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

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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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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.

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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 <0.05 was chosen for rejection of the null hypothesis.

Terminology Terminology is according to Grain et al. (1979), Foissner (1993), and Lynn (1976, 2008) unless otherwise specified. We use the term “buccal plate” for the V-shaped structure delimiting the buccal overture in the new species in order to avoid confusion with the term “buccal lip”, a part of the stichotrichine oral apparatus (Foissner and Al-Rasheid 2006). We apply the term “in situ soil percolates” to aspirates taken directly from saturated but non-flooded soils in distinction to the term “soil percolates” often used in reference to in vitro effluent obtained from dried and subsequently rewetted soils in the non-flooded Petri dish method (Foissner et al. 2002; Foissner and Xu 2007). Classification and vernacular group names are according to Foissner et al. (2011) unless otherwise noted.

Results Description of Bryophryoides ocellatus n. g., n. sp. (Figs 1–75). Body length and width variable, but with comparable ranges in vivo (66–101 ␮m × 31–55 ␮m), in protargol (68–113 ␮m × 33–86 ␮m), and Chatton-Lwoff (69–116 ␮m × 34–69 ␮m) preparations. Shape broadly reniform to elliptical, dorsal margin convex, ventral margin straight, meet to form an indistinct blunt left anterior rostrum. Laterally compressed ∼2:1; ellipsoidal in cross section (Figs 1, 8a–e, 12–14). Macronucleus location near cell center in vivo, homomerous, globular, about 18–22 ␮m across in vivo (∼16 ␮m across after protargol and Chatton-Lwoff impregnation); nucleoli inconspicuous, ∼3 ␮m in diameter, numerous, finely granular. Several (2–4) ellipsoidal to lenticular micronuclei; on

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Figs. 1–11. Bryophryoides ocellatus, from life (1, 8a–e, 9–11), after temporary silver carbonate (2, 3, 6, 7a–f), dry silver nitrate (4), and composite dry silver nitrate and silver carbonate impregnation (5). (1) Ventral view of representative specimen. (2, 3) Ventral and dorsal view of ciliary pattern (same individual). (4) Silverline pattern of posterior ventral surface. (5) Semi-schematic diagram of oral and perioral structures, right anteroapical view. The white and black asterisks mark the right and left parts of the long scoop-shaped right oral ciliary field. Black arrowhead marks the cup-like expansion of the right oral ciliary field. (6) Left anterior ventral dikinetid and associated structures after silver carbonate, arrow shows kinety axis. (7a–f) Variations of adoral organelles (“paves”) in the preoral suture. Black arrowheads indicate

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average 4 ␮m × 2 ␮m in vivo; at least one micronucleus adjacent to macronucleus, location of other(s) variable; impregnate with silver carbonate but only inconsistently with protargol (Figs 19, 22–25, 31). Contractile vacuole terminal with single excretory pore slightly dorsal; collecting canals absent (Figs 1, 3, 15, 16, 24, 45, 47). Cortex flexible; thickened (∼2–3 ␮m); conspicuously cobbled both in vivo and in the SEM due to clusters of large cortical alveoli surrounding ciliary pits (Figs 12, 13, 17, 47–49, 53). Mucocysts numerous but inconspicuous in vivo as usually obscured by dilated cortical alveoli; discoidal and ∼1.5 ␮m across; do not impregnate with protargol, inconsistently stain with silver carbonate; do not extrude or stain with methyl-green pyronin Y (Fig. 35). Cortical granules absent. Cytoplasm colorless to slightly pinkish-orange with scattered minute shiny lipid droplets and shard-like crystals that sometimes concentrate in defecation vacuoles (Fig. 16). Cells often packed with ∼10 ␮m × 5 ␮m ovoid food vacuoles containing fine filamentous cyanobacteria in tightly packed whorls (Figs 1, 18, 20–22). Swimming motion moderately rapid, rotating about long body axis, occasionally lingering over debris to feed on filamentous cyanobacteria. Somatic cilia ∼8 ␮m long in vivo, arranged in an average of 43 left-spiraling sigmoid kineties (Table 2; Figs 2, 3, 23–26). Ciliary rows consist of dikinetids doubly ciliated in about anterior third of cell while only posterior basal body ciliated over posterior two thirds of cell (Figs 44–46). Interkinetidal distance increases posteriorly. Right somatic kineties originate on preoral suture, two to five kineties interrupted at the level of the buccal apparatus, sometimes with one or more proximal monokinetids (Figs 2, 23, 25, 52). Vestibular kineties absent (Figs 2, 23, 25, 51, 52, 60). Four to nine postoral kineties abut left margin of oral apparatus; isolated dikinetids sometimes interspersed between postoral kineties (Figs 1, 23, 25). Left anterior somatic kineties (11 on average) abut preoral suture (Figs 2, 23, 25). Dikinetids in anterior cell have two conspicuous narrowly triangular fibrillar associates in silver carbonate preparations, smaller anterior one lies parallel to kinety, larger right posterior one almost perpendicular to kinety (Figs 6, 28). Two granules to left of dikinetids in silver carbonate preparations, corresponding pores in SEM preparations consistent with parasomal sacs (Figs 6, 33, 53). Silverline pattern kreyellid in all cells (n = 172), ∼3 ␮m irregular meshes, pattern occuring over entire cell (Figs 4, 5, 34). Vestibulum absent (Figs 44, 46, 51, 52). Buccal overture small (∼8% of the cell length), narrowly triangular, positioned at base of a shallow unciliated

