of the family Oxytrichidae were reported to possess transverse cirri; however, the number is reduced in some cases, as in some species of Oxytricha, Sterkiella, and Urosomoida (now nonoxytrichid Dorsomarginalia). This is the first report of a species without transverse cirri in the family Oxytrichidae. Detailed descriptions of the morphology, morphogenesis, and phylogeny based on the SSU rDNA sequence of P. sibillinensis n. gen., n. sp. are presented. MATERIALS AND METHODS Description of the Gualdo beech forest and analyses of the main physico-chemical and pedological parameters of soil samples The Gualdo beech forest (42°52′55.46″N; 13°10′24.40″E) is located within the protected areas of the National Park of the Sibillini Mountains (http://www.sibillini.net/en/index. html) (Nardoni 1999). The park spans an area of approximately 70,000 ha between the Marche and Umbria regions in Central Italy. All physico-chemical analyses were performed at the laboratory of Agrochemistry of ASSAM (Agenzia per i Servizi nel Settore Agroalimentare delle Marche, http://www.assam.marche.it) according to the standard procedures provided by D.M 13/09/99 GU SO n° 248 del 21/10/1999 “Metodi Ufficiali di Analisi Chimica del Suolo”. Pedological analyses were performed at the Osservatorio Regionale Suoli (http://suoli.regione. marche.it/) according to U.S. Soil Taxonomy (Soil Survey Staff 2006). Sampling and sample processing Soil samples were collected twice in 2009, in spring (May) and autumn (October), at the same topographic position in the Gualdo forest. Ten soil samples (0–10 cm deep), including fine plant roots and litter, were randomly collected from an area of approximately 100 m2, mixed to obtain a composite sample (weighing approximately 1 kg) and transferred to the laboratory. Ciliates were made to excyst and emerge from 1-mo-dried soil samples (approximately 300 g) by employing the nonflooded Petri dish method (Foissner 1987). Twenty to 30 cells were isolated with the help of a glass micropipette from both samples to raise established cultures of P. sibillinensis n. gen., n. sp., these cultures

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were used to study the morphology, ontogenesis, and molecular phylogeny. Cells were found to thrive well when maintained at a temperature of 18 °C 2 °C in Pringsheim’s medium with the green alga Chlorogonium elongatum as the food organism (Ammermann et al. 1974). Live observations were made using a microscope with bright-field illumination at a magnification of 100–1,000X. The body shape and flexibility of the cells were studied without coverslip pressure, and protargol staining (Kamra and Sapra 1990) was used to reveal their infraciliature. Biometric characterization was carried out at a magnification of 1,000X using the Optika Vision Lite software (DFM - Distribuzioni Fototecniche Molinari, Venezia, Italy). An Optika microscope camera was employed for photomicrography, and line diagrams were prepared using free-hand sketches. To demonstrate the changes during morphogenesis, old (parental) structures were depicted by contour, while newly formed structures were shaded in black. The numbering of the FVT cirri is according to Eigner (1999), Berger (1999), and Blatterer and Foissner (2003). DNA extraction, PCR amplification, and sequencing DNA extraction was performed according to Thomas et al. (2005). Thirty to 40 cells from a culture that was starved overnight were collected with the help of glass micropipettes and washed three times with autoclaved distilled water. For DNA extraction, 50 ll of 5% (w/v) Chelex100 and 2 ll of Proteinase K (20 mg/ml) solution were added to the sample and then incubated at 37 °C for 30 min, followed by incubation at 98 °C for 5 min. The reaction mixture was cooled immediately on ice and centrifuged in a microfuge tube for 2–3 s at a speed of 16,000 g. Without disturbing the Chelex100 beads, 5 ll of the supernatant was carefully drawn from the top of the sample and stored at 4 °C until PCR amplification or immediately used for PCR. Extracted DNA (5 ll) was dispensed into a PCR tube containing 5 ll of distilled water, and amplifications were carried out using high-fidelity Pfx50TM DNA polymerase (Invitrogen S.r.l., San Giuliano Milanese, Italy) in a total volume of 50 ll with the universal eukaryotic primers (Medlin et al. 1988) Euk A (FW 5′-AACCTGGTTGATCCTGC CAGT-3′) and Euk B (RV 5′-TGATCCTTCTGCAGGTTCAC CTAC-30). In addition, the newly designed, nested primer pairs Eup 18S (FW 5′-TAG AGG GAC TTT GTG TGC AAC C-3′) and Eup 18S (RV 5′-ATC TCC CTG AAA CAC ACG TTG G-3′) were used in combination with the universal primers for amplification and sequencing. The PCR program for 18S rDNA amplification included an initial denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 1 min, 55 °C for 45 s and 72 °C for 80 s, with a final extension step at 72 °C for 10 min. After confirmation of the appropriate size, the PCR products were purified using the Nucleospin gel extraction kit (QIAGEN S.r.l., Milan, Italy) and were inserted into a PGEM-T easy vector system (Promega Italia S.r.l., Milan, Italy). Five independent, positive clones were then sequenced on both strands at StarSEQ GMBH, Germany.



