A New Subspecies of Oxytricha Granulifera (Hypotrichia: Oxytrichidae) from Mexico, with Notes on Its Morphogenesis and Phylogenetic Position

Journal of Eukaryotic Microbiology ISSN 1066-5234

ORIGINAL ARTICLE A New Subspecies of Oxytricha granulifera (Hypotrichia: Oxytrichidae) from Mexico, with Notes on its Morphogenesis and Phylogenetic Position

Daniel Mendez-S anchez a , Rosaura Mayen-Estrada a, Xiaotian Luob & Xiaozhong Hub a Laboratorio de Protozoologıa, Facultad de Ciencias, Universidad Nacional Autonoma de Mexico, Circuito Ext. s/num. Ciudad Universitaria, Av. Universidad 3000, Coyoacan, 04510 Ciudad de Mexico, Mexico b Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

Keywords ABSTRACT Cell division; Chiapas; dorsal kineties; Oxytri- cha granulifera chiapasensis; SSU rRNA; The genus Oxytricha Bory de Saint-Vincent in Lamouroux, Bory de Saint-Vin- undulating membranes. cent and Deslongchamps, 1824 comprises about 38 species distributed worldwide and has been considered to be a nonmonophyletic group. Based on Correspondence living observations, protargol preparations, and a small subunit ribosomal RNA D. Mendez-S anchez, Laboratorio de Proto- (SSU rRNA) gene sequence, we describe a new subspecies Oxytricha granulif- zoologıa, Facultad de Ciencias, Universidad era chiapasensis n. subsp. This new taxon is morphologically characterized by Nacional Autonoma de Mexico, Circuito Ext. undulating membranes basically in a Stylonychia-pattern, six dorsal kineties, s/num., Av. Universidad 3000 Ciudad size in vivo ca. 60–120 9 20–40 lm, 21–30 right and 21–31 left marginal cirri, Universitaria, C. P. 04510, Coyoacan, Ciudad 22–29 adoral membranelles, and spherical cortical granules arranged in longitu- de Mexico, Mexico dinal rows on the dorsal side. In terms of the SSU rRNA gene sequence, the Telephone number: +(525)56224924; new subspecies differs from populations of O. granulifera from GENBANK by FAX number: +(525)56224828; 7–35 nucleotides. Phylogenetic analyses showed that Oxytricha granulifera e-mail: [email protected] gene sequences were nested into three groups, with the new subspecies included in one of them. Oxytricha granulifera chiapasensis n. subsp. is differ- Received: 31 March 2017; revised 2 ent from Oxytricha granulifera granulifera Foissner and Adam, 1983 and Oxytri- October 2017; accepted October 6, 2017. cha granulifera quadricirrata Blatterer and Foissner, 1988 based on: Early View publication November 10, 2017 (i) undulating membranes in Stylonychia-pattern, (ii) formation of a sixth dorsal kinety during morphogenesis, (iii) the adoral membranelles number, and doi:10.1111/jeu.12479 (iv) inhabiting freshwater habitats.

OXYTRICHIDS are a species-rich group of ciliates with marine environments, and also soil, mosses, decaying high morphological and molecular variation (Foissner 2016; wood mass, and water of bromeliad tanks worldwide (Ber- Hu et al. 2011). Ciliates belonging to the genus Oxytricha ger 1999; Berger and Foissner 1989; Duran-Ram ırez et al. Bory de Saint-Vincent in Lamouroux, Bory de Saint-Vincent 2015; Foissner 1997, 1999, 2016; Kwon and Shin 2004, and Deslongchamps, 1824, have a flexible body, and are 2008, 2013; Tirjakova and Bartosov a 2004; Weisse et al. characterized by an adoral zone of membranelles resem- 2013). To our knowledge, only five species, i.e. Oxytricha bling a question mark, undulating membranes arranged in discifera (Aladro-Lubel et al. 2006), O. fallax (Aladro-Lubel an Oxytricha-pattern (slightly curved and intersected spa- et al. 2006; Lugo-Vazquez et al. 1991; Moreno-Baron and tially), 18 frontal–ventral–transverse cirri (with frontoventral Lopez-Ochoterena 1976; Rico-Ferrat and Lopez-Ochote- cirri in a V-shape pattern), one right and one left row of rena 1976; Sokoloff and Ancona 1937), O. hymenostoma marginal cirri, usually five or six dorsal kineties in Oxytri- (Sokoloff 1936), O. opisthomuscorum (Duran-Ram ırez cha-pattern, and caudal cirri present (Berger 1999; Shao et al. 2015), and Oxytricha sp. (Aladro-Lubel et al. 2006; et al. 2011, 2015). Duran-Ram ırez et al. 2015; Lugo-Vazquez et al. 1998) have Within this family, Oxytricha is a species-rich genus, been recorded in Mexico. and, so far, comprises around 38 nominal species (Berger The genus Oxytricha has been considered to be a non- 1999; Foissner 1999, 2016; Shao et al. 2011, 2014; monophyletic group (Hu et al. 2011; Paiva et al. 2009; Weisse et al. 2013). They inhabit freshwater, brackish and Schmidt et al. 2007; Shao et al. 2014) by using

