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ScienceDirect European Journal of Protistology 50 (2014) 47–67

Morphology and phylogeny of Bryophryoides ocellatus n. g., n. sp. (Ciliophora, Colpodea) from in situ soil percolates of Idaho, U.S.A. William A. Bourlanda,∗ , Laura Wendella , Greg Hampikiana , Peter Vd’aˇcn´yb a b

Department of Biological Sciences, Boise State University MS-1515, 1910 University Avenue, Boise, ID 83725-1515, USA Department of Zoology, Comenius University, Mlynská dolina B-1, Bratislava SK-84215, Slovak Republic

Received 1 May 2013; received in revised form 31 August 2013; accepted 18 September 2013 Available online 1 October 2013

Abstract We describe the morphology and 18S rDNA phylogeny of Bryophryoides ocellatus n. g., n. sp., a bryophryid ciliate inhabiting in situ soil percolates from Idaho, U.S.A. The new genus is distinguished from other bryophryid genera by a combination of the following features: (1) kreyellid (irregularly meshed) silverline pattern, (2) polymorphic adoral organelles in the preoral suture, (3) absence of vestibular kineties. In phylogenetic analyses, Bryophryoides ocellatus is most closely related to Bryophrya gemmea. The 18S rDNA sequence pairwise distance of 2% between these genera, while similar to that between many colpodidan species, exceeds that between some colpodidan genera (e.g. Mykophagophrys and Pseudoplatyophrya, 1.1%), further supporting establishment of the new genus. Topology hypothesis testing strongly supports the monophyly of the Colpodida including the bryophryids. Despite weak nodal support, tests of topology constraints narrowly reject the non-monophyly of the sequenced Bryophryidae (Bryophrya + Bryophryoides + Notoxoma). Likewise, the monophyletic origin of the sequenced Bryophryidae is indicated in the phylogenetic networks though with low support. © 2013 Elsevier GmbH. All rights reserved. Keywords: Bryophryidae; Cortical alveoli; Silverline pattern; Single-cell PCR; 18S rRNA gene; Vestibulum

Introduction The order Colpodida (class Colpodea) comprises a group of primarily terrestrial, cyst-forming ciliates of amazing morphologic diversity (Foissner 1993; Puytorac et al. 1974). Two new families (Sandmanniellidae and Ilsiellidae) and four new genera (Bromeliothrix, Dragescozoon, Pseudomaryna, and Sandmanniella) including ten new species have been added in the 20 years since Foissner’s (1993) seminal monograph of the Colpodea (Bourland et al. 2011; Foissner 2003; Foissner 2010; Foissner and Stoeck 2009; Foissner

∗ Corresponding

author. Tel.: +1 208 861 4449; fax: +1 815 301 8958. E-mail addresses: [email protected], [email protected] (W.A. Bourland). 0932-4739/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ejop.2013.09.001

et al. 2002). Six of the ten recently added species are assignable to clades arising from nodes basal to the “classic colpodas” based on their nuclear small subunit ribosomal DNA (18S rDNA) phylogeny (Bourland et al. 2011; Foissner et al. 2011). However, the topology of the large colpodid clade in general, and its deeper nodes in particular, remains poorly resolved despite expanded molecular character sampling (Dunthorn et al. 2011). This underscores the continuing importance of increased taxon sampling in efforts to resolve relationships in this group. The monogeneric families, Bardeliellidae, Ilsiellidae, and Sandmanniellidae comprise only five species. The Marynidae comprise at least 20 species, however only four 18S rDNA sequences are currently available. The Bryophryidae comprise four genera and ten species of which only two have been sequenced (Bourland et al. 2011). In this report we describe the

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W.A. Bourland et al. / European Journal of Protistology 50 (2014) 47–67

morphology, morphometrics and molecular phylogeny of Bryophryoides ocellatus n. g., n. sp., a bryophryid inhabiting in situ soil percolates from Idaho, U.S.A.