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Phylogenetic analyses For phylogenetic analyses, the newly sequenced SSU rRNA gene sequence of P. sibillinensis n. gen., n. sp., was aligned with SSU rRNA gene sequences of 35 hypotrichs that were retrieved from the GenBank database using the MATTF 7.047 software (choosing the iterative refinement methods L-INS-i) (Katoh and Standley 2013). The final alignment was then used for subsequent phylogenetic analyses after converting the FASTA (.fas) file to NEXUS (.nex) format using the open web-based tool ALTER (Alignment Transformation EnviRonment) ~a et al. 2010). A Bayesian inference (BI) (Glez-Pen analysis was performed using Mr.Bayes v.3.2.1 (Ronquist and Huelsenbeck 2003) and the GTR+I+G model, as selected by the jModel Test v.2.1.3 software (Posada 2008) under the Akaike Information Criterion corrected (AICc). Markov chain Monte Carlo (MCMC) simulations were run, with two sets of four chains using the default settings, for 8,000,000 generations with trees sampled every 100 generation. A prior burn-in of 25%, i.e., the first 20,000 sampled trees was discarded. The remaining trees were used to generate a consensus tree and to calculate the posterior probabilities. A maximum likelihood (ML) tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA) v.5.2.2 (Tamura et al. 2011), and the topology of the trees was inferred by running 1,000 bootstrap replicates and was expressed as a percentage. Phylogenetic trees were visualized using the free software package FigTree v1.4 by A. Rambaut at http://tree.bio.ed.ac.uk/software/figtree/. RESULTS Description of P. sibillinensis n. sp. (Fig. 1, 2, Table 1) On average 130 9 60 lm in protargol preparations, about 145 9 65 lm in vivo. Body shape ellipsoidal with narrowly to broadly rounded posterior end. Body semirigid, flattened dorso-ventrally about 2:1. Four to eight macronuclear nodules aligned left of cell median, accompanied by 2–5 micronuclei, 2.6 lm across (Fig. 2A). Contractile vacuole left of mid-body behind the proximal end of AZM. Cortical granules absent, cytoplasm colorless, filled with cytoplasmic granules 1–2 lm in diam., and few fat droplets (Fig. 2A, E). Specimen in established cultures with around 100–150 food vacuoles containing the green alga C. elongatum. Slow movement, creeping, and gliding on the substratum (Fig. 1A–C, 2A–E). Adoral zone of membranelles about 40% of body length composed of 40 membranelles on average, cilia in membranelles about 20 lm long in vivo. Undulating membranes (UMs) slightly curved, paroral optically intersects endoral in anterior third; both membranes terminate slightly anterior of proximal end of AZM. Usually six, rarely seven FV rows (=anlagen; the rows are numbered according to the anlagen formation from which they originate during ontogenesis), row 1 consists of the first frontal cirrus; row 2 consists of the second frontal cirrus and buccal

cirri; row 3 consists of the third frontal cirrus plus some cirri behind; row 4 has a distinct break, with the first anterior cirrus of row 4 aligned beneath row 3 and the rest to the left of row 5; row 5 commences near the buccal vertex and extends posteriorly. FV row 3, first anterior cirrus of row 4, and row 5 aligned to form a mixed row extending to near rear end of cell; row 6 commences near rightmost frontal cirrus and extends posteriorly. Ventral cirri about 12 lm long in vivo. One left and one right marginal row with about 28 and 29 cirri, respectively; rows confluent posteriorly (Fig. 1A–C, 2A, F, G). Marginal cirri about 17 lm long in vivo. Dorsal bristles in vivo 2.5–3.0 lm long. Six dorsal kineties (Fig. 1C, 2G): first and second bipolar with 26–39 and 19–36 bristles, respectively; dorsal kineties 3 and 4 of almost same length, composed of 17–26 and 17–25 bristles, respectively. Two dorsomarginal rows with about 7–18 and 3–8 bristles. One or two caudal cirri, at posterior end of dorsal kinety 4 (Fig. 1C, 2G). Resting cysts 50 lm in diam. in vivo; cyst’s surface with hyaline ridges, about 4 lm thick. Cyst wall only about 0.5 lm. Cyst content close to the wall composed of lipid droplets about 4 lm across and 4–6 globular macronuclear nodules (Fig. 1D–F). 18S rDNA sequence and phylogeny (Fig. 7) The 18S rDNA sequence of P. sibillinensis has a length of 1,667 bp and a GC content of 45%. It has been deposited in the NCBI database under accession number KF184655. Phylogenetic analyses inferred from the SSU rDNA sequences using BI and ML present similar topologies; therefore, only the BI tree is presented here (Fig. 7). Phylogenetic trees consistently place the new genus within the stylonychines, close to the species Pattersoniella vitiphila and Gastrostyla steinii. Morphogenesis of P. sibillinensis (Fig. 2H–6) Morphogenesis commences with the appearance of small groups of basal bodies that develop postorally from three or four cirri and one or two cirri of FV rows 4 and 5, respectively (Fig. 2H, 5A). The groups of basal bodies proliferate and join to form the oral primordium (OP) (Fig. 2I, J, 5B, C). The OP broadens by increasing its number of kinetosomes, the anterior end of the OP develops three streaks (opisthe’s anlagen I–III), and the portion below the streaks differentiates to form the adoral membranelles (AMs) of the opisthe. Simultaneously, the second and third cirri of FV row 3 disaggregate to form anlagen III and IV of the proter (Fig. 2K, L, 5D, E). During the next stage, buccal cirri disaggregate to form anlage II of the proter, and the anterior-most cirrus of FV row 4 disaggregates to produce anlagen V and VI. Some kinetosomes to the right of anlagen I–III of the opisthe extend toward FV row 5, and these basal bodies produce anlagen IV and V of the opisthe and the UMs (Fig. 3A, B, 5F). Subsequently, the newly formed anlagen V and VI of the proter and anlagen IV, V, and VI of the