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists Journal of Eukaryotic Microbiology 2018, 65, 357–371 357 New Mexican Subspecies of Oxytricha granulifera Mendez-S anchez et al. phylogenetic analyses based on the SSU rRNA gene and Blood and Tissue kit (Qiagen, Hilden, Germany). For PCR other molecular markers; however, the incongruence amplification, two universal primers were used: Euk A (50- between morphology and molecular information suggests AACCTGGTTGATCCTGCCAGT-30) and Euk B (50- that Oxytricha is a genus with high genetic divergence TGATCCTTCTGCAGGTTCACCTAC-30) (Medlin et al. 1988). (Foissner 2016) and, thus, has been considered to be an The cycles were started at 98 °C for 30 s, followed by 35 artificial genus (Berger 1999; Hu et al. 2011). cycles of 10 s each at 94 °C, 30 s at 48 °C, and 1 min Oxytricha granulifera Foissner and Adam, 1983, type- and 30 s at 65 °C, and finally 1 cycle for 5 min at 65 °C. species of the genus (Berger and Foissner 1997; Foissner The PCR product was sequenced directly using primers 1989), has been well-characterized both morphologically Euk A, Euk B, and three internal primers (Wang et al. and morphogenetically (Foissner and Adam 1983). This 2016). species is more often found in terrestrial environments (Berger 1999), but has also been isolated from freshwater Phylogenetic analysis environments (Kwon and Shin 2013). Geographically, O. granulifera has been recorded in the Neotropical, The SSU rRNA gene sequence obtained was aligned with Palearctic, Ethiopian, and Australian biogeographical 67 hypotrichid sequences available from GENBANK by regions (Berger 1999; Foissner 1997, 1999, 2016; Foissner using the MUSCLE algorithm (Edgar 2004) on the GUI- and Adam 1983; Foissner et al. 2002, 2005, 2008; Kwon DANCE web server. Ambiguous columns with a confi- and Shin 2013; Shao et al. 2014; Tirjakova and Bartosov a dence score below 0.93 were removed (Penn et al. 2010), 2004). and the alignment was then manually edited in MEGA 7.0 The goal of this study was to describe the morphology (Kumar et al. 2016) to remove ambiguous gaps (both pri- of a new subspecies, Oxytricha granulifera chiapasensis n. mers were cut off). The resulting alignment was 1,718 subsp., which is first documented from Montebello, a characters. For phylogenetic analysis, the GTR + I + G Mexican lake, based on its morphological features, mor- model was used according to Weisse et al. (2013) and phogenetic pattern, and its unique SSU rRNA gene Shao et al. (2014). Maximum likelihood (ML) analysis was sequence. carried out using RAxML-HPC2 on XSEDE v. 8.2.9 (Sta- matakis 2014) with 1,000 bootstraps replication. Bayesian Inference (BI) was performed with MrBayes 3.2 (Ronquist MATERIALS AND METHODS et al. 2012), with four chains of 10,000,000 generations, with trees sampled every 100 generations, of which the Sampling, morphology and morphogenesis first 25,000 generations were discarded as burn-in. Both Freshwater samples were collected in June of 2015 in the ML and BI were performed via the CIPRES Science Gate- littoral zone of Montebello Lake, in Tziscao Ecoturistic way website. Novistrombidium orientale FJ422988, National Park within the southern portion of Chiapas, Mex- Stombidium apolatum DQ662848, Strombidinopsis acumi- ico (16°6018.22″N, 91°4204.79″W) (Fig. S1). The lake is sur- nate FJ790209, and Parastrombidinopsis minima rounded by a pine-oak forest at 1,500 masl. Specimens DQ393786 were selected as the outgroup taxa. To visua- were isolated and maintained as nonclonal cultures (pure lize tree topologies, MEGA 7.0 was used (Kumar et al. cultures) in Petri dishes at room temperature using mi- 2016). The gene sequence obtained was aligned with the neral water with nonsterile rice grains to enrich bacteria Oxytricha granulifera gene sequences used for the phylo- as food resource. Living observations were made with genetic tree, the alignment was manually edited in MEGA bright field and differential interference contrast (DIC) 7.0 and both primers were cut off, in order to generate microscopy. Cells were stained with in-house synthesized the similarity structural matrix (pairwise analysis) and the protargol (Pan et al. 2013), following the protocol of Wil- number of nucleotides differences (absolute distance bert (1975) to reveal the infraciliature and nuclear appara- matrix) using MEGA 7.0 (Kumar et al. 2016). tus. We observed several well-impregnated dividers in different stages to partially characterize the morpho- RESULTS genetic pattern. Measurements and counts of stained cells were carried Morphological description of Oxytricha granulifera out with an ocular micrometer at 1,000X magnification, chiapasensis n. subsp including stages of morphogenesis. Drawings are based on photomicrographs. Terminology and systematic classifi- Morphological description cation are according to Berger (1999, 2006), Shao et al. Live cells about 60–120 9 20–40 lm, with an adoral zone (2011, 2015), and Lynn (2008). of membranelles occupying around 30% of the cell length. Cell shape ellipsoidal with left border slightly convex (Fig. 1A, 2A–C), and body soft and flexible (Fig. 2C); cyto- DNA extraction and PCR amplification plasm colorless (Fig. 2A), highly vacuolated and with ye- Several cells were isolated from nonclonal cultures that llowish crystals bars (Fig. 2B, D). Spherical cortical were used for morphological studies (pure cultures), granules, < 1 lm in diameter, arranged in longitudinal washed several times with distilled water and transferred rows on dorsal side (Fig. 1E, 2E) and irregularly arranged to microfuge tubes. DNA was extracted with DNeasy on ventral side. Contractile vacuole spherical (about 10 lm

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists 358 Journal of Eukaryotic Microbiology 2018, 65, 357–371 Mendez-S anchez et al. New Mexican Subspecies of Oxytricha granulifera

Figure 1 Schematic drawings of Oxytricha granulifera chiapasensis n. subsp. from life (A) and after protargol impregnation (B–E). (A) Ventral view. (B–D) Paratype specimen. Ciliature in ventral view (B), arrange of frontoventral cirri and undulating membranes, arrow marks the rightmost frontal cirrus (C), dorsal view showing dorsal kineties (1–6) and nuclear apparatus (D). (E) Dorsal view showing pattern and shape of cortical granules from a paratype not well-impregnated specimen. AZM = adoral zone of membranelles; BC = buccal cirrus; CC = caudal cirri; E = endoral membrane; FC = frontal cirri; LMC = left marginal cirri row; Ma = macronucleus; Mi = micronucleus; P = paroral membrane; PTVC = pretransverse ventral cirri; PVC = postoral ventral cirri; RMC = right marginal cirri row; TC = transverse cirri. Scale bar = 25 lm.

in diameter) and located at mid-body near left margin of dikinetids; kinety 3 terminating at 80% of body length and cell, diastole time 10–13 s (Fig. 1A, 2B, F), collecting comprising 8–18 (on average 15, n = 23) dikinetids. Kinety canals were not observed, however, they could be over- 4 starting behind mid-body and comprising three or four looked. Locomotion by fast crawling on the substrate. dikinetids. Kinety 5 beginning at anterior region and ending Two macronuclear nodules with one micronucleus adja- at half of body with 6–10 dikinetids, and kinety 6 with 1–3 cent to each nodule (Fig. 1A, D, 2D, 3A, C, D). (usually two) dikinetids (Fig. 1D, 3D, E); bristles about 3- Adoral zone composed of 22–29 membranelles lm long. One caudal cirrus each at posterior end of kin- (Fig. 1A–C, 3A, B). Paroral and endoral membranes eties 1, 2, and 4 (Fig. 1B, D, 3D, G and Table 1). arranged in Stylonychia-pattern (Fig. 1B, C, 3A, B, F), sometimes crossed at proximal region. Invariably 18 Notes on morphogenesis frontal–ventral–transverse cirri (Fig. 1B, 3A). Three frontal Stomatogenesis begins with the formation of the oral pri- cirri, ca. 10-lm long in live specimens, one buccal cirrus at mordium, which occurs as an elongated dense ﬁeld of basal anterior end of endoral, and four frontoventral cirri bodies below the parental adoral zone of membranelles to (Fig. 1B, C, 3A, B). Cirrus III/2 to left of level of cirri VI/3 the anteriormost transverse cirri, between left marginal cirri and VI/4; cirrus IV/3 located at level of rear portion of the and postoral ventral cirri (Fig. 4A, 5A). At this stage, the oral endoral membrane (Fig. 1C, 3B). Three postoral ventral apparatus, somatic ciliature, and nuclear apparatus remain cirri distinctly separated from two pretransverse ventral intact. At an early middle state (Fig. 4B, 5B), with the prolif- cirri; anterior pretransverse ventral cirrus distinctly sepa- eration of basal bodies, the oral primordium begins to rated from posterior one (Fig. 1B, 3A). Five transverse cirri develop and differentiate into a new adoral zone of mem- forming a “check mark” (Fig. 1B, 3A), with cilia ca. 16-lm branelles. Meanwhile, the frontal–ventral–transverse cirral long in vivo. One left and one right marginal cirri row anlagen and undulating membranes anlagen are generated slightly separated posteriorly, composed of 21–31 and 21– as six streaks (I–VI anlagen) (Fig. 4B). In the proter, the indi- 30 cirri, respectively; cilia in vivo ca. 10-lm long (Fig. 1B, vidual anlagen I is formed from undulating membranes 3A), right marginal cirri row begins slightly behind level of (Fig. 4B, 5B), while in the opisthe, the new adoral zone of rightmost frontal cirrus (Fig. 1B, C, 3A, B). membranelles is almost completed. At the same time, the Six dorsal kineties, of which the two rightmost are dor- marginal cirri rows anlagen are formed within the parental somarginal kineties. Kinety 1 and 2 bipolar with ca. 20 marginal cirri rows in both proter and opisthe (Fig. 4B, 5B).

