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Rigidosticha italiensis n. gen., n. sp. (Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy
Accepted Manuscript Title: Rigidosticha italiensis n. gen., n. sp. (Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy Author: Daizy Bharti Santosh Kumar Antonietta La Terza PII: DOI: Reference:
S0932-4739(16)30064-5 http://dx.doi.org/doi:10.1016/j.ejop.2016.08.004 EJOP 25446
To appear in: Received date: Revised date: Accepted date:
14-4-2016 11-8-2016 15-8-2016
Please cite this article as: Bharti, Daizy, Kumar, Santosh, La Terza, Antonietta, Rigidosticha italiensis n.gen., n.sp.(Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy.European Journal of Protistology http://dx.doi.org/10.1016/j.ejop.2016.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rigidosticha italiensis n. gen., n. sp. (Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy Daizy Bharti*, Santosh Kumar, Antonietta La Terza* School of Bioscience and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy
_______ *Corresponding authors. Tel.: +39 0737 403272; Fax: +39 0737 403290 E-mail addresses: [email protected] (D. Bharti); [email protected] (A. La Terza)
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Abstract
The morphology of Rigidosticha italiensis n. gen., n. sp., which was found in a soil sample collected from an uncultivated field in Lombardia, Italy, was investigated using live observation and protargol staining. Rigidosticha n. gen. is characterised by a rigid body, undulating membranes resembling a Steinia pattern, oxytrichid frontal ciliature, distinct mid-ventral cirral pairs, transverse cirri, one right and one left row of marginal cirri, absence of dorsal kinety 3 fragmentation, more than two dorsomarginal rows, and caudal cirri. The new species shows the following features: size in vivo 230–330 × 100–170 µm, on average 230 × 115 µm in protargol preparations; two ellipsoidal macronuclear nodules; 51 adoral membranelles; one buccal cirrus; one parabuccal cirrus; two frontoterminal cirri; 16 mid-ventral cirral pairs; and three transverse cirri. Rigidosticha mainly differs from Rigidothrix, Afrophrya, Uroleptus, and Territricha, in having the undulating membranes in Rigidosticha (vs. oxytrichid and cyrtohymenid) pattern. The oxytrichid frontal ciliature and midventral pattern in the present species further support the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis.
Keywords: Lombardia; Rigidosticha italiensis n. gen., n. sp.; Rigidosticha pattern; soil ciliates; Steinia pattern.
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Introduction The family Rigidotrichidae Foissner and Stoeck, 2006 was erected for a curious species, possessing a rigid body and a distinct mid-ventral pattern; the species broke the flexibility dogma that all stichotrichs with mid-ventral rows are flexible (Berger 1999, 2006). Further characteristics of the family are frontal cirri in oxytrichid pattern, oral apparatus in Oxytricha or Cyrtohymena like pattern, absence of kinety fragmentation, and more than two dorsomarginal rows. At present, the family includes three genera; namely, Rigidothrix Foissner and Stoeck, 2006, Afrophrya Foissner and Stoeck, 2006, and Territricha Berger and Foissner, 1988 (Foissner and Stoeck 2008). The present study adds another genus to the family which possesses a rigid body, oxytrichid frontal ciliature, mid-ventral pattern, and undulating membranes resembling a Steinia pattern. It has been reported that the shape and the arrangement of paroral and endoral membrane are useful features for separating genera and species and understanding their evolution (Berger 1999; Blatterer and Foissner 1988; Foissner 1989; Kumar and Foissner 2015; Voss and Foissner 1996). The undulating membranes in the present species show a novel arrangement, i.e., Rigidosticha pattern, and thus could serve as an important feature for elucidating phylogenies. A detailed description based on the examination of specimens in vivo and after protargol impregnation from raw culture is presented. This is the second report on the identification of a novel ciliate species from the Parona sampling site, Lombardia, Italy, under the interdisciplinary “Soil Mapping” project funded by Lombardia Region (Bharti et al. 2015; La Terza et al. 2015).
Material and Methods
Description of the P10R sampling site and sample processing
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The P10R site (45°17′11.74″N 8°48′43.24″E), Parona, Lombardia region, Italy, represents one of the eight sampling sites selected, in the frame of the project “Soil Mapping, Lombardia”, for the monitoring of the quality/health of the agricultural soils surrounding incinerator plant of Parona, a village in the Pavia Province, 30 km southwest from Milan. As described in Bharti et al. (2015), five out of eight sampling sites in the area of Parona were represented by cultivated rice fields with the exception of the sites P3R and P12R which were cultivated with corn and soybean, respectively. At the time of sampling, the P10R site was uncultivated and in set-aside. The field was cultivated with soybean in the previous year. P10R is located about 2 km to the east of the incinerator plant at an elevation of 107 m a.s.l. Soil samples (0–10 cm depth) were collected in March 2013 as previously described in Bharti et al. (2015). Main chemical-physical parameters (pH, moisture, temperature) were measured at P10R site, using a soil pH meter (PH-212, Lutron, UK), a moisture meter (PMS-714 Lutron, UK), and a thermometer (HI-98501, Hanna, UK), respectively. Soil texture analyses were performed according to the United State Department of Agriculture classification system (Gee and Or 2002). Ciliates were reactivated from resting cyst from one-month-dried soil samples (approximately 300 g) by employing the non-flooded Petri dish method (Foissner 1987). Live observations were made using a microscope with bright-field illumination. The protargol staining method described by Kamra and Sapra (1990) was used with some modification to reveal the infraciliature. Measurements of impregnated specimens were performed out at a magnification of 1000× using the Optika Vision Lite software. An Optika microscope camera was employed for photomicrography. The illustration of the live specimen was prepared using free-hand sketches, while those of impregnated specimens were made with a drawing device. Terminology is according to Berger (1999, 2006).
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Results
Rigidosticha n. gen. Diagnosis: Rigid Rigidotrichidae with oxytrichid frontal ciliature and conspicuous midventral complex composed of cirral pairs. Buccal, parabuccal, frontoterminal, and transverse cirri present. One right and 1 left row of marginal cirri. Three bipolar dorsal kineties and more than two dorsomarginal rows; caudal cirri present. Undulating membranes slightly curved, not intersecting. Type species: Rigidosticha italiensis n. sp. Etymolgy: Rigidosticha is a composite of the Latin adjective rigidus (rigid) and the Greek substantive stich (row, line) and the inflectional ending –a, meaning a rigid ciliate with midventral cirri forming rows.