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Figure 1 Photomicrographs of Paraparentocirrus sibillinensis from life (A, D–F) and after protargol impregnation (B, C). A. Slightly compressed specimen due to coverslip pressure with food vacuoles containing the green alga Chlorogonium elongatum. B, C. Ventral and dorsal surface of the holotype specimen showing the ciliature in six fronto-ventral rows 1–6 on the ventral surface; dorsal kineties 1–4 (DK 1–4) and dorsomarginal rows 1, 2 (DM 1, 2) on the dorsal surface. Arrowhead marks the caudal cirrus at the end of dorsal kinety 4. D. Surface view of a resting cyst, 50 lm across, showing the hyaline structure that forms the fine ridges (opposed arrowheads). E. Enlarged optical section showing numerous lipid droplets and globular macronuclear nodules. Opposed arrowheads mark the thin cyst wall (about 0.5 lm). F. Optical section of a complete cyst; opposed arrowheads mark the thick hyaline layer (about 4 lm). FV = food vacuole; L = lipid droplets; MA = macronucleus. Scale bars = 10 lm (E); 25 lm (D–F); and 40 lm (A–C).

opisthe lengthen by the addition of basal bodies. Later, anlage VI of the opisthe increases in length by incorporating one or two cirri of parental FV row 5 (Fig. 3C, 5G).

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Subsequently, the anterior portion of the parental UMs begins to reorganize and produces anlage I of the proter, and the fourth or fifth cirrus of parental FV row 6 begins to disaggregate and is later joined by the proliferating



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Figure 2 Line diagrams of Paraparentocirrus sibillinensis from life (A–E) and after protargol impregnation (F–L). A. A representative cell with length of 145 lm. B–D. Specimens observed without a cover slip. E. Section showing the cytoplasmic granules. F, G. Ventral and dorsal view of the holotype specimen. H–L. Segments showing the morphogenetic events on the ventral surface. H–J. OP formation by disaggregation of 3–5 cirri of fronto-ventral row 4 and 2–3 cirri of row 5 (H, I), which later combines and proliferates (J). K. Anterior end of the OP begins to differentiate and forms two streaks (I and II) for the opisthe. L. A third streak arises and forms anlage III of the opisthe, and the second and third cirri of parental fronto-ventral row 3 begin to disaggregate. Numbers 1–6 denote the six fronto-ventral rows on the ventral surface, I–VI the newly formed anlagen for the proter and opisthe. AZM = adoral zone of membranelles; CC = caudal cirri; C = cytoplasmic granules; DK(1–4) = dorsal kineties; DM (1, 2) = dorsomarginal rows; EM = endoral membrane; FC (1, 3) = frontal cirrus; LM = left marginal row; OP = oral primordium; PM = paroral membrane; RM = right marginal row. Scale bars = 30 lm (H–K); 40 lm (F, G, L); and 60 lm (A).

anlage VI of the proter. Further involvement of one or two more cirri takes place during lengthening. In the opisthe, all anlagen are formed and begin to separate from the

anlage of the UM; and two-thirds of the new AMs in the OP are organized from anterior to posterior in oblique rows (Fig. 3D, E, 5H, 6A).


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Table 1. Morphometric data on Paraparentocirrus sibillinensis Characteristicsa Body, length Body, width Body length: width, ratio AM, no. AZM, length AZM/body length% Ma, no. Ma, length Ma, width Mi, no. Mi, diam. Buccal cirri, no. Frontal cirri, no. FV row 1, no. of cirri FV row 2, no. of cirri FV row 3, no. of cirri FV row 4, no. of cirri FV row 5, no. of cirri FV row 6, no. of cirri RMR, no. RMR, no. of cirri LMR, no. LMR, no. of cirri DK, no. DK 1, no. of dikinetids DK 2, no. of dikinetids DK 3, no. of dikinetids DK 4, no. of dikinetids DM, no. DM 1, no. of dikinetids DM 2, no. of dikinetids CC, no.