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists Journal of Eukaryotic Microbiology 2018, 65, 357–371 359 New Mexican Subspecies of Oxytricha granulifera Mendez-S anchez et al.

Figure 2 Oxytricha granulifera chiapasensis n. subsp. from life. (A) Ventral view showing the body shape. (B) Ventral view showing several yel- lowish crystal-bars, contractile vacuole (arrowhead) and adoral zone (arrow). (C) Ventral view showing two marginal cirri rows (arrowheads) and transverse cirri (arrow). (D) Dorsal view showing two macronuclear nodules (arrows), and lipid vacuoles (arrowheads). (E) Dorsal view showing longitudinal rows of cortical granules (arrows). (F) Contractile vacuole. Scale bars = 20 lm.

Figure 3 Infraciliature of Oxytricha granulifera chiapasensis n. subsp. after protargol impregnation. (A) Holotype specimen in ventral view showing 18 frontal–ventral–transverse cirri and one right and one left marginal cirri rows, postoral ventral cirri (arrows), and transversal cirri (encircled). (B) Holotype specimen showing the disposition of frontoventral cirri, frontal cirri (encircled), and undulating membranes (arrowheads), the arrow shows the rightmost frontal cirrus which is not at the same level of fronto-terminal cirrus. (C) Holotype specimen in dorsal view showing two macronuclear nodules with one micronucleus (arrows) attached to each one. (D, E) Holotype (D) and paratype (E) in dorsal view showing the infraciliature, dikinetids of each kinety connected by dotted lines (E). (F) Ventral view showing buccal cirrus (arrowhead), and paroral and endoral membranes. (G) Paratype specimen in dorsal view showing three caudal cirri. AZM = adoral zone of membranelles; BC = buccal cirrus; CC = caudal cirri; E = endoral membrane; FTC = fronto-terminal cirrus; LMC = left marginal cirri row; Ma = macronucleus; P = paroral membrane; RMC = right marginal cirri row. Scale bar = 50 lm.

In middle stages (Fig. 4C, D, 5C), in both proter and new cirri and the marginal cirri rows still lengthen. Paroral opisthe, all the frontal–ventral–transverse cirral anlagen (II– and endoral membranes occur from anlage I. At this time, VI) lengthen to long streaks and begin to differentiate into in opisthe, the adoral zone of membranelles is completed.

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists 360 Journal of Eukaryotic Microbiology 2018, 65, 357–371 Mendez-S anchez et al. New Mexican Subspecies of Oxytricha granulifera

Table 1. Morphometric data of Oxytricha granulifera chiapasensis n. subsp.

Mean M SD CV Min Max N

Body length 100.9 100.1 13.3 13.3 79.2 128.7 27 Body width 45.1 40.7 11.2 27.5 30.8 78.0 27 Length/width body proportion 2.3 2.3 0.3 16.0 1.2 2.9 27 Adoral zone of membranelles length 34.0 33.0 4.2 12.9 25.3 43.5 27 AZM/BODY proportion 2.9 2.9 0.3 11.6 2.3 3.5 26 AE to proximal end of endoral, distance 8.8 8.8 1.9 22.4 5.5 12.0 27 AE to proximal end of paroral, distance 13.0 12.1 2.3 19.1 8.8 17.6 27 Endoral length 22.9 23.1 2.2 9.9 18.7 30.0 27 Paroral length 18.5 18.7 1.9 10.6 15.4 27.0 27 AE to buccal cirrus, distance 11.1 11.0 1.8 16.4 7.7 14.3 27 AE to cirrus VI/4, distance 16.2 15.4 2.1 14.0 12.1 21.0 27 AE to cirrus III/2, distance 16.7 16.5 2.1 13.2 12.1 25.0 27 AE to cirrus VI/3, distance 20.6 20.9 2.5 12.2 15.4 30.0 27 AE to cirrus IV/3, distance 28.3 28.6 3.6 12.7 22.0 36.0 27 AE to anterior postoral ventral cirrus, distance 34.9 35.6 5.4 15.3 17.5 44.0 26 AE to rearmost postoral ventral cirrus, distance 47.3 46.2 4.7 10.3 38.5 59.4 25 AE to left marginal row, distance 29.2 28.6 3.2 11.4 22.0 37.5 27 AE to right marginal row, distance 10.9 9.9 2.2 23.0 7.7 15.4 27 PE to anteriormost PTVC, distance 20.6 19.8 3.4 17.1 15.4 28.6 27 PE to rearmost transverse cirrus, distance 6.7 5.5 3.3 61.0 3.0 16.5 27 PE to anteriormost transverse cirrus, distance 13.4 12.1 3.6 30.0 5.5 20.9 27 Left CC to middle caudal cirrus, distance 4.2 4.4 1.1 26.8 2.2 7.7 27 Middle CC to right caudal cirrus, distance 4.7 4.4 1.6 36.9 2.2 8.8 27 AE to anterior macronuclear nodule, distance 26.6 26.4 3.0 11.4 19.8 36.0 27 Anterior macronuclear nodule length 19.4 19.8 2.9 14.9 13.2 25.3 27 Anterior macronuclear nodule width 10.2 9.9 2.2 22.9 6.6 17.7 27 Posterior macronuclear nodule length 23.5 22.0 5.5 25.3 16.5 38.5 27 Posterior macronuclear nodule width 9.81 9.9 1.9 19.6 5.5 14.3 27 Macronuclear nodules distance in between 10.1 9.9 3.6 36.8 3.3 18.7 27 Anterior micronucleus diameter 2.8 3.3 0.5 15.7 2.2 3.3 22 Posterior micronucleus diameter 2.8 3.0 0.7 23.8 2.2 4.4 25 Macronuclear nodules, number 2.0 2.0 0.0 0.0 2.0 2.0 27 Micronucleus, number 1.8 2.0 0.2 14.4 1.0 2.0 25 Adoral membranelles, number 26.2 26.0 1.9 7.4 22.0 29.0 27 Frontal cirri, number 3.0 3.0 0.0 0.0 3.0 3.0 27 Buccal cirri, number 1.0 1.0 0.0 0.0 1.0 1.0 27 Frontoventral cirri, number 4.0 4.0 0.0 0.0 4.0 4.0 27 Postoral ventral cirri, number 3.0 3.0 0.4 13.6 3.0 5.0 26 Pretransverse ventral cirri, number 2.0 2.0 0.4 20.0 2.0 4.0 27 Transversal cirri, number 5.0 5.0 0.0 0.0 5.0 5.0 27 Left marginal cirri, number 25.3 25.0 2.5 10.1 21.0 31.0 27 Right marginal cirri, number 25.1 25.0 1.9 7.8 21.0 30.0 27 Dorsal kineties total, number 5.6 6.0 0.5 8.3 5.0 6.0 20 Dikinetids on dorsal kinety 1, number 15.5 17.0 3.3 19.8 8.0 20.0 27 Dikinetids on dorsal kinety 2, number 15.6 16.0 2.2 14.3 12.0 21.0 25 Dikinetids on dorsal kinety 3, number 13.5 14.0 2.8 20.4 7.0 18.0 23 Dikinetids on dorsal kinety 4, number 3.7 4.0 0.4 11.3 1.0 4.0 13 Dikinetids on dorsal kinety 5, number 7.8 8.0 1.0 13.0 6.0 10.0 26 Dikinetids on dorsal kinety 6, number 1.9 2.0 0.4 22.3 1.0 3.0 22 Total bristles, number 62.5 64.0 3.9 6.1 56.0 68.0 9 Caudal cirri, number 3.0 3.0 0.0 0.0 3.0 3.0 27