Rigidosticha italiensis n. sp. (Figs 1A, B, 2A, B, 3A–C, 4A, B, 5A–C; Table 1) Diagnosis: Size in vivo about 265 μm × 115 μm. Body ellipsoidal. Two ellipsoidal macronuclear nodules and two micronuclei. On average, three frontal, one buccal, one parabuccal, two frontoterminal, and three transverse cirri. Mid-ventral complex composed of an average of 16 cirral pairs. Left marginal row on average composed of 26 cirri, right of 29. Adoral zone occupies 38% of body length, on average composed of 51 membranelles. Undulating membranes separate, paroral curved in the middle and endoral curved towards paroral anteriorly. Three bipolar dorsal kineties and five to seven dorsomarginal rows; three caudal cirri at the end of dorsal kineties 1, 2, and 3. Type locality: Soil from an uncultivated field (the P10R site; 45°17′11.74″N 8°48′43.24″E) located about 2 km to the east of the incinerator plant of the city of Parona, Lombardia region (Italy), at an elevation of 107 m a.s.l. 5
Type material: The slide containing the holotype specimen (Figs 2A, B, 4A, B) and two paratype slides with protargol-impregnated specimens have been deposited in the Natural History Museum, London, UK (Registration numbers: NHMUK 2016.8.9.1, NHMUK 2016.8.9.2, NHMUK 2016.8.9.3). Relevant specimens have been marked by black ink circles on the back of the slides. Etymology: The species group name italiensis refers to the country where the species was discovered, i.e., Italy. Description: Size in vivo 230–330 × 100–170 μm, on average 230 × 115 μm in protargol preparations; length:width ratio on average 2.1:1 in protargol preparations. Body narrowly ellipsoid to ellipsoid, i.e., anterior end broad, posterior more or less narrowly rounded (Figs 1A, B, 2A, B, 3A–C, 4A, B; Table 1). Nuclear apparatus within central quarters of cell slightly left or in midline, composed of two macronuclear nodules and two to four, on average two micronuclei (Figs 1A, B, 2B, 3B, C, 4A, B, 5C; Table 1). Macronuclear nodules ellipsoid to broadly ellipsoid, on average 30 × 16 µm in protargol preparations; containing many globular nucleoli. Micronuclei attached to or near to macronuclear nodules, globular, on average 5 µm across in protargol preparations. Contractile vacuole in mid-body at left cell margin (Fig. 1A). Cortex rigid, specific cortical granules absent. Cytoplasm colourless, with some lipid droplets 4–7 μm in size. Food vacuoles scattered throughout body, in vivo up to 40 µm across, contain naked and testate amoebae (e.g. Trinema lineare), ciliates (Vorticella sp.), and bacteria (Fig. 1A). Movement moderately fast. Cirral pattern urostyloid, number of fronto-ventral-transverse cirri rather variable. Usually three slightly enlarged, in vivo about 25 µm long frontal cirri, right cirrus anterior of parabuccal cirrus, middle cirrus anterior of buccal cirrus, left cirrus anterior of distal end of paroral membrane. Invariably one parabuccal and one buccal cirrus about 14 µm posterior from anterior end of paroral in protargol preparations. Two frontoterminal cirri posterior of distal end of adoral zone. On average 16 mid-ventral cirral pairs extending slightly obliquely and sigmoidally from right anterior end of body to transverse cirri, right cirri of pairs larger than left, two or three additional right cirri of pairs and one or two less hypertrophied pretransverse (?) cirri present at rear end difficult to 6
designate without ontogenetic data. Usually three obliquely arranged transverse cirri, about 28 µm long in vivo, posterior-most cirrus on average 10 µm from posterior body end in protargol preparations (Figs 1A, 2A, 3A, 4A, 5A, B; Table 1). Marginal cirri arranged in two non-confluent rows, about 20 µm long in vivo; left row composed of an average of 26 cirri, right row ends subterminally in body midline, composed of an average of 29 cirri (Figs 1A, 2A, 3A, 4A; Table 1). On average nine dorsal kineties with bristles about 5 µm long in protargol preparations: kineties 1, 2, 3 bipolar; kineties 4–9 slightly or distinctly shortened anteriorly and posteriorly (Figs 2B, 3B, C, 4B, 5C; Table 1). Three caudal cirri in midline of cell, one each at the posterior end of dorsal kineties 1, 2, and 3, about 23 µm long in protargol preparations, easily confused with last cirri of left marginal row (Figs 2B, 3B, C, 4B, 5C; Table 1). Adoral zone occupies about 38% of body length, commences far posteriorly on right margin of cell (DE-value 0.4), on average composed of 51 ordinary membranelles with up to 20 µm long cilia, bases of largest membranelles on average 20 µm long in protargol preparations (Figs 1A, B, 2A, 3A, 4A; Table 1). Buccal cavity conspicuous, wide and deep; buccal lip narrow and hyaline. Undulating membranes in body‟s midline, resemble a Steinia pattern, however, without buccal cavity and fragmentation of endoral, paroral slightly curved, composed of dikinetids; endoral membrane extends parallel to adoral zone across buccal area and recurved towards paroral anteriorly, extends to buccal vertex and composed of very narrowly spaced monokinetids, both membranes never intersect; pharyngeal fibres about 50 µm long, extend obliquely backwards (Figs 1A, 2A, 3A, 4A, 5A, B; Table 1). Occurrence and ecology: Thus far, Rigidosticha italiensis has been found only from the type location. Although, soil samples were collected from seven other fields around the incinerator of Parona, the novel species was exclusively identified from the soil samples collected at P10R site. The main physical-chemical parameters that were measured at the time of sampling were the following: soil moisture 18.6%; soil temperature 7.6 °C (at 5 cm); pH 6.49; soil texture sandy loam. 7
Discussion Note on the undulating membranes pattern A recent investigation on the shape of the paroral membrane resulted in identification of a new pattern among oxytrichids, i.e., the Australocirrus pattern which was often confused with the Cyrtohymena pattern (paroral strongly curved and recurved anteriorly as in Cyrtohymena vs. not recurved anteriorly as in Australocirrus) (Kumar and Foissner 2015). The undulating membranes pattern of Afrophrya Foissner and Stoeck, 2006 resembles that of Rigidosticha, however, in the former the paroral is strongly curved and recurved anteriorly. Thus, Rigidosticha italiensis presents a novel pattern of undulating membranes as a generic character, where the paroral is slightly to moderately curved, but not recurved anteriorly and the endoral extends parallel to the adoral zone of membranelles across the buccal area and recurved towards paroral anteriorly. Furthermore, the Rigidosticha pattern resembles that of the oxytrichid genus Steinia Diesing, 1866, with some differences in having a continuous (vs. discontinuous) endoral membrane and lacks (vs. present) a buccal depression. This indicates that the Rigidosticha pattern possibly has evolved from species of the oxytrichid genus Steinia. Comparison of Rigidosticha n. gen. with similar genera Rigidosticha n. gen. belongs to the family Rigidotrichidae because it has a Uroleptus-like midventral complex and dorsomarginal kineties and lacks kinety 3 fragmentation. The family at present comprises three genera; namely, Rigidothrix Foissner and Stoeck, 2006, Afrophrya Foissner and Stoeck, 2006, and Territricha Berger and Foissner, 1988, which should be compared with the new genus. Rigidothrix can be separated from Rigidosticha n. gen. in having undulating membranes in Oxytricha (vs. Rigidosticha) pattern and the presence (vs. absence) of stylonychid frontal area. Afrophrya differs from Rigidosticha mainly in having undulating membranes in Cyrtohymena (vs. 8