Min

Max

Mean

SD

SE

CV

n

104.0 47.0 1.5

149.9 75.4 2.7

127.4 57.0 2.3

13.1 7.1 0.2

2.6 1.4 0.1

10.3 12.4 10.6

25 25 25

34.0 43.7 34.1

47.0 63.8 44.3

39.8 51.2 40.2

3.0 5.5 2.7

0.6 1.1 0.5

7.4 10.7 6.6

25 25 25

4.0 8.2 6.5 2.0 2.2 1.0 3.0 1.0 2.0 3.0 5.0 11.0 10.0 1.0 24.0 1.0 23.0 4.0 26.0

8.0 17.6 11.2 5.0 3.1 2.0 3.0 1.0 3.0 3.0 10.0 18.0 20.0 1.0 33.0 1.0 33.0 4.0 39.0

5.9 10.8 8.6 3.2 2.7 1.8 3.0 1.0 2.8 3.0 8.0 14.1 15.8 1.0 29.3 1.0 28.2 4.0 30.2

1.3 2.4 1.0 0.8 0.3 0.4 0.0 0.0 0.4 0.0 1.3 2.0 2.6 0.0 2.3 0.0 2.6 0.0 3.1

0.3 0.5 0.2 0.2 0.1 0.1 0.0 0.0 0.1 0.0 0.3 0.4 0.5 0.0 0.5 0.0 0.5 0.0 0.6

22.1 22.1 12.0 25.5 9.5 22.7 0.0 0.0 14.6 0.0 16.1 14.1 16.7 0.0 7.9 0.0 9.3 0.0 10.3

25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25

19.0

36.0

26.2

4.4

0.9

16.8

25

17.0

26.0

20.3

2.5

0.5

12.4

25

17.0

25.0

20.6

2.3

0.5

11.0

25

DISCUSSION

2.0 7.0

2.0 18.0

2.0 12.5

0.0 2.8

0.0 0.6

0.0 22.1

25 25

Comparison with related genera and species

3.0

8.0

6.2

1.1

0.2

17.4

25

1.0

2.0

1.0

0.2

0.0

19.2

25

a

Data based on mounted, protargol-impregnated, and randomly selected specimens from established cultures. Measurements in lm. AM = adoral membranelles; AZM = adoral zone of membranelles; CC = caudal cirri; CV = coefficient of variation in %; DK = dorsal kineties; FV = fronto-ventral; DM = dorsomarginal rows; LMR = left marginal rows; M = median; Ma = macronuclear nodules; Max = maximum; Mi = micronuclei; Min = minimum; n = number of individuals investigated; RMR = right marginal rows; SD = standard deviation; SE = standard error of arithmetic mean.

Progressively, the anterior disorganized portions of the UMs of the proter and opisthe generate the first frontal cirrus, and the FV anlagen I–VI for both daughter cells develop into new cirri and produce an average of 1, 3, 3, 8, 14, and 16 cirri, respectively. The formation of new AMs for the opisthe is complete, and the anterior portion of the adoral zone begins to curve toward the right.

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Migration of FV rows 4 (except for the first anterior cirrus) and 5 toward the cell posterior end takes place. FV row 3, anterior first cirrus of FV row 4, and row 5 align to form a mixed row (Fig. 3F, G, 6B, C). The unused cirri of the parental FV rows are completely resorbed. The AZM for the opisthe is formed from the OP while that of the parental cell is retained unchanged for the proter. The marginal primordia arise at each of the two levels by “within-row” anlagen formation by utilizing one or two of the parental cirri. The first cirrus of the left marginal row and fourth cirrus of the right marginal row disaggregate to form the anlagen for the proter, and, similarly, develop anlagen for the left and right marginal rows of the opisthe in their mid-region. These anlagen then lengthen and completely replace the old parental structure (Fig. 3C–G, 5G, H, 6A–C). On the dorsal surface, three anlagen are formed within-row from dorsal kineties 1–3 at two levels (one set each for the proter and the opisthe). The third dorsal anlage fragments in the middle and gives rise to the third and fourth dorsal kineties, which are of nearly equal length. The two dorsomarginal rows arise near the anterior end of the new right marginal row and move from the lateral to the dorsal surface. One or, rarely, two caudal cirri are formed at the posterior end of the new kinety 4, and the short caudal cirrus, which is located in the gap between the two marginal rows, becomes inconspicuous (Fig. 4A–C, 6D–F). Nuclear division proceeds as usual. The macronuclear nodules fuse to form a single mass in middle dividers, which divides two to three times to produce the typical four to eight nodules in the late dividers. The micronuclei undergoes mitotic division as usual (Fig. 3E–G).

Paraparentocirrus n. gen. belongs to the group “Stylonychine Oxytrichidae” (Berger 2008) of the family Oxytrichidae because it possesses a semirigid body (Fig. 2B–D). Paraparentocirrus is a sensu lato oxytrichid ciliate with ventral ciliature in FV rows and is a unique “Stylonychine Oxytrichidae” without transverse cirri. The family Oxytrichidae comprises three genera which are morphologically similar to Paraparentocirrus: Paraurostyla (Borror 1972), Apoamphisiella (Foissner 1997), and Parentocirrus (Voß 1997). Paraurostyla differs from Paraparentocirrus in having more than six ventral primordia (vs. six) and the presence of transverse cirri (vs. absent). However, during morphogenesis both Paraurostyla and Paraparentocirrus utilize the rightmost ventral row for primordia formation. Paraurostyla granulifera (Berger and Foissner 1989a), P. polymicronucleata (Berger 1999), and P. weissei (Berger 1999) have two macronuclear nodules; however, their numbers of ventral rows are similar to that of P. sibillinensis. The multinucleate species Paraurostyla polynucleata (Alekperov 1993) can be separated from P. sibillinensis in that nine macronuclear nodules (vs. 4 to 7) are present in the latter.