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U-shaped trough situated in anterior body third, angled left by ∼30◦ . Buccal plate V-shaped, borders buccal overture covering right and left oral ciliary fields, cilia of which protrude between its edges; proximal vertex of buccal plate fimbriated (Figs 5, 46, 51, 52). Right and left oral ciliary fields completely contained within deep tubular buccal cavity (Figs 1, 5, 14, 15, 52, 57). Right oral ciliary field (ROCF) a continuous curved sheet of densely-spaced ciliated kineties carpeting right, posterior, and part of left walls of buccal cavity; superficial end of ROCF cup-like expanded just beneath buccal plate (Figs 5, 14, 23–25, 60, 69). Single file of 5–8 dikinetids inclined at ∼90◦ to axis of the buccal overture extends anteriorly from right anterior margin of ROCF in 10 of 44 (22.7%) of silver carbonate-impregnated specimens, file contained within buccal cavity thus hidden by overlying buccal plate in SEM (Figs 32, 46, 51, 52, 60). Left oral ciliary field (LOCF) comprises about 7 closely spaced rectangular organelles on left wall of buccal cavity; abuts left edge of ROCF near its cup-like expansion (Figs 5, 25, 32, 69). Six to eleven adoral organelles (AOs) occupy short preoral suture not extending onto dorsal surface; inconspicuous in vivo and in the SEM but easily recognizable after silver carbonate impregnation. Proximal-most two or three bipartite (i.e., composed of a small anterior triangular group of basal bodies and a separate more or less rectangular array). Fourth and fifth AOs usually ocellate or eye-like (i.e. a circular array of basal bodies enclosing a central basal body). Distally smaller and highly variable in shape as composed of fewer and more or less irregularly arranged basal bodies (Figs 2, 7a–f, 23, 25, 27–30, 50, 51, 60, 66–68). Resting cysts (Figs 10, 11, 36–42). Encystment begins within 24 h after isolation in filtered site water. Cells initially rotate within the cyst wall but are motionless by 48 h. Numerous food vacuoles are visible at 24 h but disappear by 48 h (Figs 37, 38). Extruded material visible between the inner and middle layer at 24 h cysts disappeared by 48 h (Figs 10, 38). The wall of young resting cysts comprises three layers. At 14 d only an inner layer and a thin irregular outer mucous coat remained (Figs 11, 40). As cysts aged, numerous cytoplasmic crystals coalesced around the central nuclear mass and cysts decreased to half of initial size (Figs 10, 11, 36–40, 42). Excystment was not observed. Division cysts (Figs 9, 43). Reproduction occurs in division-cysts. Division cysts are digenic but this is a preliminary observation since only two division cysts were found. Three cyst layers envelop tomites. Extruded material is compressed between the inner and middle layers and rod-shaped

distal organelle. Open arrowheads indicate eye-shaped organelles and the arrows mark bipartite proximal organelles. (8a–e) Variations in cell shape. (9) One-day-old digenic division cyst has 3 layers (numerals 1–3). (10) Two-day-old resting cyst. (11) Two-week-old resting cysts. AB – anterior basal body, AO – adoral organelles, BO – buccal overture, BP – buccal plate, C – perinuclear crystals, CA – cortical alveoli, CV – contractile vacuole, DK – dikinetids, EM – extruded material, EP – excretory pore, FA – fibrillar associate, Ma – macronucleus, Mi – micronuclei, NM – nuclear material, OA – oral apparatus, P – left oral ciliary field (“palette”), PB – posterior basal body, PS – parasomal sac, ROCF – right oral ciliary field, T – tomite. Scale bars: 25 ␮m (1–3, 8a–f, 9–11), 10 ␮m (4–7a–e), and 1 ␮m (6).

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Figs. 12–22. Bryophryoides ocellatus in vivo (DIC). (12, 13) Ventral and dorsal views (two different specimens). Enlarged cortical alveoli give cortex its cobbled appearance. (14) Optical section showing right oral ciliary field (white and black arrowhead) which has C-shape in cross section. (15) Optical section of lightly squashed specimen. The left oral ciliary field descends at angle into the upper buccal cavity to abut the left part of the right oral ciliary field (black arrow). Two micronuclei are marked by black arrowheads. An asterisk marks the terminal contractile vacuole. (16) Squashed specimen with two crystal-filled defecation (?) vacuoles (arrowheads). (17) Ventral view of slightly squashed specimen showing external details of the oral apparatus. The buccal shelf (right side marked by black arrowhead) delimits the buccal overture (white arrowhead). Cilia of the left oral ciliary field (black arrow) are protruding from the buccal overture medial to the left margin of the buccal shelf. Protuberant cortical alveoli form rosettes around cortical ciliary pits (white arrows). (18) Flexible cell cortex deformed by surrounding

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Table 2. Morphometric data on Bryophryoides ocellatus n. g., n. sp. Characteristica

Method

Mean

M

SD

CV

Min

Max

n

Body, length (A)

IV CL P

81.0 88.4 84.7

80.0 88.0 84.5

10.47 10.81 11.26

16.0 12.2 13.3

66.0 69.0 68.0

101.0 116.0 113.0

17 52 40

Body, width

IV CL P

40.0 47.2 52.2

39.0 47.0 49.0

6.51 8.28 11.90

16.3 17.5 22.8

31.0 34.0 33.0

55.0 69.0 86.0

17 52 40

Body, length:width ratio

IV CL P

2.0 1.9 1.7

2.1 1.9 1.7

0.21 0.17 0.20

10.3 8.9 12.1

1.7 1.6 1.3

2.4 2.3 2.1

17 52 40

Anterior end to proximal end of buccal overture, distance (B)

IV CL P

26.4 24.2 27.1

26.0 24.0 26.0

4.64 3.40 5.22

17.6 14.0 19.3

21.0 18.0 17.0

32.0 30.0 40.0

9 20 36

B/A, ratio

CL P

0.04 0.07

13.7 21.9

0.2 0.2

0.3 0.5

20 36

Buccal overture, length

IV CL P

11.3 6.3 7.9

11.3 6.3 7.7

1.62 1.06 1.65

14.4 16.8 20.8

9.2 4.8 6.2

14.7 8.0 13.5

9 19 26

Buccal overture, width

IV CL P

6.5 5.7 5.9

6.6 5.6 5.5

1.52 0.78 1.44

23.4 13.7 24.4

4.6 4.7 3.4

9.4 7.0 10.0

9 19 20

Anterior end to Ma, distance Macronucleus, number Macronucleus, length

P MGP, P, SC P SC

37.3 1.0 16.1 16.2

37.5 1.0 15.5 15.6

7.50 0.00 3.35 2.43

20.1 0.0 20.8 15.0

24.0 1.0 11.0 12.3

55.0 1.0 28.0 21.7

36 112 38 25

Macronucleus, width

P SC

14.2 14.4

13.6 14.4

3.05 1.55

21.5 10.8

10.9 11.2

25.8 17.5

38 25

Micronucleus, number Micronucleus, length Micronucleus, widthb Somatic kineties, number Postoral kineties, numberc Left somatic kineties terminating on preoral suture, number Adoral organelles in preoral suture, number ROCF, length in lateral view Dikinetids in mid-dorsal kinety, number Interrupted right anterior kineties, number