Rigidosticha) pattern i.e., paroral strongly curved and recurved anteriorly. Territricha differs from Rigidosticha in having a flexible (vs. rigid) body, an indistinct (vs. distinct) midventral complex, extrusomes (vs. lacking) and multiple (vs. no fragmentation) dorsal kinety fragmentation. Rigidosticha n. gen. differs from Uroleptus Ehrenberg, 1831, by the rigid (vs. flexible) body, round (vs. tailed) posterior body end, and undulating membranes resembling Steinia (vs. Oxytricha) pattern. The presence of an oxytrichid frontal ciliature and a midventral pattern in Rigidosticha and Uroleptus is in accordance with the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis (Foissner et al. 2004), which clarifies that the midventral pattern evolved at least two times, i.e., first one caused the separation of oxytrichid and urostylid lineages, while the second one caused the development of the midventral pattern among several oxytrichid lineages. Comparison of Rigidosticha italiensis n. gen., n. sp. with similar species Three species possess a rather similar morphology like Rigidosticha italiensis namely, Afrophrya camerounensis (Dragesco, 1970) Foissner and Stoeck, 2006; A. macrostoma (Dragesco, 1970) Foissner and Stoeck, 2006; and Caudiholosticha stueberi (Foissner, 1987) Berger, 2003. Rigidosticha italiensis differs from A. camerounensis sensu Borror and Wicklow (1983) in body length (229 vs. 108 µm), number of left (26 vs. 41) and right marginal cirri (29 vs. 46) and number of adoral membranelles (51 vs. 40) (values of A. camerounensis from revision by Berger 2006). The population studied by Dragesco and Dragesco-Kernéis (1986) differs from R. italiensis in body size (130–160 × 43–58 µm in protargol preparations vs. 200–284 × 84–147 µm), and number of transverse cirri (12–13 and 9–13 vs. 2–4). The endoral membrane of A. camerounensis sensu Dragesco and Dragesco-Kernéis (1986) is very similar to that of R. italiensis, however, the paroral membranes differ significantly (i.e., strongly curved and recurved anteriorly as in Cyrtohymena species vs. moderately curved and not recurved in R. italiensis). Afrophrya macrostoma differs from R. italiensis by the reduced number of midventral pairs (4 or 5 vs. 16). Rigidosticha italiensis can be distinguished from Caudiholosticha stueberi by the number of transverse cirri (2–4 vs. invariably 9
3), number of dorsal kineties (8–10 vs. 5–7), number of bristles in dorsal kineties 1 and 2 (57 and 44 vs. 33 and 31), and the undulating membranes (Oxytricha vs. Rigidosticha pattern). Soil Ciliate diversity from Italy The discovery of Rigidosticha italiensis, a very large and probably endemic soil species from Italy, suggests that despite being explored over the years, ciliate diversity, particularly in soil, appears to be still far from being satisfactorily evaluated. It is therefore possible that still hundreds of undescribed ciliate species remain to be discovered in Europe as well as in other areas with restricted geographical distribution. This argument, considering Italian studies as an example, is supported not only by the study on soil samples from the Lombardia region where more than ten new ciliate species were recorded, including the present species, but also from the study on soil samples from Marche (MOSYSS - MOnitoring SYstem of Soils at multi-Scale - project) and Umbria (BioPrint project) Regions where a similar number of new species were discovered (Santosh Kumar, Daizy Bharti, and Antonietta La Terza, unpublished results; Kumar et al. 2014; Bharti et al. 2014, 2015). Thus, we agree with Foissner et al. (2008) and Foissner (2016) in that only a tiny fraction of the diversity of ciliates has been identified up to now. Further sampling campaigns are needed to discover new species in order to contribute to deepen the knowledge about ciliates diversity and their phylogenetic relationships, as well as to possibly unveil their functional roles in both agroecosystems and natural systems. Overall, our studies have shown a better insight into the diversity of soil ciliates in this so far unexplored areas.
Acknowledgements This study is part of a larger project denominated as “Soil Mapping – Lombardia” funded by Lombardia region to ALT and from which financial support was provided to the co-authors DB and SK. The authors wish to greatly thank Dr. Silvia Marinsalti and Prof. Emilio Insom, School of 10
Bioscience and Veterinary Medicine, University of Camerino (Italy) for help in sampling and for supporting us through all the stages of the research. A special thank to Prof. Roberto Cenci, retired employee from JRC, EC, Institute for Environment and Sustainability, Ispra (Italy) for the planning of the “Soil Mapping” project. Furthermore, the authors wish to greatly thank two anonymous reviewers and the Associate Editor Helmut Berger for improving a first version of the manuscript with their helpful suggestions and constructive criticisms.
References 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. Bharti, D., Kumar, S., La Terza, A., 2014. Morphology, morphogenesis and molecular phylogeny of a novel soil ciliate, Pseudouroleptus plestiensis n. sp. (Ciliophora, Oxytrichidae), from the uplands of Colfiorito, Italy. Int. J. Syst. Evol. Microbiol. 64, 2625–2636. Bharti, D., Kumar, S., La Terza, A., 2015. Two gonostomatid ciliates from the soil of Lombardia, Italy; including note on the soil mapping project. J. Eukaryot. Microbiol. 62, 762–772. Blatterer, H., Foissner, W., 1988. Beitrag zur terricolen Ciliatenfauna (Protozoa: Ciliophora) Australiens. Stapfia 17, 1–84. Borror, A. C., Wicklow, B. J., 1983. The suborder Urostylina Jankowski (Ciliophora, Hypotrichida): morphology, systematics and identification of species. Acta Protozool. 22, 97–126. Dragesco, J., Dragesco-Kernéis, A., 1986. Ciliés libres de l‟Afrique intertropicale. Introduction à la connaissance et à l‟étude des ciliés. Faune tropicale 26, 1–559. Foissner, W., 1987. Soil protozoa: fundamental problems, ecological significance, adaptations in ciliates and testaceans, bioindicators, and guide to the literature. Progr. Protistol. 2, 69–212. 11
Foissner, W., 1989. Morphologie und Infraciliatur einiger neuer und wenig bekannter terrestrischer und limnischer Ciliaten (Protozoa, Ciliophora). Sber öst. Akad. Wiss., Mathematischnaturwissenschaftliche Klasse, Abt. I 196 (year 1987), 173–247. Foissner, W., 2016. Terrestrial and semiterrestrial ciliates (Protozoa, Ciliophora) from Venezuela and Galápagos. Denisia 35, 1–912. Foissner, W., Stoeck, T., 2006. Rigidothrix goiseri nov. gen., nov. spec. (Rigidothrichidae nov. fam.), a new "flagship" ciliate from the Niger floodplain breaks the flexibility-dogma in the classification of stichotrichine spirotrichs (Ciliophora, Spirotrichea). Eur. J. Protistol. 42, 249–267. Foissner, W., Moon-van der Staay, S. Y., van der Staay, G. W. M., Hackstein, J. H. P., Krautgartner, W. -D., Berger, H., 2004. Reconciling classical and molecular phylogenies in the stichotrichines (Ciliophora, Spirotrichea), including new sequences from some rare species. Eur. J. Protistol. 40, 265–281. Foissner, W., Chao, A., Katz, L., 2008. Diversity and geographic distribution of ciliates (Protista: Ciliophora). Biodivers. Conserv. 17, 345–363. Gee, G. W., Or, D., 2002. Particle-size analysis. In: Dane, J. H., Topp, G. C., (Eds.), Methods of Soil Analysis, Part 4, Physical Methods, Vol. 5. Soil Science Society of America Book Series, Madison, WI. pp. 255–293. Kamra, K., Sapra, G. R., 1990. Partial retention of parental ciliature during morphogenesis of the ciliate Coniculostomum monilata (Dragesco and Njine, 1971) Njine, 1978 (Oxytrichidae, Hypotrichida). Eur. J. Protistol. 25, 264–278. Kumar, S., Foissner, W., 2015. Biogeographic specializations of two large hypotrich ciliates: Australocirrus shii and A. australis and proposed synonymy of Australocirrus and Cyrtohymenides. Eur. J. Protistol. 51, 210–228. Kumar, S., Bharti, D., Marinsalti, S., Insom, E., La Terza, A., 2014. Morphology, morphogenesis, and molecular phylogeny of Paraparentocirrus sibillinensis n. gen., n. sp., a „„Stylonychine 12
Oxytrichidae‟‟ (Ciliophora, Hypotrichida) without transverse cirri. J. Eukaryot. Microbiol. 61, 247–259. La Terza, A., Bharti, D., Kumar, S., Marinsalti, S., Molina, M. G., Insom, E., 2015. Struttura delle Comunità dei Protozoi Ciliati. In: Beone, G. M., Cenci, R. M., Guidotti, L., Sena, F., Umlauf, G., (Eds.), Progetto di Monitoraggio Ambientale su tutto il Territorio della Regione Lombardia (Progetto Soil): Indagine conoscitiva della qualità e dello stato di salute dei suoli lombardi, Report EUR 27161 IT, Luxembourg: Publications Office of the European Union, pp. 259–271. Voss, H. J., Foissner, W., 1996. Divisional morphogenesis in Steinia sphagnicola (Ciliophora, Hypotrichida): a comparative light and scanning electronic microscopic study. Eur. J. Protistol. 32, 31–46.