AE = anterior end of cell; AZM = adoral zone of membranelles; CC = caudal cirri; PE = posterior end of cell; PTVC = pretransverse ventral cirri. Measurements in lm. All data are based on protargol-prepared specimens, including holotype. CV = Coefﬁcient of variation; M = Median; Max = Maximum; Min = Minimum; N = Number of individuals; SD = Standard deviation.

The two macronuclear nodules are fused into a single kineties and dorsomarginal kineties. Dorsal kineties 1, 2, mass. From middle to late stages, the dorsal kineties are and 3 anlagen develop within each parental kinety (dorsal formed from two groups in both proter and opisthe: dorsal kineties 1, 2, and 3) by intrakinetal proliferation of basal

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists Journal of Eukaryotic Microbiology 2018, 65, 357–371 361 New Mexican Subspecies of Oxytricha granulifera Mendez-S anchez et al.

Figure 4 Schematic drawings of morphogenetic pattern of Oxytricha granulifera chiapasensis n. subsp. after protargol impregnation. Early stage (A), early middle stage (B), middle stage (C, D), late stages (E, F), very late stages (G–L). (A) Ventral view of an early divider showing oral primordium. (B) Ventral view of an early middle divider showing anlagen I–VI (six streaks), marginal cirri row anlagen, and a new adoral zone. (C, D) Left lateral view of a middle divider showing a single fused macronuclear mass, and elongation of marginal cirri row anlagen and dorsal kineties anlagen. (E, F) Ventral (E) and dorsal (F) view of the same late divider, showing 18 frontal–ventral–transverse cirri and undulating membranes in proter and opisthe, and elongation of dorsal kineties anlagen. (G, H) Ventral (G) and dorsal (H) view of the same very late divider showing a new set of cirri in proter and opisthe (G); and dorsal kinety 3 fragmentation (arrows), and new caudal cirri (H). (I, J) Ventral (I) and dorsal (J) view of same very late divider showing dorsomarginal kinety 6 anlage (arrows) (I); kinety 3 fragmentation (arrows) (J). (K, L) Ventral (K) and dorsal (L) view of the same very late divider, proter and opisthe with a new set of 18 cirri, undulating membranes in Stylonychia-pattern, and six dorsal kineties. aDK = dorsal kinety anlage; AZM = adoral zone of membranelles; DK1, 2, 3 = dorsal kinety 1, 2, 3; LMA = left marginal cirri row anlage; Ma = macronucleus; Mi = micronucleus; nAZM = new adoral zone of membranelles; nD5, 6 = new dorsomarginal kinety 5, 6; oD5, 6 = old dorsomarginal kinety 5, 6; OP = oral primordium; oAZM = old adoral zone of membranelles; oDK4 = old dorsal kinety 4; nDK 4 = new dorsal kinety 4; PTVC = pretransverse ventral cirri; PVC = postoral ventral cirri; RMA = right marginal cirri row anlage; TC = transverse cirri.

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists 362 Journal of Eukaryotic Microbiology 2018, 65, 357–371 Mendez-S anchez et al. New Mexican Subspecies of Oxytricha granulifera

Figure 5 Morphogenetic pattern of Oxytricha granulifera chiapasensis n. subsp. after protargol impregnation. Early stage (A), early middle stage (B), middle stage (C), late stages (D, E), very late stages (F–L). (A) Ventral view of an early divider showing oral primordium (arrow). (B) Ventral view of an early middle divider showing six streaks (anlagen I–VI) in proter and opisthe (black arrowheads) and formation of marginal cirri anlagen (white arrowheads). (C) Left lateral view of a middle divider showing formation of dorsal kineties anlagen (arrows), and a completely fused macronuclear mass. (D, E) Ventral (D) and dorsal (E) views of the same late divider, showing formation of dorsomarginal kineties anlagen (arrows), and two sets of new cirri in opisthe and proter (encircled) (D) and elongation of dorsal kineties 1, 2, and 3 (arrows); the old kineties 4, 5, and 6 remain (arrowheads) (E). (F–I) Ventral (F–H,) and dorsal views (I) of the same very late divider; proter (G) and opisthe (H) showing the dorsal kinety 5 (arrows) and formation of kinety 6 (arrowheads). Dorsal kineties (black arrowheads), dorsal kinety 3 fragmentation (arrows) and caudal cirri (white arrowheads) (I). (J, K) Dorsal (J) and ventral (K) views of the same very late divider. Dorsal kineties 1, 2, 3, and 4 (J); and cirri reorganizing in frontoventral (encircled by lines), postoral ventral (encircled by continuous line), and transverse (encircled by spaced lines) (K). (L, M, N) Ventral (M, N) and dorsal (N) views of the same very late divider, showing the undulating membranes in Stylonychia-pattern in proter (L) and opisthe (M), and dorsal kinety 3 fragmentation (N). (O, P) Ventral (O) and dorsal (P) view of an opisthe. AZM = adoral zone of membranelles; DK5 = dorsal kinety 5; E = endoral membrane; Ma = macronucleus; nD5, 6 = new dorsomarginal kinety 5, 6; nDK4 = new dorsal kinety 4; nM = new membranelles; P = paroral membrane; RMC = right marginal cirri row.