MGP, SC SC SC P, SC CL, P, SC CL, P, SC

2.4 4.0 2.0 43.6 6.6 11.4

2.0 4.0 2.0 43.0 7.0 11.0

0.53 0.80 0.44 3.53 1.01 2.20

21.8 19.8 22.1 8.1 15.3 19.3

2.0 2.9 1.3 34.0 4.0 7.0

4.0 6.1 2.8 52.0 9.0 17.0

66 48 48 41 72 68

SC P SC SC

8.0 21.5 28.7 3.3

8.0 22.0 29.0 3.0

1.05 2.64 2.28 0.93

13.0 12.3 7.9 28.5

6.0 16.0 24.0 2.0

11.0 25.0 33.0 5.0

42 10 18 34

0.28 0.32

0.27 0.32

a Data based on randomly selected specimens from type locality. CL, Chatton-Lwoff silver nitrate; CV, coefficient of variation (%); IV, in vivo; M, median; Ma, macronucleus; Max, maximum; Mean, arithmetic mean; MGP, methyl green-pyronin Y; Min, minimum; n, number of individuals investigated; P, protargol Wilbert method; ROCF, right oral ciliary field; SC, silver carbonate. b Measurement of the micronucleus when seen edge-on. c Kineties terminating on left margin of the oral apparatus.

silt particles. The cytoplasm is packed with food vacuoles containing tightly folded filamentous cyanobacteria (black arrowheads). White arrowhead marks the oral apparatus. (19) Squashed specimen with a coronal optical section of the ciliated buccal cavity (black arrowheads). White arrowhead marks the cup-shaped expansion of the superficial end of the buccal cavity. Black arrows mark two micronuclei adjacent to the macronucleus (Ma). (20) Left ventrolateral view of a well-fed uncompressed specimen. Black arrowhead marks left oral ciliary field. Food vacuoles contain tightly-packed Jaaginema-like cyanobacteria (white arrowheads). (21) Ingested (black arrowheads) cyanobacteria. White arrowhead marks uningested filaments. (22) Ventral view. Left oral ciliary field (“palette”) marked by white arrowhead. Black arrowheads mark micronuclei adjacent to the macronucleus (Ma). AO – adoral organelles, Ma – macronucleus. Scale bars: 25 ␮m (15, 16, 18–20, 22) and 10 ␮m (12–14, 17, 21).

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Figs. 23–26. Bryophryoides ocellatus after silver carbonate impregnation. (23, 25) Ventral view of ciliary pattern. Adoral organelles in preoral suture lie to viewer’s left of asterisks (23). White arrows mark proximal ends of shortened right somatic kineties and black arrow (25) marks group of five dikinetids associated with ROCF (asterisk). Arrowheads denote two micronuclei. (24) Dorsal view of ciliary pattern. (26) Apical view of ciliary pattern. White arrow marks the distalmost adoral organelle. White arrowheads mark margins of preoral suture. Asterisk denotes right oral ciliary field. EP – excretory pore, LK – left somatic kineties, Ma – macronucleus, Mi – micronucleus, OA – oral apparatus, RK – right somatic kineties, ROCF – right oral ciliary field. Scale bars: 25 ␮m (23–26).

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bacteria adhere to the outer mucous layer. Details of stomatogenesis were not observed. Occurrence and ecology. We found B. ocellatus in circumneutral (pH 7.47) oligohaline (conductivity 863 ␮S) in situ soil percolates from sprinkler-irrigated lawns in Boise, Idaho, during summer months. The new species occurred at two neighboring sites (∼300 m apart) in Fort Boise Park. Regular irrigation with city water during the sampling period kept soil at the sites saturated but, with the exception of a few small ephemeral puddles, not flooded. Bryophryoides ocellatus was found as part of a highly diverse community of metazoans, protists and cyanobacteria, including a variety of other colpodeans (e.g. Bryophrya, Colpoda, Etoschophrya, Kuklikophrya, Maryna, Tectohymena, and Woodruffides). Bryophryoides ocellatus feeds exclusively on very pale green, almost colorless, fine filamentous cyanobacteria that are tightly packed in food vacuoles (Figs 18, 20). Although not definitively identified, they are consistent with Jaaginema sp. (Komárek et al. 2003). Molecular characteristics. The macronuclear 18S rDNA sequence of B. ocellatus is 1735 bp in length (excluding primer sites) with a GC content of approximately 44% (GenBank accession number KF569684). Four 18S rDNA sequences from single cells (three cells from Site 1 and one cell from Site 2) were identical. Phylogenetic analyses (Fig. 76). Trees recovered by all three phylogenetic methods (ML, BI, MP) had identical topologies but varying nodal support levels. The loglikelihood of the best ML tree was −8191.18. Bryophryoides ocellatus and Bryophrya gemmea form a fully supported clade. Notoxoma is the sister group to this clade but with only weak support from all three methods. The pairwise distance between Bryophryoides ocellatus and Bryophrya gemmea is 2.0%, exceeding that between the colpodidan genera Mykophagophrys and Pseudoplatyophrya (1.1%). Our molecular analysis, like that of Foissner et al. (2011), shows Bresslauides clustering within Colpodidae rather than Hausmanniellidae in which it was placed by Foissner (1993) based on morphologic characters. Phylogenetic networks (Fig. 77). Network analyses of the 18S rDNA dataset were qualitatively similar to phylogenetic trees, but they revealed more complex relationships among the colpodean orders and among the main