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Figure explanations Fig. 1A, B. Rigidosticha italiensis, line diagrams from life (A) and after protargolimpregnation (B). A: Ventral view of a representative specimen, length 265 μm. B: Specimen, showing the undulating membranes and nuclear apparatus. Note the slightly curved paroral and the slightly recurved endoral anteriorly. AZM – adoral zone of membranelles, E – endoral membrane, MA – macronucleus, MI – micronucleus, P – paroral membrane. Scale bars 100 μm. Fig. 2A, B. Rigidosticha italiensis, line diagrams of the holotype specimen after protargolimpregnation. A: Ventral view, showing the infraciliature. Note the additional right cirri of pairs (arrowheads) and pretransverse (?) cirri at the rear end, difficult to designate in the absence of ontogenetic data. B: Dorsal view, showing the ciliature and nuclear apparatus. The caudal cirri are present at the posterior end of dorsal kineties 1, 2, and 3. AZM – adoral zone of membranelles, BC – buccal cirrus, CC – caudal cirri, DK1,3 – dorsal kineties, DM – dorsomarginal rows, E – endoral membrane, FC1, 3 – frontal cirri, FT – frontoterminal cirri, LM – left marginal row, MA – macronucleus, MI – micronucleus, MP – midventral cirral pairs, P – paroral membrane, PTC – pretransverse cirri, RM – right marginal row, TC – transverse cirri. Scale bar 80 μm. Fig. 3A–C. Rigidosticha italiensis, line diagrams of specimens after protargol-impregnation. A: A specimen with slightly shortened right marginal row and without additional right cirri of midventral pairs and pretransverse cirri at the rear end. The three enlarged frontal cirri and the undulating membranes in Rigidosticha pattern are often associated with fibres. B, C: Dorsal view of paratype specimens, showing the infraciliature and nuclear apparatus. Note that the caudal cirri are present at the posterior end of dorsal kineties 1, 2, and 3. AZM – adoral zone of membranelles, CC – caudal cirri, DK1,3 – dorsal kineties, DM – dorsomarginal rows, E – endoral membrane, F – fibres, FC1, 3 – frontal cirri, FT – frontoterminal cirri, MP – midventral cirral pairs, LM – left
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marginal row, MA – macronucleus, MI – micronucleus, P – paroral membrane, RM – right marginal row, TC – transverse cirri. Scale bars 100 μm.
Fig. 4A, B. Rigidosticha italiensis, photomicrographs of holotype specimen after protargol impregnation. A: Ventral view, showing the infraciliature and the clearly separated, i.e., never intersecting, paroral and endoral membrane. B: Dorsal view, showing the caudal cirri at the end of bipolar dorsal kineties 1, 2, and 3. AZM – adoral zone of membranelles, C – paroral cilia, CC – caudal cirri, DK1 – dorsal kinety 1, DM – dorsomarginal rows, E – endoral membrane, FC3 – frontal cirrus 3, FT – frontoterminal cirri, LM – left marginal row, MA – macronucleus, MI – micronucleus, MP – midventral cirral pairs, P – paroral membrane, RM – right marginal row, TC – transverse cirri. Scale bars 80 μm. Fig. 5A–C. Rigidosticha italiensis, photomicrograph of specimens after protargol impregnation. A, B: Detail of oral apparatus, showing paroral cilia, associated fibres (A), and undulating membranes in Rigidosticha pattern (B). C: A right lateral view, showing the posteriorly shortened dorsomarginal rows (arrowheads) and caudal cirri at the end of dorsal kineties (broken lines). AZM – adoral zone of membranelles, BC – buccal cirrus, C – paroral cilia, CC – caudal cirri, DK3 – dorsal kinety 3, DM – dorsomarginal rows, E – endoral membrane, FC1 – frontal cirrus 1, MA – macronucleus, P – paroral membrane; RM – right marginal row. Scale bars 15 μm (A), 20 μm (B), and 100 μm (C).
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Table 1. Morphometric data on Rigidosticha italiensis n. gen., n. sp. Characteristica
Mean
Body, length
229.1 221.0 20.6 4.5
Body, width
115.5 110.0 14.6 3.2 13.1
Body length:width, ratio
M
SD SE
2.1
2.1
Anterior body end to proximal end of adoral zone, distance
87.2
88.0
7.0 1.6
Anterior body end to distal end of adoral zone, distance
34.2
Anterior body end to proximal end of adoral zone, percentage of body length DE-value
CV
Min
Max
n
9.0 200.0 284.0 21
1.7
2.5 21
8.1
75.0
98.0 19
34.0
8.2 2.0 24.0
24.0
56.0 17
38.2
37.8
3.9 0.9 10.2
32.1
45.2 19
0.4
0.4
0.1 0.0 17.7
0.3
0.6 17
Adoral membranelles, longest membranellar base
19.7
20.0
1.8 0.4
8.9
17.0
24.0 17
Adoral membranelles, number
51.4
51.0
5.5 1.3 10.8
40.0
62.0 19
Anterior end of paroral membrane to buccal cirrus, distance
13.8
14.0
4.3 1.0 31.0
7.0
25.0 17
Anterior body end to first macronuclear nodule, distance
72.9
70.5 11.4 2.8 15.7
53.0 100.0 17
Posterior body end to second macronuclear nodule, distance
79.1
78.0
9.2 2.5 11.6
61.0
95.0 13
Anterior macronuclear nodule, length
30.1
28.5
5.6 1.2 18.5
24.0
43.0 21
Anterior macronuclear nodule, width
16.4
16.0
1.8 0.4 11.2
14.0
20.0 21
Macronuclear nodules, number
2.0
2.0
0.0
2.0
2.0 21
Anteriormost micronucleus, diameter
5.1
5.0 0.7
0.2 12.9
4.0
6.0 19
Micronuclei, number
2.3
2.0
0.6 0.1 24.8
2.0
4.0 19
Anterior body end to right marginal row, distance
40.7
40.0
7.6 2.0 18.6
34.0
63.0 15
Right marginal row, number of cirri
29.3
30.0
2.6 0.7
9.0
24.0
33.0 15
Anterior body end to left marginal row, distance
76.9
80.0
8.7 2.3 11.4
57.0
90.0 15
Left marginal row, number of cirri
26.2
28.0
4.2 1.1 15.9
21.0
32.0 15
Gap between last cirri of marginal rows
9.4
8.0
3.9 1.2 41.7
6.0
20.0 11
Frontal cirri, number
3.3
3.0 0.5
0.1 14.0
3.0
4.0 15
Frontoterminal cirri, number
2.0
2.0 0.0
0.0
2.0
2.0 15
26.0
52.0 17
Anterior body end to buccal cirrus, distance
0.2 0.1 11.0
84.0 147.0 21
0.0 0.0
0.0
34.0
34.5
Buccal cirrus, number
1.0
1.0
0.0 0.0
0.0
1.0
1.0 15
Parabuccal cirrus, number
1.0
1.0
0.0 0.0
0.0
1.0
1.0 15
16
6.0 1.4 17.6
Characteristica
Mean
M
15.9
16.0
10.1
10.5 1.9
Transverse cirri, number
2.9
3.0 0.6
Dorsal kineties, number
8.9
9.0
0.8 0.2
Dorsal kinety 1, number of bristles
57.3
56.0
Dorsal kinety 2, number of bristles
43.8
42.0
Midventral cirral pairs, number
b
Posterior body end to rearmost transverse cirrus, distance
Caudal cirri, number a
3.0
SD SE
CV
Min
Max
2.7 0.7 16.7
12.0
20.0 13
0.6 18.5
6.0
13.0 11
0.2 21.9
2.0
4.0 13
8.8
8.0
10.0 17
5.4 1.8
9.5
51.0
70.0
9
3.7 1.2
8.5
40.0
51.0
9
3.0 0.0
0.0
0.0
3.0
3.0 15
Data based on mounted, protargol-impregnated, and randomly selected specimens from non-flooded Petri dish
cultures. Measurements in µm. CV – coefficient of variation in %, M – median, Max – maximum, Mean – arithmetic mean, Min – minimum, n – number of individuals investigated, SD – standard deviation, SE – standard error of arithmetic mean. b
Two or three right cirri of the pairs and one or two pretransverse cirri (?) at the rear end not included. See Fig. 2A.