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Figure 3 Line diagrams of protargol-impregnated mid and late dividers of Paraparentocirrus sibillinensis showing morphogenetic events on the ventral surface. Numbers 1–6 denote the six parental fronto-ventral rows and I–VI the newly formed anlagen for the proter and opisthe. A. Buccal cirri disaggregate to form anlage II of the proter, and the arrowhead points to the disaggregating first cirrus of fronto-ventral row 4. Kinetosomes proliferate to form anlage IV and V of the opisthe. B. Segment showing the formation of anlagen IV, V, and VI of the proter and opisthe. C. Anlage IV of the proter lengthens. Similarly, anlage VI of the opisthe proliferates toward fronto-ventral row 5 and incorporates some cirri. The arrowheads mark the anlagen of the marginal rows at two levels. D. Anlage VI of the proter incorporates some of the disaggregating cirri of parental fronto-ventral row 6 (arrowhead), and anlage VI of the opisthe increases in length. E. The macronucleus fuses to a single mass (inset E). Arrowheads point to the marginal row anlagen. F. Splitting of anlagen into cirri begins. The arrowhead points to the dorsomarginal anlagen. Macronucleus and micronucleus division takes place (inset F). G. Fronto-ventral rows 4 and 5 migrate posteriorly. Arrowhead points to the dorsomarginal rows. Macronucleus divides a second time (inset G). MA = macronucleus; MI = micronucleus; OP = oral primordium. Scale bars = 40 lm.


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Figure 4 Line diagrams of protargol-impregnated specimens of Paraparentocirrus sibillinensis showing morphogenetic events on the dorsal surface. A. Three anlagen are formed within-row from dorsal kineties 1–3 at two levels, the left two give rise to kineties 1 and 2, and the third kinety fragments to give rise to the third and fourth dorsal kineties. B, C. Caudal cirri are formed at the posterior end of dorsal kinety 4. Two dorsomarginal rows arise at the anterior ends of the right marginal anlagen and shift to the dorsal surface (arrowheads). CC = caudal cirri; DK(1–4) = dorsal kineties; DM(1, 2) = dorsomarginal rows. Scale bars = 40 lm.

Apoamphisiella can be distinguished from Paraparentocirrus by the presence of transverse cirri (vs. absent) and dorsal morphogenesis with multiple fragmentation (vs. rt and Tam simple). Apoamphisiella tihanyiensis (Gelle as 1958) differs mainly from P. sibillinensis in having two macronuclear nodules (vs. 4–7), the presence of cortical granules (vs. absent), and an increased number of caudal cirri (3–14 vs. 1–2). Apoamphisiella hymenophora (Stokes 1886) is similar to A. tihanyiensis except for the absence of cortical granules (Berger 1999). In terms of ciliature, living morphology, and resting cyst structure, Parentocirrus closely resembles Paraparentocirrus. However, the two can be clearly separated by the presence of transverse cirri (vs. absent). In addition, during morphogenesis, Paraparentocirrus involves FV row 6 in primordia formation (vs. not involved). Furthermore, the species P. hortualis (Blatterer and Foissner 2003; Voß 1997) differs from P. sibillinensis in having 5–16 macronuclear nodules (vs. 4–8) and more caudal cirri (2–4 vs. 1–2). A reduced number of caudal cirri have also been reported in the oxytrichid genera Rubrioxytricha (Berger 1999); however, lack of dorsal kinety fragmentation makes its classification uncertain. Rubrioxytricha differs from Paraparentocirrus in having the cirral pattern in 18 FVT cirri (vs. FV rows), colored cytoplasm (vs. colorless), and the presence of transverse cirri (vs. absent).

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Three other genera that are morphologically similar to Paraparentocirrus but not in the family Oxytrichidae are: Parakahliella (Berger et al. 1985), Afrokahliella (Berger 2011), and Fragmocirrus (Foissner 2000). Parakahliella can be distinguished from Paraparentocirrus by the formation of the dorsal ciliary pattern and no kinety fragmentation (vs. present); however, both genera lack transverse cirri. Their ventral morphogenesis is similar with some minor differences. Paraparentocirrus sibillinensis differs from Parakahliella macrostoma (Berger et al. 1985; Foissner 1982) in having six macronuclear nodules (vs. 11) and one left marginal row (vs. four), and it differs from Parakahliella haideri (Berger and Foissner 1989b) by having 1–2 caudal cirri (vs. 3–6) and six dorsal kineties (vs. five). Paraparentocirrus sibillinensis differs from P. terricola (Berger 2011; Buitkamp 1977) in the number of macronuclear nodules on average six (vs. 8), AMs on average 40 (vs. 28), dorsal kineties (six vs. five), and left marginal cirral rows (one vs. three). Afrokahliella can be distinguished from Paraparentocirrus by the following features: flexible body (vs. semirigid), UMs that are straight or parallel (vs. Oxytricha pattern), the absence of kinety fragmentation (vs. present), one dorsomarginal row (vs. two), and caudal cirri at kineties 1 and 2 (vs. at kinety 4); nevertheless, both genera lack transverse cirri. Afrokahliella binucleata (Berger 2011;