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colpodid lineages. Monophylies of all colpodean orders were supported by long parallel edges as well as by the 100% bootstrap values. The Colpodida is separated from all other colpodean orders by a prominent set of fully supported parallel edges. The genera Ilsiella and Bardeliella branch off first within the colpodid clade (83% bootstrap). The next diverging group is represented by the bryophryids (Notoxoma + Bryophrya + Bryophryoides) whose monophyletic origin is, however, only poorly supported by the 57% bootstrap (not shown). On the other hand, a sister relationship of Bryophrya and Bryophryoides is supported by two distinct parallel edges and the 92% bootstrap. Any closer relationship of Sandmanniella and the bryophryids is not recognized, but a sister relationship of Sandmanniella and the clade Maryna + Pseudomaryna is indicated by several short parallel edges (54% bootstrap; not shown). The families Colpodidae and Tillinidae represent the crown colpodids which are separated, as a whole, by a set of parallel edges (90% bootstrap) from the families Grossglockneriidae (100% bootstrap) and the Maryna + Pseudomaryna clade (100% bootstrap) as well as from Hausmanniella discoidea. However, the NeighborNet graph shows considerable conflict in the phylogenetic signal within the Colpodidae, as indicated by several short edges and the lack of treeness. Constrained analyses (Table 3). Despite only weak ML, BI and MP support for a clade containing Notoxoma, Bryophrya and Bryophryoides, the non-monophyly of this clade was narrowly rejected by the AU test (p = 0.047). It should be noted that there is currently no 18S rDNA sequence for the type species of Bryophrya (Bryophrya bavariensis), thus the genus is represented only by B. gemmea in the phylogenetic analyses. Exclusion of the bryophryid clade from the Colpodida was strongly rejected (p = 0.002). A sister group relationship of Sandmanniella with the bryophryids was rejected (p = 0.039) but a topology in which Sandmanniella is sister to the Maryna + Pseudomaryna clade was not rejected (p = 0.696).

Discussion The new genus Bryophryoides. The combination of the following characters in Bryophryoides distinguishes it from other genera in the Bryophryidae (1) kreyellid

Table 3. Analyses of topological constraints. Constraint

−Log-likelihood

AU test (p)

Unconstrained Sandmanniellidae sister to Maryna spp. + Pseudomaryna Colpodida excluding B. ocellatus, Bryophrya gemmea, Notoxoma parabryophryides Bryophrya gemmea + Bryophryoides excluding Notoxoma parabryophryides Bryophrya + Notoxoma parabryophryides excluding Bryophryoides Sandmanniellidae sister to B. ocellatus + Bryophrya gemmea + Notoxoma parabryophryides

−8191.18 −8192.21 −8259.16 −8202.47 −8222.43 −8204.48

0.696 0.459 0.002 0.047 0.007 0.039

AU – approximately unbiased test.

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Figs. 27–35. Bryophryoides ocellatus from type population after silver carbonate (27–33, 35) and dry silver nitrate (34) impregnation. (27) Perioral ciliature. White arrowheads mark the curious eye-shaped adoral organelles. The black arrow marks a proximal bipartite organelle. The white arrow marks the shortened right somatic kinety. (28) Apicoventral view. Bipartite adoral organelles may include an eye-shaped component (white arrowhead). The white arrow marks the distalmost adoral organelle. The back arrow marks the distal end of the first right somatic kinety. Anterior dikinetids bear short stout nematodesmata arising from the posterior basal body (black arrowheads. The asterisk indicates the right oral ciliary field. (29) Ventral view. The horseshoe-shaped buccal plate (white arrowheads) delimits the buccal overture (asterisk). The black arrowheads mark three eye-shaped adoral organelles and the black arrows mark bipartite membranelles. The white arrow marks the distal end of the left oral ciliary field on the left anterior wall of the buccal cavity. (30) The black arrow marks the medial triangular component of a bipartite adoral organelle. The white asterisk marks the buccal overture and the white arrow marks the densely packed basal

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Figs. 36–43. Bryophryoides ocellatus. Resting cysts (36–42) and division cyst (43) of type population in vivo (brightfield 36, 37, 39, 40, 43; DIC 38, 41, 42). (36) Eighteen hours post isolation. Trophont has settled to bottom of dish and has rounded up. White arrowhead marks single cyst layer. (37) Twenty-four hour-old cyst. White arrowheads mark extruded material between endocyst and mesocyst. Bacteria and detritus adhere to the outer layer (black arrowhead). Food vacuoles (white asterisks) are still present at this stage. (38) Forty-eight hour-old cyst. The diameter of the cell and cyst layers has decreased by ∼30%. Food vacuoles and extruded material have disappeared. The contractile vacuole (white arrowhead) is still active. Cytoplasmic crystals (black arrowheads) appear. (39) Seven-day-old cyst. Cell size the same as at 48 h Only two cyst layers are discernible (black arrowhead and white arrow). Cytoplasmic crystals (white arrowheads) appear dark in brightfield. White asterisk marks condensing mass of nuclear material. (40) Fourteen-day-old cyst further decreases in size to <50% of the one-day old cyst (37). The cytoplasmic crystals (white arrowhead) form a dense envelope around the nuclear material (white asterisk). Only two cyst layers are discernible (black and white arrow). (41) Forty-eight-hour old cyst with rod-shaped bacteria (arrowheads) embedded in outer cyst layer. (42) Ruptured seven-day-old cyst. Black arrow marks site of rupture. Escaping cell contents (black asterisks) including numerous crystals (white arrows) distend the mesocyst layer (white arrowhead). The white asterisk marks nuclear material. Black arrowhead denotes the endocyst layer. (43) One-day-old division cyst with two tomites (T). Black arrowheads mark extruded material. T – tomite. Scale bars: 25 ␮m (36–43).

silverline pattern (2) polymorphic (i.e. bipartite and ocellate) adoral organelles in the preoral suture (3) absence of vestibular kineties (Foissner 1993). Bryophryoides has considerably more complex oral structures in comparison with the bryophryid genera Notoxoma and Parabryophrya. Notoxoma also lacks vestibular kineties (Foissner 1993). However, Bryophryoides differs from Notoxoma in its silverline pattern (kreyellid vs. platyophryid) and adoral organelles (multiple

polymorphic in preoral suture vs. simple single, not in preoral suture). Foissner (1993) describes two vestibular kineties in Parabryophrya penardi but does not mention the vestibulum or its kineties in his description of Parabryophrya etoschensis (Foissner et al. 2002). However several figures suggest that it has one or two vestibular kineties (Foissner et al. 2002; Fig. 206b and d). Bryophryoides is also distinguished from Parabryophrya by the silverline pattern

bodies of the right oral ciliary field. (31) Four micronuclei (white arrowheads) clustered around the macronucleus (Ma). (32) Ventral view. The arrowheads mark the distal (black) and proximal (white) end of “palette” or left oral ciliary field. The white arrow marks the internal (dorsal end of the trough-like right oral ciliary field (ROCF). The black arrow marks the file of kinetids associated with ROCF (cf. Fig. 25). (33) One large posterior and one smaller anterior parasomal sac lie to the left of ciliated anterior dikinetids giving the appearance of tetrads in optimally impregnated specimens (white arrowheads). (34) Ventral silverline pattern consisting of polygonal rosettes surrounding dikinetids (white asterisks). (35) Dorsal view of over-impregnated specimen showing loosely spaced 1.5 ␮m discoid mucocysts (white arrowheads) within and between kineties. Ma – macronucleus. Scale bars 25 ␮m (35), 10 ␮m (27–32), and 5 ␮m (33, 34).