17
n
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
S0932-4739(16)30064-5 http://dx.doi.org/doi:10.1016/j.ejop.2016.08.004 EJOP 25446
To appear in: Received date: Revised date: Accepted date:
14-4-2016 11-8-2016 15-8-2016
Please cite this article as: Bharti, Daizy, Kumar, Santosh, La Terza, Antonietta, Rigidosticha italiensis n.gen., n.sp.(Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy.European Journal of Protistology http://dx.doi.org/10.1016/j.ejop.2016.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Rigidosticha italiensis n. gen., n. sp. (Ciliophora, Spirotricha), a novel large hypotrich ciliate from the soil of Lombardia, Italy Daizy Bharti*, Santosh Kumar, Antonietta La Terza* School of Bioscience and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy
_______ *Corresponding authors. Tel.: +39 0737 403272; Fax: +39 0737 403290 E-mail addresses: [email protected] (D. Bharti); [email protected] (A. La Terza)
1
Abstract
The morphology of Rigidosticha italiensis n. gen., n. sp., which was found in a soil sample collected from an uncultivated field in Lombardia, Italy, was investigated using live observation and protargol staining. Rigidosticha n. gen. is characterised by a rigid body, undulating membranes resembling a Steinia pattern, oxytrichid frontal ciliature, distinct mid-ventral cirral pairs, transverse cirri, one right and one left row of marginal cirri, absence of dorsal kinety 3 fragmentation, more than two dorsomarginal rows, and caudal cirri. The new species shows the following features: size in vivo 230–330 × 100–170 µm, on average 230 × 115 µm in protargol preparations; two ellipsoidal macronuclear nodules; 51 adoral membranelles; one buccal cirrus; one parabuccal cirrus; two frontoterminal cirri; 16 mid-ventral cirral pairs; and three transverse cirri. Rigidosticha mainly differs from Rigidothrix, Afrophrya, Uroleptus, and Territricha, in having the undulating membranes in Rigidosticha (vs. oxytrichid and cyrtohymenid) pattern. The oxytrichid frontal ciliature and midventral pattern in the present species further support the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis.
Keywords: Lombardia; Rigidosticha italiensis n. gen., n. sp.; Rigidosticha pattern; soil ciliates; Steinia pattern.
2
Introduction The family Rigidotrichidae Foissner and Stoeck, 2006 was erected for a curious species, possessing a rigid body and a distinct mid-ventral pattern; the species broke the flexibility dogma that all stichotrichs with mid-ventral rows are flexible (Berger 1999, 2006). Further characteristics of the family are frontal cirri in oxytrichid pattern, oral apparatus in Oxytricha or Cyrtohymena like pattern, absence of kinety fragmentation, and more than two dorsomarginal rows. At present, the family includes three genera; namely, Rigidothrix Foissner and Stoeck, 2006, Afrophrya Foissner and Stoeck, 2006, and Territricha Berger and Foissner, 1988 (Foissner and Stoeck 2008). The present study adds another genus to the family which possesses a rigid body, oxytrichid frontal ciliature, mid-ventral pattern, and undulating membranes resembling a Steinia pattern. It has been reported that the shape and the arrangement of paroral and endoral membrane are useful features for separating genera and species and understanding their evolution (Berger 1999; Blatterer and Foissner 1988; Foissner 1989; Kumar and Foissner 2015; Voss and Foissner 1996). The undulating membranes in the present species show a novel arrangement, i.e., Rigidosticha pattern, and thus could serve as an important feature for elucidating phylogenies. A detailed description based on the examination of specimens in vivo and after protargol impregnation from raw culture is presented. This is the second report on the identification of a novel ciliate species from the Parona sampling site, Lombardia, Italy, under the interdisciplinary “Soil Mapping” project funded by Lombardia Region (Bharti et al. 2015; La Terza et al. 2015).
Material and Methods
Description of the P10R sampling site and sample processing
3
The P10R site (45°17′11.74″N 8°48′43.24″E), Parona, Lombardia region, Italy, represents one of the eight sampling sites selected, in the frame of the project “Soil Mapping, Lombardia”, for the monitoring of the quality/health of the agricultural soils surrounding incinerator plant of Parona, a village in the Pavia Province, 30 km southwest from Milan. As described in Bharti et al. (2015), five out of eight sampling sites in the area of Parona were represented by cultivated rice fields with the exception of the sites P3R and P12R which were cultivated with corn and soybean, respectively. At the time of sampling, the P10R site was uncultivated and in set-aside. The field was cultivated with soybean in the previous year. P10R is located about 2 km to the east of the incinerator plant at an elevation of 107 m a.s.l. Soil samples (0–10 cm depth) were collected in March 2013 as previously described in Bharti et al. (2015). Main chemical-physical parameters (pH, moisture, temperature) were measured at P10R site, using a soil pH meter (PH-212, Lutron, UK), a moisture meter (PMS-714 Lutron, UK), and a thermometer (HI-98501, Hanna, UK), respectively. Soil texture analyses were performed according to the United State Department of Agriculture classification system (Gee and Or 2002). Ciliates were reactivated from resting cyst from one-month-dried soil samples (approximately 300 g) by employing the non-flooded Petri dish method (Foissner 1987). Live observations were made using a microscope with bright-field illumination. The protargol staining method described by Kamra and Sapra (1990) was used with some modification to reveal the infraciliature. Measurements of impregnated specimens were performed out at a magnification of 1000× using the Optika Vision Lite software. An Optika microscope camera was employed for photomicrography. The illustration of the live specimen was prepared using free-hand sketches, while those of impregnated specimens were made with a drawing device. Terminology is according to Berger (1999, 2006).