Material and Methods Sampling and cultivation Sampling sites. Bryophryoides ocellatus n. g., n. sp. was recovered from in situ soil percolates of two lawns, both sprinkler-irrigated with city water and located 300 m apart in Fort Boise Park (Site 1: 43◦ 37 0.47 N, 116◦ 11 20.56 W; Site 2: 43◦ 36 5.65 N, 116◦ 11 16.53 W, elev. 835 m) during June and July 2012. Results from both populations are pooled for this report since the sites were in proximity did not differ in conditions, and both populations were morphologically indistinguishable and had identical 18S rDNA sequences. Bryophrya gemmea was isolated from ephemeral puddles on a flood-irrigated grass lawn in Boise, Idaho (43◦ 38 010.82 N, 116◦ 13 50.5 W, elev. 813 m) from June through July 2008. Puytoraciella dibryophryis was isolated from ephemeral puddles on a grass lawn in Boise, Idaho (43◦ 36 49.63 N, 116◦ 13 23.31 W, elev. 816 m) in May 2006. An undescribed Tectohymena sp. was recovered from grass lawn soil percolates in Boise, Idaho (43◦ 36 52.33 N 116◦ 11 21.75 W, elev. 832 m) in June 2007. In situ soil percolate collection. Collections were carried out as follows: about 200 ml of soil percolate was directly aspirated from the saturated sod using a 50-ml bulb syringe aided by gentle foot pressure near the syringe tip. Percolates were clear to slightly cloudy and greenish in color with very little sediment. Raw samples were maintained for up to one week at room temperature (18–21 ◦ C). Conductivity measurements were made directly using an ExStik EC meter (Spectrum Technologies, Inc. Plainfield, IL, USA). Attempts to establish pure cultures of the new species were unsuccessful. Encystment was induced by isolating trophonts in filtered (0.22 ␮m pore size) percolate on slides kept in a moist chamber at room temperature and examined at two-week intervals.

Morphologic methods Trophonts and resting cysts were studied at magnifications of 40–1000×, with brightfield, phase and differential interference contrast using a Zeiss Axioskop 2 plus microscope. In vivo measurements were made from microphotographs of uncompressed cells except as noted. Impregnations including protargol, temporary and permanent silver carbonate and Klein-Foissner dry silver nitrate methods and staining with methyl green-pyronin Y were done as previously described (Augustin and Foissner 1984; Foissner 1991). Chatton-Lwoff wet silver nitrate preparations were done using recently described modifications (Vd’aˇcn´y and Foissner 2012). Counts of adoral organelles and paroral membrane

dikinetids were taken only from silver carbonate preparations because these features are often obscured in other types of impregnation. Counts and measurements of impregnated specimens were performed at a magnification of 630–1000×. For scanning electron microscopy, specimens were fixed for 30 min in a mixture of equal parts 2% OsO4 and 2.5% glutaraldehyde. Mounted specimens were dried in a K850 critical-point drier (Electron Microscopy Sciences, Hatfield, PA, U.S.A.) and sputtered with gold (∼20 nm) in a CRC 150 sputterer (Torr International, New Windsor, NY, U.S.A.) using ≤11 mA sputter current for 10–15 periods of 30 s each with 90 s “rests” between sputterings to avoid heat damage. Specimens were examined at 10–15 kV in a Hitachi S3400N scanning electron microscope. Drawings were based on microphotographs.

DNA extraction, amplification and sequencing Cells were selected using a stereomicroscope (90×), washed three times in filtered (0.22 ␮m pore size) Eau de Volvic mineral water. Single cells were placed in 0.2 ml PCR tubes with 50 ␮l of EB buffer (Qiagen, Valencia, CA, USA) and stored at −20 ◦ C. DNA was extracted from single cells using a modified Chelex method (Strüder-Kypke and Lynn 2003) and 18S rDNA was amplified and sequenced as previously described (Bourland et al. 2012). Puytoraciella and Tectohymena have not yet been sequenced.