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Figs. 44–49. Bryophryoides ocellatus. Scanning electron microscopy of specimens from type location. (44, 45) Ventral and dorsal overview showing the broadly reniform shape and the relatively small oral apparatus. (46) Ventral view of anterior body portion. Black arrowheads mark shortened kineties to right of oral apparatus. The buccal shelf lies deep to the cortical alveoli (white arrowhead). (47) Posterior pole view showing the single excretory pore of the contractile vacuole. (48) Ventral view. Protuberant cortical alveoli (white asterisks) form rosettes around the cortical ciliary pits (white arrowheads). (49) Specimen in which a patch of cortex has broken away revealing underlying epiplasmic material (white asterisks) and the inner (white arrowheads) and outer (black arrowheads) alveolar membranes. EP – excretory pore, OA – oral apparatus. Scale bars: 25 ␮m (44, 45), 10 ␮m (46, 47), and 2 ␮m (48, 49).

(kreyellid vs. platyophryid) and adoral organelles (multiple polymorphic in preoral suture vs. single, not in preoral suture). Bryophryoides is distinguished from Bryophrya by the silverline pattern (kreyellid, vs. platyophryid), type of adoral organelles in the preoral suture (polymorphic vs. monomorphic), and absent vs. present vestibular kineties (Foissner 1993). Bryophryoides is distinguished from Puytoraciella by vestibular kineties (absent vs. present), adoral organelles (polymorphic, bipartite and ocellate vs. monomorphic, irregular polygonal), length (∼80 ␮m vs. ∼250 ␮m),

and number (∼44 vs. ∼160) of somatic kineties. The silverline pattern of Puytoraciella is not known (Bourland 2008). The silverline pattern and adoral organelles in the preoral suture as generic characters in Bryophryidae are discussed in greater detail below. Comparison of Bryophryoides ocellatus with similar species. Bryophryoides ocellatus most closely resembles Bryophrya gemmea in vivo, especially when the latter is feeding on fine pale green Jaaginema-like cyanobacteria (Figs 54, 55). Since the adoral organelles of both species

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Figs. 50–53. Bryophryoides ocellatus. Scanning electron microscopy of specimens from type location. (50) Ventral view of anterior body portion. White arrowheads mark adoral organelles in preoral suture. The white asterisk marks the oral apparatus. (51) Detail of oral apparatus. Arrowheads mark cilia of the right (white) and left (black) oral ciliary fields protruding between the margins of the buccal shelf (white asterisks). The white arrows mark cilia of adoral organelles. (52) Detail of buccal plate and the surrounding ciliature. White arrowhead marks the bluntly serrated vertex of the buccal plate. Cilia of the right (white arrow) and left (black arrowhead) oral ciliary fields protrude through the buccal overture. Black arrows mark monokinetids to right of oral aperture. (53) Posterior dorsal cortex. Stomata of parasomal sacs (white arrowheads) are seen to the right of dikinetids within cortical ciliary pits. Scale bars: 5 ␮m (50–52) and 2 ␮m (53).

are inconspicuous in vivo, the most important distinctions recognizable in life (i.e. absence vs. presence of refractive right paroral interkinetal cortical granules and relatively small buccal overture [≤15% of body length vs. larger, ∼20% of body length]) require careful examination (Figs 54, 55). Distinguishing characteristics requiring silver impregnation include: the silverline pattern (kreyellid vs. platyophryid), the number of micronuclei (≥2 vs. 1), the structure of the adoral organelles (bipartite and ocellate vs. irregularly pentagonal), vestibular kineties (absent vs. present), postoral ciliary rows deviating rightwards around the proximal vertex of oral apparatus (absent vs. present), and the presence vs. absence of shortened right anterior somatic kineties (Figs 60, 61, 66–72). Bryophryoides ocellatus is

distinguished from Bryophrya bavariensis by the mucocysts (inconspicuous discoidal vs. prominent rod-shaped), vestibular kineties (absent vs. present), contractile vacuole collecting canals (absent vs. present) and the polymorphic vs. monomorphic (rectangular) adoral organelles in the preoral suture (Grain et al. 1979). Both Bryophrya rubescens and B. flexilis have single micronuclei (Foissner 1993). Bryophryoides ocellatus is unlikely to be confused with Puytoraciella dibryophryis which is much larger (∼65–100 ␮m vs. ∼250 ␮m) with significantly more somatic kineties (∼44 vs. ∼160), larger number of micronuclei (≤4 vs. ≥5), and absent vs. present vestibular kineties (Bourland 2008). Oral structures in the Bryophryidae. The taxa of many colpodean groups have multiple adoral organelles (e.g.