4
Results
Rigidosticha n. gen. Diagnosis: Rigid Rigidotrichidae with oxytrichid frontal ciliature and conspicuous midventral complex composed of cirral pairs. Buccal, parabuccal, frontoterminal, and transverse cirri present. One right and 1 left row of marginal cirri. Three bipolar dorsal kineties and more than two dorsomarginal rows; caudal cirri present. Undulating membranes slightly curved, not intersecting. Type species: Rigidosticha italiensis n. sp. Etymolgy: Rigidosticha is a composite of the Latin adjective rigidus (rigid) and the Greek substantive stich (row, line) and the inflectional ending –a, meaning a rigid ciliate with midventral cirri forming rows.
Rigidosticha italiensis n. sp. (Figs 1A, B, 2A, B, 3A–C, 4A, B, 5A–C; Table 1) Diagnosis: Size in vivo about 265 μm × 115 μm. Body ellipsoidal. Two ellipsoidal macronuclear nodules and two micronuclei. On average, three frontal, one buccal, one parabuccal, two frontoterminal, and three transverse cirri. Mid-ventral complex composed of an average of 16 cirral pairs. Left marginal row on average composed of 26 cirri, right of 29. Adoral zone occupies 38% of body length, on average composed of 51 membranelles. Undulating membranes separate, paroral curved in the middle and endoral curved towards paroral anteriorly. Three bipolar dorsal kineties and five to seven dorsomarginal rows; three caudal cirri at the end of dorsal kineties 1, 2, and 3. Type locality: Soil from an uncultivated field (the P10R site; 45°17′11.74″N 8°48′43.24″E) located about 2 km to the east of the incinerator plant of the city of Parona, Lombardia region (Italy), at an elevation of 107 m a.s.l. 5
Type material: The slide containing the holotype specimen (Figs 2A, B, 4A, B) and two paratype slides with protargol-impregnated specimens have been deposited in the Natural History Museum, London, UK (Registration numbers: NHMUK 2016.8.9.1, NHMUK 2016.8.9.2, NHMUK 2016.8.9.3). Relevant specimens have been marked by black ink circles on the back of the slides. Etymology: The species group name italiensis refers to the country where the species was discovered, i.e., Italy. Description: Size in vivo 230–330 × 100–170 μm, on average 230 × 115 μm in protargol preparations; length:width ratio on average 2.1:1 in protargol preparations. Body narrowly ellipsoid to ellipsoid, i.e., anterior end broad, posterior more or less narrowly rounded (Figs 1A, B, 2A, B, 3A–C, 4A, B; Table 1). Nuclear apparatus within central quarters of cell slightly left or in midline, composed of two macronuclear nodules and two to four, on average two micronuclei (Figs 1A, B, 2B, 3B, C, 4A, B, 5C; Table 1). Macronuclear nodules ellipsoid to broadly ellipsoid, on average 30 × 16 µm in protargol preparations; containing many globular nucleoli. Micronuclei attached to or near to macronuclear nodules, globular, on average 5 µm across in protargol preparations. Contractile vacuole in mid-body at left cell margin (Fig. 1A). Cortex rigid, specific cortical granules absent. Cytoplasm colourless, with some lipid droplets 4–7 μm in size. Food vacuoles scattered throughout body, in vivo up to 40 µm across, contain naked and testate amoebae (e.g. Trinema lineare), ciliates (Vorticella sp.), and bacteria (Fig. 1A). Movement moderately fast. Cirral pattern urostyloid, number of fronto-ventral-transverse cirri rather variable. Usually three slightly enlarged, in vivo about 25 µm long frontal cirri, right cirrus anterior of parabuccal cirrus, middle cirrus anterior of buccal cirrus, left cirrus anterior of distal end of paroral membrane. Invariably one parabuccal and one buccal cirrus about 14 µm posterior from anterior end of paroral in protargol preparations. Two frontoterminal cirri posterior of distal end of adoral zone. On average 16 mid-ventral cirral pairs extending slightly obliquely and sigmoidally from right anterior end of body to transverse cirri, right cirri of pairs larger than left, two or three additional right cirri of pairs and one or two less hypertrophied pretransverse (?) cirri present at rear end difficult to 6
designate without ontogenetic data. Usually three obliquely arranged transverse cirri, about 28 µm long in vivo, posterior-most cirrus on average 10 µm from posterior body end in protargol preparations (Figs 1A, 2A, 3A, 4A, 5A, B; Table 1). Marginal cirri arranged in two non-confluent rows, about 20 µm long in vivo; left row composed of an average of 26 cirri, right row ends subterminally in body midline, composed of an average of 29 cirri (Figs 1A, 2A, 3A, 4A; Table 1). On average nine dorsal kineties with bristles about 5 µm long in protargol preparations: kineties 1, 2, 3 bipolar; kineties 4–9 slightly or distinctly shortened anteriorly and posteriorly (Figs 2B, 3B, C, 4B, 5C; Table 1). Three caudal cirri in midline of cell, one each at the posterior end of dorsal kineties 1, 2, and 3, about 23 µm long in protargol preparations, easily confused with last cirri of left marginal row (Figs 2B, 3B, C, 4B, 5C; Table 1). Adoral zone occupies about 38% of body length, commences far posteriorly on right margin of cell (DE-value 0.4), on average composed of 51 ordinary membranelles with up to 20 µm long cilia, bases of largest membranelles on average 20 µm long in protargol preparations (Figs 1A, B, 2A, 3A, 4A; Table 1). Buccal cavity conspicuous, wide and deep; buccal lip narrow and hyaline. Undulating membranes in body‟s midline, resemble a Steinia pattern, however, without buccal cavity and fragmentation of endoral, paroral slightly curved, composed of dikinetids; endoral membrane extends parallel to adoral zone across buccal area and recurved towards paroral anteriorly, extends to buccal vertex and composed of very narrowly spaced monokinetids, both membranes never intersect; pharyngeal fibres about 50 µm long, extend obliquely backwards (Figs 1A, 2A, 3A, 4A, 5A, B; Table 1). Occurrence and ecology: Thus far, Rigidosticha italiensis has been found only from the type location. Although, soil samples were collected from seven other fields around the incinerator of Parona, the novel species was exclusively identified from the soil samples collected at P10R site. The main physical-chemical parameters that were measured at the time of sampling were the following: soil moisture 18.6%; soil temperature 7.6 °C (at 5 cm); pH 6.49; soil texture sandy loam. 7