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists Journal of Eukaryotic Microbiology 2018, 65, 357–371 363 New Mexican Subspecies of Oxytricha granulifera Mendez-S anchez et al. bodies (Fig. 4D, 5C). Old dorsal kineties 4, 5, and 6 remain granulifera gene sequences used and with Paraurostyla intact (Fig. 4F, 5E, arrowheads). Dorsomarginal kinety 5 viridis and Architricha indica. This clade, which is highly anlage develops de novo anteriorly to the right marginal supported (98 ML/1.0 BI), is named Oxytricha granulifera- cirri row forming dorsal kinety 5 (Fig. 4E, 5D, arrows). The CLADE, and is divided into three groups: Group I com- macronuclear mass then divides in two masses, migrating prises six O. granulifera gene sequences which cluster each one to proter and opisthe, respectively (Fig. 4D, F, with Architricha indica and Paraurostyla viridis as sister 5D–F). group. Group II includes two Oxytricha granulifera gene In very late stages, the anterior portion of the adoral sequences. Group III comprises the current Oxytricha zone of membranelles turns to the right in the opisthe, granulifera chiapasensis n. subsp. and a population of Oxy- while in the proter, the parental adoral zone remains intact tricha granulifera (KU715983), of which the morphological throughout the process (Fig. 5F, K). The paroral and endo- data were not provided. ral membranes in proter and opisthe are formed and The similarity structural matrix and absolute distance arranged in a Stylonychia-pattern, i.e. they run parallel to matrix between Oxytricha granulifera gene sequences and each other (Fig. 5L, M); the new sets of cirri organize in Oxytricha granulifera chiapasensis n. subsp. are shown in frontal, frontoventral, postoral ventral, pretransverse ven- Table 2. tral, and transverse groups (Fig. 4G, 5K encircled). A new dorsomarginal kinety anlage develops de novo to the left DISCUSSION side of dorsal kinety 5 anlage, forming dorsal kinety 6 (Fig. 4I, K, 5G, H). Dorsally, the new dorsal kinety 3 is Systematics, morphology, and comparison with fragmented (Fig. 4H, J, 5I, J) forming the new dorsal ki- congeners nety 4 (Fig. 5N, arrows), and one caudal cirrus is devel- oped from the end of new dorsal kineties 1, 2, and 4 Oxytricha granulifera was well-described by Foissner and (Fig. 5I, white arrowheads). The macronucleus of each fil- Adam (1983) and proposed as the type-species for the ial cell (proter and opisthe) divides into two macronuclear genus Oxytricha (Foissner 1989), which was then split by nodules (Fig. 4H, J, 5F, I, J). Blatterer and Foissner (1988) into two subspecies: Oxytri- Finally, all new structures, i.e. cirri, undulating mem- cha granulifera granulifera and Oxytricha granulifera quadri- branes, adoral zone of membranelles and dorsal kineties, cirrata. However, Berger (1999) considered O. g. move to their proper position, and the cytokinesis process quadricirrata to be an entirely different species. O. g. finishes with two new independent cells (Fig. 4K, L, 5O, quadricirrata possibly belongs to the family Urosomoidae P). because it has just four transverse cirri (Foissner 2016). Its morphology, though, is very similar to Oxytricha granulifera granulifera, but its morphogenesis pattern and SSU rRNA gene sequence and phylogenetic analyses molecular data are necessary to determine its correct sta- The partial SSU rRNA gene sequence obtained for Oxytri- tus. Thus, until such data are available we consider cha granulifera chiapasensis n. subsp. (GENBANK acce- O. quadricirrata to still be a subspecies of Oxytricha ssion number KX889988) has a length of 1,770 bp and a granulifera. G + C content of 45.2%. We compared our form with 23 oxytrichids-species (in- The ML and BI trees are similar, and, therefore, only cluding populations of O. granulifera and related species), the ML tree is shown (Fig. 5). Oxytricha granulifera chia- which resemble O. granulifera chiapasensis n. subsp. pasensis n. subsp. branches together with nine Oxytricha mainly due to the presence of cortical granules, five or six

Table 2. Absolute distance (above the diagonal) and pairwise distance (below the diagonal) between several Oxytricha granulifera gene sequence and Oxytricha granulifera chiapasensis n. subsp. (in bold)

SSU rRNA gene sequences X53486 KJ081199 AF164122 AM412772 AM412768 AM412769 AM412771 AM412770 JX899421 KU715983 KX889988

X53486 – 40 42 39 39 39 39 40 33 42 35 KJ081199 0.026 – 411111613158 AF164122 0.027 0.003 – 333318151912 AM412772 0.025 0.001 0.002 – 0001512169 AM412768 0.025 0.001 0.002 0.000 – 0 0 15 12 16 9 AM412769 0.025 0.001 0.002 0.000 0.000 – 01512169 AM412771 0.025 0.001 0.002 0.000 0.000 0.000 – 15 12 16 9 AM412770 0.026 0.010 0.011 0.010 0.010 0.010 0.010 – 72518 JX899421 0.021 0.008 0.010 0.008 0.008 0.008 0.008 0.004 – 18 11 KU715983 0.027 0.010 0.012 0.010 0.010 0.010 0.010 0.016 0.011 – 7 KX889988 0.023 0.005 0.008 0.006 0.006 0.006 0.006 0.011 0.007 0.004 –