Phylogenetic analyses An 18S rDNA alignment was created including the new species, 29 taxa representing the major colpodean lineages and two nassophorean ciliates, Obertrumia georgiana and Furgasonia blochmanni, retrieved from GenBank (Table 1). Sequences from two strains (EU039901 and JQ026521) of Ilsiella palustris are included despite their minimal pairwise distance (0.01%) since they are from geographically distant sites (Brazil and Hawaii respectively, Dunthorn et al. 2012). The alignment was created using the G-INS-i strategy in MAFFT ver. 6.5 (Katoh and Toh 2008). Consideration of secondary structure did not improve alignment scores, probably due to the relatively conserved colpodean 18S rDNA sequences. Ambiguously aligned regions were masked using Gblocks ver. 0.91b (Castresana 2000) allowing gap positions within the final blocks and then further refined by eye in MEGA5 (Tamura et al. 2011). The best model (TIM3+I+Γ , lnL −8625.8648) of nucleotide substitution was found under the Akaike information criterion using jModelTest ver. 2.1.1 (Darriba et al. 2012; Guindon and Gascuel 2003). Pairwise distances were calculated in MEGA5. Bayesian analysis was done with MrBayes ver. 3.1.2 (Huelsenbeck and Ronquist 2001) on XSEDE through the CIPRES Portal ver. 1.15 (http://www.phylo.org/) with support from two parallel runs with four MCMC chains and ten million generations, sampling every 1000 generations.

W.A. Bourland et al. / European Journal of Protistology 50 (2014) 47–67

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Table 1. List of ciliate taxa with GenBank (GB) accession numbers of corresponding 18S rDNA sequences included in the phylogenetic analyses. Taxon name

GB number

Taxon name

GB number

Taxon name

GB number

Bardeliella pulchra Bresslauides discoideus Bresslaua vorax Bryometopus atypicus Bryometopus pseudochilodon Bryophrya gemmea Bryophryoides ocellatus n.g, n. sp. Bursaria truncatella Colpoda cucullus Colpoda henneguyi Colpoda lucida

EU039884 EU039885 AF060453 EU039886 EU039887 HQ337901 KF569684

Cyrtolophosis mucicola Furgasonia blochmanni Hausmanniella discoidea Ilsiella palustris Ilsiella palustris isolate1 Maryna ovata Maryna sp.a

EU039899 X65150 EU039900 EU039901 JQ026521 HQ337902 JF747218

Ottowphrya dragescoi Platyophrya vorax Pseudocyrtolophosis alpestris Pseudomaryna sp. Pseudoplatyophrya nana Rostrophrya sp. Sagittaria sp.

EU039904 EU039906 EU264564 JF747219 AF060452 EU039907 EU039908

U82204 EU039893 EU039894 EU039895

Maryna umbrellata Mykophagophrys terricola Notoxoma parabryophryides Obertrumia georgianab

JF747217 EU039902 EU039903 X65149

Sandmanniella terricola Sorogena stoianovitchae Tillina magnac

FJ610254 AF300285 EU039896

The new species is shown in boldface. a Source organism in GenBank listed as Maryna sp. MD-2011. b Junior synonym for O. aurea (Foissner 1987). c Formerly Colpoda magna (Foissner et al. 2011)

When summarizing a consensus tree, 25% of sampled generations were discarded as burn-in. Computer programs AWTY (http://ceb.csit.fsu.edu/awty) and Tracer ver. 1.5 (http://tree.bio.ed.ac.uk/software/tracer/) were employed to assess convergence. Maximum likelihood (ML) analysis was implemented using RAxML with the default GTRGAMMA model (Stamatakis 2006) and bipartitions from 10,000 bootstrap replicates drawn onto the tree topology specified by the best-scoring ML tree from 100 replicates. The maximum parsimony (MP) analysis was carried out with MEGA5 using all sites and Close-Neighbor-Interchange (CNI) on random trees with support from 1000 bootstrap replicates.

Network analyses To visualize congruent and conflicting phylogenetic signal in the 18S rDNA dataset, phylogenetic networks were calculated with the computer program SplitsTree ver. 4 (Huson 1998; Huson and Bryant 2006). Networks were generated using the NeighborNet algorithm (Bryant and Moulton 2004) with uncorrected P-genetic distances and bootstrap analyses with 1000 replicates.

Topology hypothesis testing Constrained trees (Table 3) were generated and compared with the unconstrained (i.e. best-scoring) ML tree topology generating a file of per-site log-likelihoods using RAxML (Stamatakis 2006) for comparison of constrained and unconstrained tree topologies using the approximately unbiased (AU) test in CONSEL ver. 0.1 k (Shimodaira 2002, 2008; Shimodaira and Hasegawa 2001). A p-value of

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

We describe the morphology and 18S rDNA phylogeny of Bryophryoides ocellatus n. g., n. sp., a bryophryid ciliate inhabiting in situ soil percolates fr...
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