Discussion Note on the undulating membranes pattern A recent investigation on the shape of the paroral membrane resulted in identification of a new pattern among oxytrichids, i.e., the Australocirrus pattern which was often confused with the Cyrtohymena pattern (paroral strongly curved and recurved anteriorly as in Cyrtohymena vs. not recurved anteriorly as in Australocirrus) (Kumar and Foissner 2015). The undulating membranes pattern of Afrophrya Foissner and Stoeck, 2006 resembles that of Rigidosticha, however, in the former the paroral is strongly curved and recurved anteriorly. Thus, Rigidosticha italiensis presents a novel pattern of undulating membranes as a generic character, where the paroral is slightly to moderately curved, but not recurved anteriorly and the endoral extends parallel to the adoral zone of membranelles across the buccal area and recurved towards paroral anteriorly. Furthermore, the Rigidosticha pattern resembles that of the oxytrichid genus Steinia Diesing, 1866, with some differences in having a continuous (vs. discontinuous) endoral membrane and lacks (vs. present) a buccal depression. This indicates that the Rigidosticha pattern possibly has evolved from species of the oxytrichid genus Steinia. Comparison of Rigidosticha n. gen. with similar genera Rigidosticha n. gen. belongs to the family Rigidotrichidae because it has a Uroleptus-like midventral complex and dorsomarginal kineties and lacks kinety 3 fragmentation. The family at present comprises three genera; namely, Rigidothrix Foissner and Stoeck, 2006, Afrophrya Foissner and Stoeck, 2006, and Territricha Berger and Foissner, 1988, which should be compared with the new genus. Rigidothrix can be separated from Rigidosticha n. gen. in having undulating membranes in Oxytricha (vs. Rigidosticha) pattern and the presence (vs. absence) of stylonychid frontal area. Afrophrya differs from Rigidosticha mainly in having undulating membranes in Cyrtohymena (vs. 8
Rigidosticha) pattern i.e., paroral strongly curved and recurved anteriorly. Territricha differs from Rigidosticha in having a flexible (vs. rigid) body, an indistinct (vs. distinct) midventral complex, extrusomes (vs. lacking) and multiple (vs. no fragmentation) dorsal kinety fragmentation. Rigidosticha n. gen. differs from Uroleptus Ehrenberg, 1831, by the rigid (vs. flexible) body, round (vs. tailed) posterior body end, and undulating membranes resembling Steinia (vs. Oxytricha) pattern. The presence of an oxytrichid frontal ciliature and a midventral pattern in Rigidosticha and Uroleptus is in accordance with the CEUU (Convergent Evolution of Urostylids and Uroleptids) hypothesis (Foissner et al. 2004), which clarifies that the midventral pattern evolved at least two times, i.e., first one caused the separation of oxytrichid and urostylid lineages, while the second one caused the development of the midventral pattern among several oxytrichid lineages. Comparison of Rigidosticha italiensis n. gen., n. sp. with similar species Three species possess a rather similar morphology like Rigidosticha italiensis namely, Afrophrya camerounensis (Dragesco, 1970) Foissner and Stoeck, 2006; A. macrostoma (Dragesco, 1970) Foissner and Stoeck, 2006; and Caudiholosticha stueberi (Foissner, 1987) Berger, 2003. Rigidosticha italiensis differs from A. camerounensis sensu Borror and Wicklow (1983) in body length (229 vs. 108 µm), number of left (26 vs. 41) and right marginal cirri (29 vs. 46) and number of adoral membranelles (51 vs. 40) (values of A. camerounensis from revision by Berger 2006). The population studied by Dragesco and Dragesco-Kernéis (1986) differs from R. italiensis in body size (130–160 × 43–58 µm in protargol preparations vs. 200–284 × 84–147 µm), and number of transverse cirri (12–13 and 9–13 vs. 2–4). The endoral membrane of A. camerounensis sensu Dragesco and Dragesco-Kernéis (1986) is very similar to that of R. italiensis, however, the paroral membranes differ significantly (i.e., strongly curved and recurved anteriorly as in Cyrtohymena species vs. moderately curved and not recurved in R. italiensis). Afrophrya macrostoma differs from R. italiensis by the reduced number of midventral pairs (4 or 5 vs. 16). Rigidosticha italiensis can be distinguished from Caudiholosticha stueberi by the number of transverse cirri (2–4 vs. invariably 9
3), number of dorsal kineties (8–10 vs. 5–7), number of bristles in dorsal kineties 1 and 2 (57 and 44 vs. 33 and 31), and the undulating membranes (Oxytricha vs. Rigidosticha pattern). Soil Ciliate diversity from Italy The discovery of Rigidosticha italiensis, a very large and probably endemic soil species from Italy, suggests that despite being explored over the years, ciliate diversity, particularly in soil, appears to be still far from being satisfactorily evaluated. It is therefore possible that still hundreds of undescribed ciliate species remain to be discovered in Europe as well as in other areas with restricted geographical distribution. This argument, considering Italian studies as an example, is supported not only by the study on soil samples from the Lombardia region where more than ten new ciliate species were recorded, including the present species, but also from the study on soil samples from Marche (MOSYSS - MOnitoring SYstem of Soils at multi-Scale - project) and Umbria (BioPrint project) Regions where a similar number of new species were discovered (Santosh Kumar, Daizy Bharti, and Antonietta La Terza, unpublished results; Kumar et al. 2014; Bharti et al. 2014, 2015). Thus, we agree with Foissner et al. (2008) and Foissner (2016) in that only a tiny fraction of the diversity of ciliates has been identified up to now. Further sampling campaigns are needed to discover new species in order to contribute to deepen the knowledge about ciliates diversity and their phylogenetic relationships, as well as to possibly unveil their functional roles in both agroecosystems and natural systems. Overall, our studies have shown a better insight into the diversity of soil ciliates in this so far unexplored areas.
Acknowledgements This study is part of a larger project denominated as “Soil Mapping – Lombardia” funded by Lombardia region to ALT and from which financial support was provided to the co-authors DB and SK. The authors wish to greatly thank Dr. Silvia Marinsalti and Prof. Emilio Insom, School of 10
Bioscience and Veterinary Medicine, University of Camerino (Italy) for help in sampling and for supporting us through all the stages of the research. A special thank to Prof. Roberto Cenci, retired employee from JRC, EC, Institute for Environment and Sustainability, Ispra (Italy) for the planning of the “Soil Mapping” project. Furthermore, the authors wish to greatly thank two anonymous reviewers and the Associate Editor Helmut Berger for improving a first version of the manuscript with their helpful suggestions and constructive criticisms.
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Oxytrichidae‟‟ (Ciliophora, Hypotrichida) without transverse cirri. J. Eukaryot. Microbiol. 61, 247–259. La Terza, A., Bharti, D., Kumar, S., Marinsalti, S., Molina, M. G., Insom, E., 2015. Struttura delle Comunità dei Protozoi Ciliati. In: Beone, G. M., Cenci, R. M., Guidotti, L., Sena, F., Umlauf, G., (Eds.), Progetto di Monitoraggio Ambientale su tutto il Territorio della Regione Lombardia (Progetto Soil): Indagine conoscitiva della qualità e dello stato di salute dei suoli lombardi, Report EUR 27161 IT, Luxembourg: Publications Office of the European Union, pp. 259–271. Voss, H. J., Foissner, W., 1996. Divisional morphogenesis in Steinia sphagnicola (Ciliophora, Hypotrichida): a comparative light and scanning electronic microscopic study. Eur. J. Protistol. 32, 31–46.