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists 364 Journal of Eukaryotic Microbiology 2018, 65, 357–371 Mendez-S anchez et al. New Mexican Subspecies of Oxytricha granulifera dorsal kineties, undulating membranes in an Oxytricha-pat- granulifera (Schmidt et al. 2007), indicating that this spe- tern and a Stylonychia-pattern, infraciliature in a typical cies could not be a population of Oxytricha granulifera chi- Oxytricha-pattern, nuclear apparatus, and the size and apasensis n. subsp. body shape (Berger 1999; Berger and Foissner 1987, Oxytricha granulifera chiapasensis n. subsp. also differs 1989; Foissner 2016). to Oxytricha granulifera quadricirrata Blatterer and Foiss- Oxytricha granulifera chiapasensis n. subsp. is highly ner, 1988 (before Oxytricha quadricirrata in Berger 1999) similar to the nominotypical subspecies O. granulifera based on: (i) invariably, five transverse cirri vs. four trans- granulifera Foissner and Adam 1983, but differs based on verse cirri, (ii) undulating membranes in a Stylonychia-pa- the following characters: (i) undulating membranes in a ttern vs. an Oxytricha-pattern, (iii) higher number of adoral Stylonychia–pattern vs. an Oxytricha-pattern, (ii) six dorsal membranelles (22–29 vs. 19–21), (iv) higher number of kineties vs. five, (iii) fewer adoral membranelles (22–29 vs. cirri on the left (21–30 vs. 13–18) and right (21–31 vs. 29–32), (iv) low numbers of left marginal cirri (21–30 vs. 14–17) marginal row, and (v) anterior pretransverse cirrus 27–40) and right marginal cirri (21–31 vs. 29–41), and (v) distinctly separated from posterior one vs. not being the habitat (freshwater vs. terrestrial). distinctly separated. Despite Blatterer and Foissner (1988) Kwon and Shin (2013) described a Korean population of only considering five dorsal kineties in Oxytricha granuli- O. granulifera, which was also isolated from a freshwater fera quadricirrata, we are considering a sixth dorsal kinety environment, that also possesses six dorsal kineties (Shin, (see fig. 20B in Blatterer and Foissner 1988; and fig. 78B pers. commun.) and undulating membranes in a Stylony- in Berger 1999), as proposed by Berger (1999). chia-pattern, both distinguishing characters of O. granuli- Oxytricha paragranulifera Shao, Lv, Pan, Al-Rasheid and fera chiapasensis n. subsp.; however, even this population Yi, 2014 is also similar to O. granulifera chiapasensis n. has a lower number of cirri on the left and right marginal subsp. by having undulating membranes in a Stylonychia- cirri row (Table 3), so while it is possible that Kwon and pattern, a similar size, six dorsal kineties and a similar Shin (2013) were dealing with the same subspecies, number of adoral membranelles (Shao et al. 2014), but O. molecular data are needed to resolve the taxonomy of this granulifera chiapasensis n. subsp. differs because of the Korean population. arrangement of spherical cortical granules (longitudinal A Chinese population of Oxytricha granulifera isolated rows vs. grouped in clusters in O. paragranulifera) and the from soil (Shao et al. 2014) resembles Oxytricha granulif- disposition of marginal rows on the posterior region of the era chiapasensis n. subsp., with the exception of the num- cell (unconfluent vs. confluent). ber of adoral membranelles, which is higher in the Oxytricha islandica Berger and Foissner, 1989 is similar Chinese population (28–38 vs. 22–29 in O. g. chiapasensis to O. g. chiapasensis n. subsp. in terms of the infracilia- n. subsp.), and the undulating membranes (Oxytricha-pa- ture ventral pattern and undulating membranes, but differs ttern vs. Stylonychia-pattern). Furthermore, the phyloge- by having four dorsal kineties, four macronuclear nodules, netic analysis of SSU rRNA nested the Chinese population and lacks cortical granules (Berger 1999; Berger and Foiss- (KJ081199) within Austrian populations of Oxytricha ner 1989).

Table 3. Morphological comparison of O. granulifera chiapasensis n. subsp. with previous data of Oxytricha granulifera

O. granulifera O. granulifera O. granulifera granulifera O. granulifera O. granulifera quadricirrata chiapasensis

Body size in vivo 80–130 9 35–50 90–115 9 25–38 90–130 9 30–50 70–100 9 20–30 60–120 9 20–40 Adoral membranelles number 29–32 21–26 28–38 19–21 22–29 Left marginal cirri number 27–40 18–25 23–31 13–18 21–30 Right marginal cirri number 29–41 20–28 26–32 14–17 21–31 Transverse cirri number 5 5 5 4 5 Dorsal kineties number 5 6a 666 Dikinetids number in kinety 1 16b 16b ~20 9b 8–20 Dikinetids number in kinety 2 18b 16b ~20 12b 12–21 Dikinetids number in kinety 3 16b 16bc 9b 8–18 Dikinetids number in kinety 4 4b 4bc 4b 3–4 Dikinetids number in kinety 5 8b 4bc 5b 6–10 Dikinetids number in kinety 6 0 1–2a 2–31b 1–3 Habitat Terrestrial Freshwater Terrestrial Terrestrial Freshwater Reference Foissner and Kwon and Shao et al. (2014) Blatterer and This study Adam (1983) Shin (2013) Foissner (1988) aEven Kwon and Shin (2013) mentioned ﬁve dorsal kineties, they could ﬁnd the sixth dorsal kineties which is composed by 1–2 basal bodies pairs (Shin, pers. commun.). bData from drawings or pictures on the respective paper. cData not available.

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists Journal of Eukaryotic Microbiology 2018, 65, 357–371 365 New Mexican Subspecies of Oxytricha granulifera Mendez-S anchez et al.