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Figure 5 Photomicrographs of protargol-impregnated specimens of Paraparentocirrus sibillinensis showing morphogenetic events on the ventral surface. Numbers 4–6 denote the parental fronto-ventral rows. A. Some cirri of fronto-ventral rows 4 and 5 disaggregate (arrowheads) for OP formation. B. Kinetosomes proliferate and join to form a field (arrowheads). C, D. Anterior portion of the OP proliferates to form primordia (arrowheads). E. Cirri of parental fronto-ventral row 3 disaggregate (arrowheads), and three primordia differentiate from the OP (arrow). F. The arrow marks the proliferating kinetosomes that form anlagen IV and V of the opisthe. The arrowhead marks the disaggregation of the first cirrus of fronto-ventral row 4. G. The arrow points to the disaggregating cirri of parental fronto-ventral row 5, and arrowheads point to the marginal anlagen at two levels. H. The double arrowhead points to the disaggregating cirri of parental fronto-ventral row 6, which joins anlage VI of the proter. The arrow points to the disaggregating undulating membranes, and the arrowhead points to the within-row formed anlage IV of the opisthe. For details see Fig. 2, 3. OP = oral primordium. Scale bars = 30 lm (A–D); and 40 lm (E–H).


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Figure 6 Photomicrographs of protargol-impregnated specimens of Paraparentocirrus sibillinensis showing morphogenetic events on the ventral (A–C) and dorsal surfaces (D–F). Numbers 5, 6 denote the parental fronto-ventral rows. A. The arrow points to the disaggregating undulating membranes, and the double arrowhead points to anlage VI of the proter that lengthens by incorporating some cirri of parental fronto-ventral row 6. The arrowhead points the within-row formed anlage VI of the opisthe from parental fronto-ventral row 5. B. The arrowhead marks anlage IV of the proter, and the arrow points to the dorsomarginal primordia. C. The arrow points to the newly formed dorsomarginal rows. D. Within-row formation of the anlagen for dorsal kineties 1–3. E, F. The arrows point to the single caudal cirrus at the base of dorsal kinety 4, and the arrowheads point to the dorsomarginal rows. For details see Fig. 2, 3. Scale bars = 40 lm.

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Foissner et al. 2002) and A. namibicola (Berger 2011; Foissner et al. 2002) differ from P. sibillinensis in having two macronuclear nodules (vs. 4–8), and Afrokahliella halophila (Berger 2011; Foissner et al. 2002) differs from P. sibillinensis in having four dorsal kineties (vs. six). Paraparentocirrus can be separated from Fragmocirrus mainly by the lack of transverse cirri and in its genesis of the dorsal ciliary pattern. Fragmocirrus espeletiae (Foissner 2000) differs from P. sibillinensis in having an increased number of left and right marginal rows (vs. one), five ventral rows (vs. six), and four dorsal kineties (vs. six). Molecular sequence data and phylogenetic analyses In our analyses, P. sibillinensis clustered with P. vitiphila and G. steinii, with supports of 0.91 BI for the former and 0.87 BI for the latter (Fig. 7). These three genera show some morphological similarities that may explain a close relationship: a rigid to semi-rigid body and an increased number of cirri on their ventral surface. Paraurostyla weissei is morphologically similar to P. sibillinensis as it has ciliature in the FV rows and utilizes the rightmost ventral row during morphogenesis, as mentioned in the discussion; however, it has a flexible body and clusters in the phylogenetic tree within nonstylonychine oxytrichids.

Consequently, these analyses support the flexible/rigid body as an important, distinguishing characteristic in the phylogeny and systematics of the family Oxytrichidae. The new genus is morphologically closely related to the genera Parentocirrus and Apoamphisiella. Unfortunately, no molecular data are available for these genera. Therefore, a more robust and resolved phylogeny of P. sibillinensis and other related genera (Parentocirrus, Apoamphisiella, and Parakahliella) is restricted by the limited number of SSU rDNA sequences that are available. Note on the ciliate diversity from Italy Ciliate diversity in the soil is a still largely neglected research topic, even in Europe, where the majority of studies have been performed in Austria (Foissner 1998; Foissner et al. 2005). The authors of the present paper have discovered many new species/genera (yet to be described) from Italian natural and agricultural soils through studies that have been conducted since 2009. In this regard, Southern Europe and especially Italy represent nearly virgin territories for soil ciliate diversity studies because most Italian reports date back to the beginning of the last century (Formisano 1957; Grandori and Grandori 1934; Luzzatti 1938; Stella 1948). Thus, according to various authors (Chao et al. 2006; Foissner et al. 2008) and

Figure 7 Bayesian tree inferred from the SSU rRNA gene sequences that shows the position of Paraparentocirrus sibillinensis (bold) within the Stylonychinae. Codes following the names are GenBank accession numbers. Numbers at the nodes represent the posterior probability of Bayesian analysis and the bootstrap values of maximum likelihood out of 1,000 replicates. Values lower than 40% are replaced with asterisks (*). A hyphen (-) represents minor differences between the Bayesian and ML tree topologies. The scale bar corresponds to two substitutions per 100 nucleotide positions.