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Figs. 54–65. Bryophryoides ocellatus from life (54, 57; DIC) and after silver carbonate (60), Bryophrya gemmea from life 55, 58; DIC) and after silver carbonate (61), Puytoraciella dibryophryis from life (56, 59; DIC) and after silver carbonate (62), and Tectohymena sp. from life (65; DIC) and after silver carbonate 63, 64). (54) Ventral view of Bryophryoides ocellatus. Arrowhead marks oral apparatus. (55) Ventral view of Bryophrya gemmea. White arrowhead marks oral apparatus. Black arrowhead marks refractive interkinetal granules. (56) Ventral view of Puytoraciella dibryophryis. White arrowhead marks oral apparatus. Black arrowheads mark cilia of adoral organelles in

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Platyophryina, Cyrtolophosidida, Bursariomorphida) with a simple rectangular, brick-shaped or “heterotrichid” (e.g. Bryometopus) morphology (Foissner 1993). Adoral organelles within the buccal cavity (i.e. the “palette”) of the bryophryids are more or less rectangular in Bryophrya, Bryophryoides and Puytoraciella (Figs 32, 69, 72, 75). The markedly reduced adoral organelles of Notoxoma and Parabryophrya reside in the buccal apparatus rather than the short preoral suture. The other bryophryid genera have additional adoral organelles in the preoral suture. Although these can display variation in size and geometric shapes between species, within the species (excepting Bryophryioides), there is only one morphotype of adoral organelle with only minor variations (Figs 60–62, 66–74). Bryophryoides is unique among the Colpodida in having polymorphic (bipartite and ocellate) adoral organelles in the preoral suture. Unlike Bryophrya and Puytoraciella, they do not extend to the dorsal surface. The bipartite organelles are always situated between the left oral ciliary field and the ocellate organelles (Figs 7a–f, 23, 25, 27–30, 60, 66–68). Unlike the left oral ciliary field, the right oral ciliary field of bryophryids shows considerable variability, consisting of a single file of dikinetids in Notoxoma sigmoides but comprising a curved sheet of hundreds of densely-packed basal bodies carpeting all but a small area of the buccal cavity in Puytoraciella, Bryophrya and Bryophryoides. Here, the terms “buccal cavity” and “vestibulum” require clarification as applied to the bryophryids. Foissner (1993) removed “presence of vestibular kineties” from the familial diagnosis of the Bryophryidae when he noted their absence in Notoxoma. However, N. sigmoides is described as having a . . .very shallow vestibulum” (Foissner 1993). The vestibulum, sensu stricto, is “a ciliated oral cavity which partially or completely separates a buccal cavity from the somatic region of the cortex” while the buccal cavity “contains the bases of the oral polykinetids or compound ciliary organelles” (Lynn 1976, 2008). Accordingly, the description of Bryophrya gemmea (Bourland et al. 2011) should have placed right and left oral ciliary fields within the buccal cavity and not the vestibulum. These considerations also apply to Puytoraciella dibryophryis (Bourland 2008; Foissner 1993; Njine 1979) and Bryophryoides ocellatus and

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are consistent with the terminology of Grain et al. (1979) in describing Bryophrya bavariensis. By the same definitions, B. ocellatus lacks a vestibulum. Rather, the buccal plate and buccal overture lie in the base of a small, bare (i.e. unciliated) cortical depression circumscribed by the edge of the somatic cortex (Figs 44, 46, 50–52). All three genera with extensive right oral ciliary fields (Bryophrya, Bryophryoides and Puytoraciella) have a long tubular buccal cavity extending obliquely into the cell interior and covered by the right oral ciliary field except for a small area anteriorly (Figs 14, 15, 57–59). The buccal apparatus of Tectohymena, a much smaller species (Figs 63–65), is structurally quite similar to that of Bryophrya, Bryophryoides and Puytoraciella, having a sheet-like right oral ciliary field within a tubular buccal cavity and left oral ciliary field comprised of rectangular adoral organelles (Figs 63–65). Tectohymena is currently included in the Bryometopida but may be more closely related to the Bryophryidae (Foissner et al. 2011; Lynn 2008). Scanning electron micrographs of Bryophryoides ocellatus reveal a distinct membrane-like “buccal plate” consisting of a V-shaped structure defining the narrow buccal overture (Figs 5, 44, 46, 51, 52). No similar structure was demonstrated in sections of Bryophrya bavariensis examined by transmission electron microscopy, however the buccal plate is not clearly seen by light microscopy and a homologous structure in other bryophryids cannot be excluded (Grain et al. 1979). Although the colpodids are generally considered to be “filter feeders” (Lynn 2008), the buccal ciliature of B. ocellatus is deep to, and almost completely covered by the buccal plate, making this feeding mode difficult to envision for this species. The buccal plate may play a role in the apparent food selectivity of B. ocellatus. Silverline pattern. Bryophryoides is the only genus in the order Colpodida having a kreyellid (i.e. irregular meshes) silverline pattern (Foissner 1993). Convergent evolution of silverline patterns is rife among the Colpodea, however this character may still retain phylogenetic value at the generic level (Foissner et al. 2002; Lynn et al. 1999). For example, Foissner et al. created the genus Ottowphrya for species formerly included in Platyophryides but having a platyophryid rather than colpodid silverline pattern and division in cysts (Foissner et al. 2002). Although some

preoral suture. (57) Optical section of B. ocellatus. White arrowhead marks tubular buccal cavity. (58) Optical section of B. gemmea. White arrowhead marks tubular buccal cavity. (59) Optical section of P. dibryophryis. White arrowhead marks tubular buccal cavity. (60) Ventral view of B. ocellatus. Hite arrowhead marks short file of dikinetids associate with right oral ciliary field (asterisk). Black and white arrows mark bipartite and ocellate adoral membranelles respectively. Black arrowheads mark shortened anterior somatic kineties. (61) Ventral view of B. gemmea. Black arrowheads mark vestibular kineties. White arrowheads mark pentagonal adoral organelles in preoral suture. White and black asterisks mark right and left oral ciliary fields respectively. (62) Ventral view of P. dibryophryis. Black arrowhead marks the kinety described as “paroral” and “circumoral” kinety by Njine (1979) and as a “vestibular kinety” by Foissner (1993). Black arrow marks closely spaced dikinetids of additional vestibular kineties continuing anteriorly as normally spaced somatic kineties. White arrowheads mark proximal adoral organelles in preoral suture. Right and left oral ciliary fields are marked with white and black asterisks respectively. (63) Ventral view of Tectohymena sp. White arrowhead marks oral apparatus. (64) Lateral view of Tectohymena sp. White arrowhead marks right oral ciliary field. Black arrowhead marks left oral ciliary field. (65) Transverse optical section of compressed Tectohymena sp. (cf. Fig. 14). White arrowheads mark tubular buccal cavity, which is ciliated except for the anterior wall (black arrowhead). Ma – macronucleus. Scale bars: 25 ␮m (54–59), 10 ␮m (60–65).