Oxytricha pseudosimilis Hemberger, 1985 is different membranelles (Berger 1999; Berger and Foissner 1989; from O. g. chiapasensis n. subsp., by having just five dor- Foissner 2016). sal kineties, in addition to the fact that although the undu- Paroxytricha ottowi (Foissner, 1997) Foissner, 2016 is lating membranes are in a Stylonychia-pattern, they are also very similar to O. g. chiapasensis n. subsp. in terms shorter than in our form. Plus the right and left marginal of size, undulating membranes, presence of cortical gran- row of cirri are confluent at the posterior end of the cell ules, and six dorsal kineties, but mainly differs by having (Berger 1999). eight macronuclear nodules (vs. two nodules) (Foissner Oxytricha pulvillus Foissner, 2016 is also similar to O. g. 1997). chiapasensis n. subsp., but can be distinguished by corti- Paroxytricha quadrinucleata Foissner, 2016 is similar to cal granules size, about 0.5 lm (vs. around 1 lminO. g. O. g. chiapasensis n. subsp. based on size, six dorsal ki- chiapasensis), and undulating membranes that are slightly neties, cortical granules, and a similar number of adoral straight and crossed in or near mid portion of buccal cavity membranelles (Foissner 2016), but differs by having rod- (vs. undulating membranes side-by-side) (Foissner 2016). shape cortical granules (vs. spherical), undulating mem- Oxytricha acidotolerans Weisse, Moser, Scheffel, Sta- branes in an Oxytricha-pattern (vs. a Stylonychia-pattern), dler, Berendonk, Weithoff and Berger, 2013 could be simi- and the presence of four macronuclear nodules (vs. two lar to O. granulifera chiapasensis n. subsp. by having six nodules). dorsal kineties in an Oxytricha-pattern, a similar arrange- Some species belonging to Monomicrocaryon, like ment of transverse and pretransverse cirri, and the num- Monomacrocaryon balladyna (Song and Wilbert, 1989) ber of adoral membranelles, but it differs due to the Foissner 2016; M. granulatum Foissner, 2016; and undulating membranes (Oxytricha-pattern vs. Stylonychia- M. crassicirratum Foissner, 2016 could be similar to O. g. pattern in O. g. chiapasensis n. subsp.), the disposition of chiapasensis n. subsp. because they have six dorsal ki- frontoventral cirri, the cortical granules (absent vs. pre- neties, cortical granules, and/or undulating membranes in sent) and the habitat (acidic lakes vs. a ultraoligotrophic a Stylonychia-pattern; however, they differ by having one freshwater lake) (Weisse et al. 2013). spherical micronucleus between two macronuclear no- Oxytricha granulosa Schmitz, 1986 which has cortical dules (vs. two micronuclei which are not between both granules arranged in longitudinal rows, is bigger in size macronuclear nodules) (Foissner 2016). (155–280 lm) compared to O. g. chiapasensis n. subsp. (60–120 lm), its undulating membranes are arranged in an Morphogenesis Oxytricha-pattern (vs. Stylonychia-pattern) and it only has five dorsal kineties (vs. six dorsal kineties) (Berger 1999). The morphogenetic pattern of Oxytricha granulifera chia- Oxytricha chlorelligera Kahl, 1932 has a similar body pasensis n. subsp. is similar to O. granulifera granulifera. shape and infraciliature to O. granulifera chiapasensis n. Dorsal kinety 3 is fragmented (Fig. 5I, J) as it is typical in subsp., but differs by possessing symbiotic algae (vs. the genus Oxytricha according to Berger (1999) and Shao absent) (Berger 1999). et al. (2015), and there are six streaks that form the Oxytricha aeruginosa Wrzesniowskiego, 1866 and frontal–ventral–transverse cirri in both proter and opisthe O. auripunctata Blatterer and Foissner 1988 differ from like in O. granulifera granulifera (Berger 1999; Foissner O. granulifera chiapasensis n. subsp. by having black and and Adam 1983). The main difference between the two orange-yellow cortical granules, respectively (vs. colorless subspecies is the formation of a sixth dorsal kinety to the cortical granules) (Berger 1999). left of dorsal kinety 5 in O. granulifera chiapasensis n. Other Oxytricha species, such as O. arabica, O. fallax, subsp., (Fig. 5D, G, H), while in O. granulifera granulifera O. longicirrata, O. crassistilata, and O. opisthomuscorum only five dorsal kineties are formed (Foissner and Adam could be confused with O. granulifera chiapasensis n. 1983). In addition, the undulating membranes of O. granu- subsp. by at least one of the following features: size and lifera chiapasensis n. subsp. in proter and opisthe during body shape, undulating membranes in a Stylonychia-pa- morphogenesis are parallel like in a Stylonychia-pattern ttern, or six dorsal kineties, but they lack cortical granules (Fig. 5L, M) and not crossed as in O. granulifera granuli- (Berger 1999; Foissner et al. 2008). fera Foissner and Adam, 1983. We were not able to Paroxytricha longigranulosa longigranulosa (Berger and observe all morphogenetic stages. Some of the early Foissner, 1989) Foissner, 2016 is also similar to O. granu- stages are missing, which are relevant to finding the origin lifera chiapasensis n. subsp. by having six dorsal kineties, of the anlagen I-VI for both, proter and opisthe. From undulating membranes in a Stylonychia-pattern, but P. l. Fig. 4B, 5B, it seems that the parental frontoventral cirri longigranulosa differs due to the shape of the cortical are involved in the formation of anlagen II-VI in the proter. granules (rod vs. spherical), the arrangement of cortical A detailed documentation of the complete morphogenesis granules (short longitudinal rows vs. long longitudinal is necessary to identify possible differences between the rows), and the disposition of frontoventral cirri. Also, newly described subspecies and Oxytricha granulifera Paroxytricha longigranulosa sinensis Foissner, 2016 and P. granulifera. l. imperfecta Foissner, 2016 are similar to O. g. chiapasen- In general, paroral and endoral membranes in the genus sis, but they differ by size, 145–185 lm and 65–75 lm, Oxytricha are in the so-called Oxytricha-pattern i.e. both respectively (vs. 60–120), the shape of the cortical granu- membranes are slightly curved and intersected optically les (rod in both vs. spherical) and the number of adoral (Berger 1999; Berger and Foissner 1997; Shao et al. 2011,

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists 366 Journal of Eukaryotic Microbiology 2018, 65, 357–371 Mendez-S anchez et al. New Mexican Subspecies of Oxytricha granulifera