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Figure explanations Fig. 1A, B. Rigidosticha italiensis, line diagrams from life (A) and after protargolimpregnation (B). A: Ventral view of a representative specimen, length 265 μm. B: Specimen, showing the undulating membranes and nuclear apparatus. Note the slightly curved paroral and the slightly recurved endoral anteriorly. AZM – adoral zone of membranelles, E – endoral membrane, MA – macronucleus, MI – micronucleus, P – paroral membrane. Scale bars 100 μm. Fig. 2A, B. Rigidosticha italiensis, line diagrams of the holotype specimen after protargolimpregnation. A: Ventral view, showing the infraciliature. Note the additional right cirri of pairs (arrowheads) and pretransverse (?) cirri at the rear end, difficult to designate in the absence of ontogenetic data. B: Dorsal view, showing the ciliature and nuclear apparatus. The caudal cirri are present at the posterior end of dorsal kineties 1, 2, and 3. AZM – adoral zone of membranelles, BC – buccal cirrus, CC – caudal cirri, DK1,3 – dorsal kineties, DM – dorsomarginal rows, E – endoral membrane, FC1, 3 – frontal cirri, FT – frontoterminal cirri, LM – left marginal row, MA – macronucleus, MI – micronucleus, MP – midventral cirral pairs, P – paroral membrane, PTC – pretransverse cirri, RM – right marginal row, TC – transverse cirri. Scale bar 80 μm. Fig. 3A–C. Rigidosticha italiensis, line diagrams of specimens after protargol-impregnation. A: A specimen with slightly shortened right marginal row and without additional right cirri of midventral pairs and pretransverse cirri at the rear end. The three enlarged frontal cirri and the undulating membranes in Rigidosticha pattern are often associated with fibres. B, C: Dorsal view of paratype specimens, showing the infraciliature and nuclear apparatus. Note that the caudal cirri are present at the posterior end of dorsal kineties 1, 2, and 3. AZM – adoral zone of membranelles, CC – caudal cirri, DK1,3 – dorsal kineties, DM – dorsomarginal rows, E – endoral membrane, F – fibres, FC1, 3 – frontal cirri, FT – frontoterminal cirri, MP – midventral cirral pairs, LM – left
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marginal row, MA – macronucleus, MI – micronucleus, P – paroral membrane, RM – right marginal row, TC – transverse cirri. Scale bars 100 μm.
Fig. 4A, B. Rigidosticha italiensis, photomicrographs of holotype specimen after protargol impregnation. A: Ventral view, showing the infraciliature and the clearly separated, i.e., never intersecting, paroral and endoral membrane. B: Dorsal view, showing the caudal cirri at the end of bipolar dorsal kineties 1, 2, and 3. AZM – adoral zone of membranelles, C – paroral cilia, CC – caudal cirri, DK1 – dorsal kinety 1, DM – dorsomarginal rows, E – endoral membrane, FC3 – frontal cirrus 3, FT – frontoterminal cirri, LM – left marginal row, MA – macronucleus, MI – micronucleus, MP – midventral cirral pairs, P – paroral membrane, RM – right marginal row, TC – transverse cirri. Scale bars 80 μm. Fig. 5A–C. Rigidosticha italiensis, photomicrograph of specimens after protargol impregnation. A, B: Detail of oral apparatus, showing paroral cilia, associated fibres (A), and undulating membranes in Rigidosticha pattern (B). C: A right lateral view, showing the posteriorly shortened dorsomarginal rows (arrowheads) and caudal cirri at the end of dorsal kineties (broken lines). AZM – adoral zone of membranelles, BC – buccal cirrus, C – paroral cilia, CC – caudal cirri, DK3 – dorsal kinety 3, DM – dorsomarginal rows, E – endoral membrane, FC1 – frontal cirrus 1, MA – macronucleus, P – paroral membrane; RM – right marginal row. Scale bars 15 μm (A), 20 μm (B), and 100 μm (C).
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Table 1. Morphometric data on Rigidosticha italiensis n. gen., n. sp. Characteristica
Mean
Body, length
229.1 221.0 20.6 4.5
Body, width
115.5 110.0 14.6 3.2 13.1
Body length:width, ratio
M
SD SE
2.1
2.1
Anterior body end to proximal end of adoral zone, distance
87.2
88.0
7.0 1.6
Anterior body end to distal end of adoral zone, distance
34.2
Anterior body end to proximal end of adoral zone, percentage of body length DE-value
CV
Min
Max
n
9.0 200.0 284.0 21
1.7
2.5 21
8.1
75.0
98.0 19
34.0
8.2 2.0 24.0
24.0
56.0 17
38.2
37.8
3.9 0.9 10.2
32.1
45.2 19
0.4
0.4
0.1 0.0 17.7
0.3
0.6 17
Adoral membranelles, longest membranellar base
19.7
20.0
1.8 0.4
8.9
17.0
24.0 17
Adoral membranelles, number
51.4
51.0
5.5 1.3 10.8
40.0
62.0 19
Anterior end of paroral membrane to buccal cirrus, distance
13.8
14.0
4.3 1.0 31.0
7.0
25.0 17
Anterior body end to first macronuclear nodule, distance
72.9
70.5 11.4 2.8 15.7
53.0 100.0 17
Posterior body end to second macronuclear nodule, distance
79.1
78.0
9.2 2.5 11.6
61.0
95.0 13
Anterior macronuclear nodule, length
30.1
28.5
5.6 1.2 18.5
24.0
43.0 21
Anterior macronuclear nodule, width
16.4
16.0
1.8 0.4 11.2
14.0
20.0 21
Macronuclear nodules, number
2.0
2.0
0.0
2.0
2.0 21
Anteriormost micronucleus, diameter
5.1
5.0 0.7
0.2 12.9
4.0
6.0 19
Micronuclei, number
2.3
2.0
0.6 0.1 24.8
2.0
4.0 19
Anterior body end to right marginal row, distance
40.7
40.0
7.6 2.0 18.6
34.0
63.0 15
Right marginal row, number of cirri
29.3
30.0
2.6 0.7
9.0
24.0
33.0 15
Anterior body end to left marginal row, distance
76.9
80.0
8.7 2.3 11.4
57.0
90.0 15
Left marginal row, number of cirri
26.2
28.0
4.2 1.1 15.9
21.0
32.0 15
Gap between last cirri of marginal rows
9.4
8.0
3.9 1.2 41.7
6.0
20.0 11
Frontal cirri, number
3.3
3.0 0.5
0.1 14.0
3.0
4.0 15
Frontoterminal cirri, number
2.0
2.0 0.0
0.0
2.0
2.0 15
26.0
52.0 17
Anterior body end to buccal cirrus, distance
0.2 0.1 11.0
84.0 147.0 21
0.0 0.0
0.0
34.0
34.5
Buccal cirrus, number
1.0
1.0
0.0 0.0
0.0
1.0
1.0 15
Parabuccal cirrus, number
1.0
1.0
0.0 0.0
0.0
1.0
1.0 15
16
6.0 1.4 17.6
Characteristica
Mean
M
15.9
16.0
10.1
10.5 1.9
Transverse cirri, number
2.9
3.0 0.6
Dorsal kineties, number
8.9
9.0
0.8 0.2
Dorsal kinety 1, number of bristles
57.3
56.0
Dorsal kinety 2, number of bristles
43.8
42.0
Midventral cirral pairs, number
b
Posterior body end to rearmost transverse cirrus, distance
Caudal cirri, number a
3.0
SD SE
CV
Min
Max
2.7 0.7 16.7
12.0
20.0 13
0.6 18.5
6.0
13.0 11
0.2 21.9
2.0
4.0 13
8.8
8.0
10.0 17
5.4 1.8
9.5
51.0
70.0
9
3.7 1.2
8.5
40.0
51.0
9
3.0 0.0
0.0
0.0
3.0
3.0 15
Data based on mounted, protargol-impregnated, and randomly selected specimens from non-flooded Petri dish
cultures. Measurements in µm. CV – coefficient of variation in %, M – median, Max – maximum, Mean – arithmetic mean, Min – minimum, n – number of individuals investigated, SD – standard deviation, SE – standard error of arithmetic mean. b
Two or three right cirri of the pairs and one or two pretransverse cirri (?) at the rear end not included. See Fig. 2A.
17
n
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5