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our group, a large number of new ciliate species/genera await to be discovered (or even rediscovered) to enrich (as well as to update) the checklist of ciliate species of Italian fauna. (http://www.faunaitalia.it/checklist/) (Dini et al. 1995). TAXONOMIC SUMMARY Family Oxytrichidae Ehrenberg 1838 Paraparentocirrus n. gen Diagnosis. Stylonychines without transverse cirri. Body semirigid. Adoral zone about 40% of body length, three frontal and two buccal cirri. FV cirri arranged in six longitudinal rows. OP develops from FV rows 4 and 5. FV row six involved in anlage formation for the proter. One row each of right and left marginal cirri. Dorsal ciliature with simple fragmentation of dorsal kinety 3 and dorsomarginal rows. Caudal cirri at the end of dorsal kinety 4. Type species. Paraparentocirrus sibillinensis n. sp. Etymology. Paraparentocirrus is a composite of the Greek prefix para- (beside) and the genus-group name Parentocirrus meaning a ciliate related to Parentocirrus Voß 1997. Paraparentocirrus sibillinensis n. sp. (Fig. 1–6, Table 1) Diagnosis. Size about 145 9 65 lm in vivo; body ellipsoidal. On average, six macronuclear nodules and three micronuclei. Adoral zone composed of an average of 40 membranelles. Six FV rows composed of an average of one (first frontal cirrus), three (second frontal cirrus plus buccal cirri), three (third frontal cirrus plus cirri behind), eight (row 4), 14 (row 5), and 16 (row 6) cirri. Right and left marginal rows composed of an average of 29 and 28 cirri, respectively. Four dorsal kineties: rows 1–3 originate intrakinetally, and row 4 originates by simple fragmentation of dorsal kinety 3, two dorsomarginal rows; one or two caudal cirri. Type location. Soil from a beech forest stand (Fagus sylvatica L.) located near the small rural village of Gualdo, at an elevation of 1,263 m a.s.l., within the protected areas of the National Park of Sibillini Mountains, Marche region (Italy), 42°52′55.46″N; 13°10′24.40″E. Type material. A protargol slide with the holotype specimen circled in black ink is deposited in the Natural History Museum, London, UK, with registration number NHMUK 2013.11.13.1. Two paratype slides with protargol-stained morphostatic and dividing specimens are also deposited with registration numbers NHMUK 2013.11.13.2 and NHMUK 2013.11.13.3. Etymology. Species name sibillinensis refers to a mountain chain that is part of Italy’s central Apennine Mountains, where the homonymous National Park is based. Occurrence and ecology. As yet found from the type location, where it was moderately abundant in nonflooded Petri dish culture. Currently, the new genus has been exclusively identified in soil samples taken from the beech forest of Gualdo, even though the authors of this present

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study have surveyed several beech forest stands in the Marche region. The forest is situated at an elevation of 1,263 m a.s.l., and according to U.S. Soil Taxonomy (Soil Survey Staff 2006), the soil forest has been characterized as aridic haplustept, very fine, mixed, super active, nonacid, and mesic. The humus type has been classified as oligomull, and the geological substrate is marnous limestone. The main soil physico-chemical parameters that were measured at the time of sampling were the following: soil moisture, 28.3%; pH, 6.9; organic matter, 3.1%; total nitrogen, 1.6 g/kg; exchangeable Ca2+, 4,906 mg/kg; exchangeable K+, 263 mg/kg; exchangeable Mg2+, 76 mg/kg; exchangeable Na+, 18 mg/kg; cation exchange capacity, 37.9 meq/100 g; and C/N ratio, 11:1. Soil texture: clay. ACKNOWLEDGMENTS This study is part of a larger project denominated as “Mosyss” (Monitoring System of Soils at multiscale) that was funded by Region Marche to ALT and EI and from which financial support was provided to the co-authors DB and SM. SK was financially supported by a “Young Indian Research Fellowship” through the Italian Minister of University and Research (MIUR). The authors greatly thank the two reviewers and the Editor-in-Chief for providing constructive and helpful comments that improved the quality of the manuscript. The authors would like to thank Dr Mauro Tiberi, Dr Giovanni Ciabocco, and Dr Cristina Bernacconi from Osservatorio Regionale Suoli (http://suoli. regione.marche.it/) for their help in sampling. LITERATURE CITED Alekperov, I. K. 1993. Free-living ciliates in the soils of St. Petersburg parks (Protozoa). Zoosystematica Rossica, 2:13–28. Ammermann, D., Steinbruck, G., von Berger, L. & Hennig, W. 1974. The development of the macronucleus in the ciliated protozoan Stylonychia mytilus. Chromosoma, 45:401–429. Berger, H. 1999. Monograph of the Oxytrichidae (Ciliophora, Hypotrichia). Monogr. Biol., 78:1–1080. Berger, H. 2006. Monograph of the Urostyloidea (Ciliophora, Hypotricha). Monogr. Biol., 85:1–1304. Berger, H. 2008. Monograph of the Amphisiellidae and Trachelostylidae (Ciliophora, Hypotricha). Monogr. Biol., 88:1–737. Berger, H. 2011. Monograph of the Gonostomatidae and Kahliellidae (Ciliophora, Hypotricha). Monogr. Biol., 90:1–741. Berger, H. & Foissner, W. 1989a. Morphology and biometry of some soil hypotrichs (Protozoa, Ciliophora) from Europe and Japan. Bull. Br. Mus. nat. Hist. (Zool.), 55:19–46. Berger, H. & Foissner, W. 1989b. Morphology and morphogenesis of Parakahliella haideri nov. spec (Ciliophora, Hypotrichida). Bull. Br. Mus. nat. Hist. (Zool.), 55:11–17. Berger, H. & Foissner, W. 1997. Cladistic relationships and generic characterization of oxytrichid hypotrichs (Protozoa, Ciliophora). Arch. Protistenkd., 148:125–155. Berger, H., Foissner, W. & Adam, H. 1985. Morphological variation and comparative analysis of morphogenesis in Parakahliella macrostoma (Foissner, 1982) nov. gen. and Histriculus muscorum (Kahl, 1932), (Ciliophora, Hypotrichida). Protistologica, 21:295–311.