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Figs. 66–75. Bryophryoides ocellatus (66–69), Bryophrya gemmea (70–72) and Puytoraciella dibryophryis (73–75) after silver carbonate. (66) Ventral view of B. ocellatus. White arrowheads mark adoral organelles in preoral suture. (67) Ventral view of B. ocellatus. White and black arrowheads mark ocellate and bipartite adoral membranelles respectively. (68) Ventral view of B. ocellatus. White and black arrowheads mark ocellate and bipartite adoral membranelles respectively. (69) Lateral view of B. ocellatus. Black arrowheads mark rectangular adoral organelles comprising the left oral ciliary field abutting the left margin of the right oral ciliary field (white asterisk). (70) Ventral view of

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Fig. 76. Best-scoring Maximum Likelihood tree based on 18S rDNA sequences. The new species is shown in boldface. Nodal support is shown as Maximum Likelihood (ML), bootstrap/Bayesian inference (BI), posterior probability/Maximum Parsimony (MP), bootstrap. Values ≤50% are shown as “–”. New sequence is in bold. Scale bar: substitutions/nucleotide position.

platyophryids have composites of more than one silverline pattern (e.g. Etoschophrya, Reticulowoodruffia and Semiplatyophrya), one pattern clearly dominates. To date, no species with completely different silverline patterns belong to the same colpodean genus. Although the vast majority of

the Colpodida have one or another subtype of the rectangularly meshed “colpodid” silverline patterns, three bryophryid genera (Notoxoma, Parabryophrya and Bryophrya) have a “platyophryid” pattern with an interkinetal median subdividing the more or less rectangular meshes (Bourland et al.

B. gemmea. Black arrowheads mark pentagonal adoral organelles in preoral suture. The distal file is often shortened by one basal body. Vestibular kineties are marked by white arrow. White and black asterisks mark the right and left oral ciliary fields respectively. (71) Ventral view of B. gemmea adoral organelles (black arrowheads) in the preoral suture. Although varying in size, all have the same pentagonal shape. (72) Ventral view of B. gemmea. Black arrowheads and vestibular kineties mark adoral organelles by white arrows. White and black asterisks mark the right and left oral ciliary fields respectively. (73) Ventral view of P. dibryophryis. Black arrowheads mark irregularly polygonal adoral organelles in preoral suture. (74) Ventral view of P. dibryophryis showing fine structure of irregularly polygonal adoral organelles. (75) Right posterior view of P. dibryophryis showing adoral organelles comprising the left oral ciliary field (black arrowhead). Scale bars: 25 ␮m (70–75), 10 ␮m (66–69).

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Fig. 77. Phylogenetic network computed from the 18S rDNA sequences using the NeighborNet algorithm and the uncorrected P-genetic distances. Numbers along edges are bootstrap support values coming from 1000 replicates. Values ≤50% are not shown and some values >50% are also omitted due to spatial constraints (for details, see Phylogenetic networks in the ‘Results’ section).

2011; Foissner 1993). The silverline pattern of Puytoraciella is unknown. Among the Bryophryidae, Bryophryoides is the only genus with a non-platyophryid pattern, moreover the kreyellid silverline pattern of Bryophryoides is unique among all of the Colpodida. The more widely and regularly meshed zones to the right of somatic kineties, seen in Kreyella, are lacking in Bryophryoides (Foissner 1993). Although the silverlines of Bryophryoides ocellatus appear, at first sight, to correspond with junctions of dilated cortical alveoli, this cannot be the primary determinant of the pattern since some individuals of Bryophrya gemmea have large alveoli with a cortical texture indistinguishable from that of B. ocellatus yet their silverline pattern is exclusively platyophryid (Figs 54, 55; Bourland et al. 2011, Fig. 39). Thus cortical and subcortical structures other than alveolar junctions must contribute to the silverline pattern in bryophryids, consistent with observations in other species (Foissner and Simonsberger 1975). Collection of bryophryids from terrestrial habitats. The bryophryids inhabit terrestrial or semi-terrestrial biotopes. Foissner (1993) and Foissner et al. (2002) noted the rarity of of Notoxoma and Parabryophrya in non-flooded Petri dish preparations and did not find Bryophrya or Puytoraciella in over 1000 worldwide soil samples. We have found abundant populations of Puytoraciella dibryophryis and Bryophrya gemmea in ephemeral puddles on cultivated grass lawns

(Bourland 2008; Bourland et al. 2011). However, we have been unable to recover P. dibryophryis or B. gemmea from rewetted non-flooded Petri dish cultures of soils collected from the same sites and dried in the usual manner (WB own observ.). Attempts by others to culture bryophryids have also failed (Grain et al. 1979; Foissner 1993). We hypothesize that bryophryid resting cysts may be less desiccation resistant than their hardier, more ubiquitous colpodid cousins. The usual ≥30 day drying of non-flooded Petri dish soil samples could result in an intolerable degree of desiccation not occurring in the actual habitat of most bryophryids. Parabryophrya etoschensis, recovered from very dry soil, may represent an exception (Foissner et al. 2002). The food-selectivity of the bryophryids is another possible impediment to successful culture. All bryophryids are cyanobacterivorous (Bourland et al. 2011; Foissner 1993; Foissner et al. 2002). Bourland et al. (2011) overlooked the very pale pigmentation of filamentous bacteria uniformly packed in the food vacuoles of some Bryophrya gemmea identical to those in B. ocellatus (Figs 1, 18, 20, 21; Bourland et al. 2011, Fig. 40). This characteristic orderly packing of bacteria in the food vacuoles is also found in Notoxoma and Parabryophrya and, although described as “colorless”, the bacterial filaments in these species may be the same lightly pigmented cyanobacteria comprising the exclusive food of B. ocellatus (Foissner 1993; Foissner et al. 2002). Growth of these cyanobacteria in