2015); however, Oxytricha granulifera chiapasensis n. (Hewitt et al. 2003), is a result of a misidentification as subsp. and other species, such as O. paragranulifera, O. Berger (2006) suggested, and is closer to Oxytricha granu- fallax, O. islandica, and O. lanceolata, possess undulating lifera (Hewitt et al. 2003) instead of other Paraurostyla membranes in a Stylonychia-pattern (Berger 1999; Berger species, as in our tree (Fig. 6). Berger (2006) considered and Foissner 1989; Shao et al. 2014), with both mem- this species to resemble Onychodromopsis; however, due branes side-by-side and nearly straight (see Shao et al. to its current position in the tree, it is probably related to a 2015). This character is important to identify oxytrichids to species of Architricha, and possesses multiple marginal the species level, and even discriminate among genera, cirri rows (Luo, pers. commun.). Both species could origi- but it must be considered carefully because an impregna- nate from an Oxytricha granulifera ancestor, but more data tion procedure could alter the arrangement. This could are necessary to clarify their position and origin. lead to the correct pattern being overlooked, as in O. The X53486 Oxytricha granulifera gene sequence is lanceolata with undulating membranes being nearly super- positioned outside of the Oxytricha granulifera-CLADE, but imposed (Berger and Foissner 1987, 1989) or Oxytricha grouped with Paroxytricha species, similar to the results granulifera from Korea (Kwon and Shin 2013) with undula- of Schmidt et al. (2007). Schmidt et al. (2007) and Foiss- ting membranes sometimes crossed at distal end. Based ner et al. (2008) mentioned that Schlegel et al. (1991) on this, the individuals that we found with crossed undu- could have mixed up O. longigranulosa and O. granulifera lating membranes could be due to an impregnation proce- species, because they are morphologically similar (Berger dure, but the morphogenesis data are a strong evidence 1999; Foissner 2016), resulting in a misidentification. to confirm that the correct pattern for undulating mem- We recognize three groups in the Oxytricha granulifera- branes for Oxytricha granulifera chiapasensis n. subsp. is CLADE. Group I comprises six species, one of them belon- the Stylonychia-pattern (Fig. 5L, M). ging to the Chinese population (Shao et al. 2014) which is Thus, the unique presence of a sixth dorsal kinety com- separated from the remaining gene sequences, and per- prising one or three dikinetids and the organization of haps the sixth dorsal kinety produced this separation. The undulating membranes allow us to establish the Mexican remaining species of this group, with the exception of population as a subspecies for Oxytricha granulifera: Oxy- AF164122 (Prescott et al. direct submission to GENBANK), tricha granulifera chiapasensis n. subsp. correspond to populations that were revised by W. Foissner (Schmidt et al. 2007). Supposing that they possess five dorsal kineties and undulating membranes in an Oxytricha-pa- Habitat and ecology ttern, then they correspond to Oxytricha granulifera Oxytricha granulifera granulifera has been primarily isolated granulifera. Furthermore, AM412768 and AM412769 are from terrestrial environments, and also occasionally from isolated from the type locality from which Foissner and aquatic environments (Berger 1999; Foissner 1997, 1999, Adam (1983) originally described O. granulifera granulifera. 2016; Foissner and Adam 1983; Foissner et al. 2002, 2005, Group II includes a species isolated from a pond in Salz- 2008; Kwon and Shin 2013; Shao et al. 2014); therefore, we burg, Austria, also identified by W. Foissner (Schmidt consider this species to prefer terrestrial habitats, but is et al. 2007), as well as others that currently lack informa- able to colonize aquatic systems. In the case of O. granulif- tion on morphology and locality (Singh and Kamra, direct era chiapasensis n. subsp., it is the first record in the world submission to GENBANK), so more data are required to from Montebello Lake, which is a karstic ultraoligotrophic better understand the formation of this clade. freshwater lake (Alcocer et al. 2015). We found this sub- Group III includes Oxytricha granulifera chiapasensis n. species in a shallow area of the freshwater lake with a subsp. and another Oxytricha granulifera species water temperature of 26.3 °C, dissolved oxygen concentra- (KU715983); however, its morphological characterization tion of 9.02 mg/L, pH 7, and conductivity 175 lS/cm. was not provided (Abraham et al. direct submission to GENBANK). The small pairwise distance (d = 0.004) and only seven differing nucleotides, however, indicate that O. Phylogenetic analysis granulifera chiapasensis n. subsp. is closest to KU715983. Oxytricha granulifera chiapasensis n. subsp. grouped with The other described O. granulifera with six dorsal kine- O. granulifera species in the clade here designated as ties (KJ081199, Shao et al. 2014) also shows small Oxytricha granulifera-CLADE and is highly supported by genetic distance (d = 0.005) and only eight differing ML and BI (Fig. 6). Interestingly, two seemingly unrelated nucleotides to O. granulifera chiapasensis n. subsp., indi- species, Architricha indica and Paraurostyla viridis, were cating a possible closer relationship of Groups I and III nested in this clade and both species are morphologically than inferred in the current phylogenetic analysis. distinct from Oxytricha granulifera, and even from other We conclude that Oxytricha granulifera chiapasensis n. Oxytricha species. subsp. is closest to Oxytricha granulifera granulifera Foiss- Architricha indica (KJ000536) is well-characterized mor- ner and Adam 1983; in addition, the Mexican subspecies phologically by the presence of 18 frontal–ventral–trans- was found far away from nominotypical subspecies popu- verse cirri and with multiple right and left marginal cirri lations, the nearest strain was isolated from Venezuela rows (Xu et al. 2015). The gene sequence AF508766, (AM412772), which did not cluster together O. granulifera which is labeled as Paraurostyla viridis (Urostyla viridis in chiapasensis n. subsp., while the remaining species were Berger 2006) and lacks morphological characterization isolated in Europe and Asian continent.

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists Journal of Eukaryotic Microbiology 2018, 65, 357–371 367 New Mexican Subspecies of Oxytricha granulifera Mendez-S anchez et al.

Figure 6 Maximum likelihood (ML) phylogenetic tree inferred from SSU rRNA gene sequences of 68 hypotrichs, and phylogenetic position of Oxytricha granulifera chiapasensis n. subsp. (in blue bold). Disagreement between ML and BI (*). DK = dorsal kineties; O-UM = undulating membranes in Oxytricha-pattern; S-UM = undulating membranes in Stylonychia-pattern.

species into two subspecies, mainly by morphological TAXONOMIC SUMMARY differences. Subclass Stichotrichia Small and Lynn, 1985 Improved diagnosis. The diagnosis is based on original Order Stichotrichida Faure-Fremiet, 1961 description and redescription of the three subspecies (Ber- Family Oxytrichidae Ehrenberg, 1838 ger 1999; Blatterer and Foissner 1988; Foissner and Adam Subfamily Oxytrichinae Ehrenberg, 1838 1983; Kwon and Shin 2013; Shao et al. 2014). Cells 60–130 9 20–50 lm, with spherical cortical gran- Oxytricha granulifera Foissner and Adam, 1983. ules disposed in longitudinal rows on the dorsal side. Ado- Remarks. We reactivated the subspecies rank proposed ral zone extending to 30% of body length. Four or ﬁve by Blatterer and Foissner (1988), who already split this transverse cirri. Contractile vacuole located at mid-body of

© 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists 368 Journal of Eukaryotic Microbiology 2018, 65, 357–371 Mendez-S anchez et al. New Mexican Subspecies of Oxytricha granulifera left margin of cell, collecting canals could be present. Mar- ACKNOWLEDGMENTS ginal rows separated posteriorly. Two macronuclear nodules, each one with one micronucleus attached. Paroral To Posgrado en Ciencias Biologicas Universidad Nacional and endoral membranes are disposed in Oxytricha or Sty- Autonoma de Mexico, and CONACyT for the grant to lonychia-pattern. Five or six dorsal kineties. DMS for master’s studies. We acknowledge IRCN-BC for Subspecies included. Oxytricha granulifera granulifera the grant to DMS for a stay in the Laboratory of Protozoo- Foissner and Adam, 1983; Oxytricha granulifera quadrici- logy of Ocean University of China (OUC) at Qingdao, rrata Blatterer and Foissner, 1988; and Oxytricha granuli- China. We are thankful to the Tziscao community for gi- fera chiapasensis n. subsp. ving us permission to collect samples. Special thanks to Biol. Margarita Reyes-Santos Laboratorio de Protozoologıa, Oxytricha granulifera granulifera Foissner and Adam, 1983. UNAM for assistance in cell isolation. Special thanks to Improved diagnosis. Cells 70–127 9 28–75 lm in vivo; Miss Chundi Wang and Mr. Zhishuai Qu Laboratory of Pro- adoral zone with 29–32 membranelles; paroral and endoral tozoology, OUC, for their technical assistance in the membranes optically intersected in Oxytricha-pattern; the molecular procedures and staining protocols respectively. right marginal cirri row begins at the same level of the Special thanks also to Derek A. Woller from the United rightmost frontal cirrus; constantly ﬁve transverse cirri. States Department of Agriculture’s APHIS, PPQ, Science Rarely occurs a sixth dorsal kinety. Conﬁned to terrestrial and Technology’s Phoenix Lab, Phoenix, Arizona, U.S.A. habitats. See Berger (1999); Foissner and Adam (1983) for assisting with the English. Publication of this article and Shao et al. (2014). was supported by the R. P. Hall Fund.

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