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Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India.

Morphology, morphogenesis and molecular phylogeny of a new marine ciliate, Trichototaxis marina n. sp. (Ciliophora, Urostylida).

Morphology, morphogenesis and molecular phylogeny of a novel soil ciliate, Pseudouroleptus plestiensis n. sp. (Ciliophora, Oxytrichidae), from the uplands of Colfiorito, Italy.

Morphology and phylogeny of Bryophryoides ocellatus n. g., n. sp. (Ciliophora, Colpodea) from in situ soil percolates of Idaho, U.S.A.

Morphology and Molecular Phylogeny of Three Cyrtophorid Ciliates (Protozoa, Ciliophora) from China, Including Two New Species, Chilodonella parauncinata sp. n. and Chlamydonella irregularis sp. n.

Morphology and molecular phylogeny of Apoterritricha lutea n. g., n. sp. (Ciliophora, Spirotrichea, Hypotrichia): a putative missing link connecting Cyrtohymena and Afrokeronopsis.

Morphology and Phylogeny of Three Pleuronema Species (Ciliophora, Scuticociliatia) from Hangzhou Bay, China, with Description of Two New Species, P. binucleatum n. sp. and P. parawiackowskii n. sp.

Morphology and ontogenesis of Psilotrichides hawaiiensis nov. gen., nov. spec. and molecular phylogeny of the Psilotrichidae (Ciliophora, Hypotrichia).

Morphology, morphogenesis and molecular phylogeny of a soil ciliate, Pseudouroleptus caudatus caudatus Hemberger, 1985 (Ciliophora, Hypotricha), from Lhalu Wetland, Tibet.

Morphology of Clapsiella magnifica gen. n., sp. n., a new hypotrichous ciliate with a curious dorsal ciliary pattern.

Malalcahuelloocaresi gen. & sp. n. (Elateridae, Campyloxeninae).

The morphology of three Loxophyllum species (Ciliophora, Pleurostomatida) from southern China, L. lembum sp. n., L. vesiculosum sp. n. and L. perihoplophorum Buddenbrock, 1920, with notes on the molecular phylogeny of Loxophyllum.

Morphology and Molecular Phylogeny of Pseudocyrtohymena koreana n. g., n. sp. and Antarctic Neokeronopsis asiatica Foissner et al., 2010 (Ciliophora, Sporadotrichida), with a Brief Discussion of the Cyrtohymena Undulating Membranes Pattern.

Morphology of three species of Amphileptus (Protozoa, Ciliophora, Pleurostomatida) from the South China Sea, with note on phylogeny of A. dragescoi sp. n.

Muscular sarcocystosis in two arctic foxes (Vulpes lagopus) due to Sarcocystis arctica n. sp.: sarcocyst morphology, molecular characteristics and phylogeny.

Two New Brackish Ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. from Mangrove Wetlands in Southern China, with Notes on the Molecular Phylogeny of the Family Amphileptidae (Protozoa, Ciliophora, Pleurostomatida).

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

Sinolatindia petila gen. n. and sp. n. from China (Blattodea, Corydiidae, Latindiinae).

Leptojacobus dorci n. gen., n. sp. (Nematoda: Diplogastridae), an Associate of Dorcus Stag Beetles (Coleoptera: Lucanidae).

Paratrichosoma crocodilus n. gen. n. sp. (Nematoda: Trichosomoididae) from the skin of the New Guinea crocodile.

Macuahuitloides inexpectans n. gen., n. sp. (Molineidae: Anoplostrongylinae) from Mormoops megalophylla (Chiroptera: Mormoopidae).

Eremonidiopsis aggregata, gen. n., sp. n. from Cuba, the third West Indian Dioptinae (Lepidoptera, Notodontidae).

Morphology, ontogenesis and molecular phylogeny of Platynematum salinarum nov. spec., a new scuticociliate (Ciliophora, Scuticociliatia) from a solar saltern.

A new genus and species (Cornucollis gen. n. masoalensis sp. n.) of praying mantis from northern Madagascar (Mantodea, Iridopterygidae, Tropidomantinae).