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non-flooded Petri dish cultures may be insufficient to support populations of the relatively fastidious bryophryids even if viable cysts are present. Large Nostocaceae, Oscillatoriaceae and diatoms are commonly ingested by Puytoraciella and Bryophrya also ingests large cyanabacteria and green algae that are notably absent from the food vacuoles of B. ocellatus, Notoxoma and Parabryophrya (Bourland 2008; Bourland et al. 2011; Foissner 1993; Foissner et al. 2002). Directly aspirated (i.e. in situ) soil percolates contain the active interstitial soil ciliates as well as encysted forms and may improve recovery of hard-to-culture terrestrial taxa such as the bryophryids. We plan to test this hypothesis in future studies comparing species composition in samples obtained by direct soil percolate aspiration, traditional non-flooded Petri dish soil cultures and samples of ephemeral puddles at the same sites. Phylogenetic relationships of the early-branching colpodids. Despite increased taxon sampling and use of multi-gene alignments, the resolution of relationships within the order Colpodida remains an elusive goal (Bourland et al. 2011; Dunthorn et al. 2008; Foissner et al. 2011). Among the early branching taxa, two families comprise a single genus and species each (Bardeliellidae and Sandmanniellidae) and most genera are either monotypic (e.g. Puytoraciella and Bryophryoides) or consist of fewer than five species (Foissner 1993; Foissner et al. 2002). Further discovery of new taxa belonging to the early-branching groups is likely and, together with more detailed morphologic and molecular study of poorly-known species, should allow improved phylogenetic resolution in this region of the colpodid tree. In accordance with phylogenetic trees, the present network analyses also support an early branching position of the Bardeliellidae and Ilsiellidae within the order Colpodida. Further, they show the Bryophryidae branch off earlier than the “core” colpodids (Fig. 77). This corroborates the evolutionary scenario for the formation of oral ciliature in the Colpodida proposed by Bourland et al. (2011). The monophyly of the suborder Bryophryina (Sandmanniellidae + Bryophryidae) as suggested by Foissner et al. (2011) is rejected by the AU test, but monophyly of the Sandmanniellidae and the Marynidae could not be rejected (Table 3). Although statistical analysis of our molecular data rejects the non-monophyly of the Bryophryidae (Notoxoma + Bryophrya + Bryophryoides), caution is warranted in interpreting test of topologic constraints involving small numbers of taxa (Rosenberg 2007).

Taxonomic Summary Class Colpodea, Small and Lynn (1981) Order Colpodida, Puytorac et al. (1974) Emended diagnosis. Very small to large (∼10–600 ␮m), oblong, ellipsoidal, or reniform Colpodea with oral apparatus subapical, in mid-body, or in posterior body end. Oral cavity small or large, in some groups absent. Right oral ciliary field a single row of monokinetids, dikinetids, or a

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complex organelle including roof kineties and/or monokinetidal ciliary fields. Left oral ciliary field composed of one to several brick-shaped polykinetids and/or a comparatively large polykinetid comprising few to many rows of monokinetids. Macronucleus and micronucleus each with a separate membrane. Silverline pattern colpodid, platyophryid or kreyellid. Usually divide in reproductive cysts, very rarely in freely motile condition. Stomatogenesis merotelokinetal or in a pleuromerotelokinetal mode, parental ciliature usually reorganized. Without sex. Most terrestrial, some limnetic. Family Bryophryidae, Puytorac et al. (1979) Emended diagnosis. Small to large Colpodida with distinctly subapical oral apparatus. Paroral formation (right oral ciliary field) composed of single row of dikinetids or of many regularly spaced longitudinal kineties extending over right slope of buccal cavity and sometimes curving onto its left wall. Left oral ciliary field composed of one to many organelles. Adoral organelles of highly variable morphology, usually extending into preoral suture. Silverline pattern platyophryid or kreyellid. Macronucleus and micronucleus each with separate nuclear membrane. Division in reproductive cysts. Usually inhabiting terrestrial or semi-terrestrial biotopes. Bryophryoides n. g. Diagnosis. Medium size Bryophryidae with polymorphic adoral organelles in preoral suture, Vestibulum absent. Kreyellid silverline pattern. Type species. Bryophryoides ocellatus n. sp. Etymology. Bryophryoides is a composite of the stem of the generic name Bryophrya, the thematic vowel “·o” and the Latin suffix “-oides” (like, resembling) denoting the general similarity of this new genus to Bryophrya. Masculine gender according to the Article 30.1.4.4 of the ICZN (1999). Species included. Bryophryoides ocellatus n. sp. Diagnosis. Size about 80 ␮m × 40 ␮m in vivo; shape broadly reniform to elliptical. Average of 43 left-spiraling somatic kineties. Several interrupted right anterior somatic kineties. Short preoral suture with average of 8 ocellate and bipartite adoral organelles not extending to dorsal surface. Type locality. Soil from Fort Boise Park, Boise, Idaho, U.S.A., 43◦ 37 0.47 N, 116◦ 11 20.56 W. Type material. A slide with a permanent silver carbonate impregnated specimen, fixed as the holotype and 12 paratype slides (six protargol impregnated, five dry silver nitrate preparations and one permanent silver carbonate preparation) are deposited in the Biologiezentrum of the Oberösterreichische Landesmuseum in Linz (LI), Austria, designated as “Bryophryoides ocellatus n. g., n. sp.” (collection accession numbers not yet assigned). Relevant specimens are marked with black ink circles on the cover glass. Remarks. Like many colpodeans, details of the ciliature of B. ocellatus are best demonstrated in well-compressed, temporary silver carbonate-impregnated cells (Figs 3, 4, 24–27). Permanent silver carbonate preparations are often of poorer quality (Augustin and Foissner 1984). This is also the case

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for the B. ocellatus holotype specimen, however, the main diagnostic features (except, of course the silverline pattern) can still be seen. The silverline pattern is seen only in the silver nitrate-impregnated paratype slides. Gene sequence. The 18S-rDNA sequence, obtained from a single specimen (accession number KF569684) has been deposited in GenBank (http://www.ncbi.nlm.nih.gov/nucleotide/). Etymology. From the Latin adjective ocellat·us, -a, -um [m, f, n] meaning “having little eyes” in reference to the distinctive eye-like adoral organelles.

Acknowledgements The senior author thanks Prof. Dr. Wilhelm Foissner and Mag. Barbara Harl for their kind patience in teaching him scanning electron microscopy techniques at the University of Salzburg. This work was partially supported by startup funds from the Boise State University Department of Biological Sciences and the Slovak Scientific Grant Agency (VEGA Projects 1/0600/11 and 1/0248/13) and the DoE (National Nuclear Security Administration) under award number DENE0000338. We also thank the anonymous reviewers for valuable insights that helped improve the quality of the manuscript.

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