Lamtostyla ovalis, Luo & Gao & Yi & Pan & Al-Farraj & Warren, 2017

GENUS LAMTOSTYLA BUITKAMP, 1977 View in CoL View at ENA LAMTOSTYLA OVALIS SP. NOV.

( FIGS 3A – I View Figure 3 , 4A – H; TABLE 2)

Diagnosis

Body 70 – 110 9 40 – 60 lm in vivo; generally oval in outline, colourless to slightly grey-greenish; about 22 adoral membranelles; three frontal cirri; one buccal cirrus; amphisiellid median cirral row comprises about 11 cirri and terminates ahead of mid-body; three to seven frontoventral cirri; two pretransverse ventral cirri; five transverse cirri; 19 left and 24 right marginal cirri on average; three dorsal kineties; two macronuclear segments; two micronuclei; brackish water habitat.

Type locality and ecology

A small puddle near Zhan Qiao Pier in Qingdao (36 ° 06 0 N, 120 ° 31 0 E), Shandong Province, China. Water temperature 21 ° C, salinity 20 psu GoogleMaps .

Type specimens

The protargol slide containing the holotype specimen (see Fig. 3B, C, G View Figure 3 ; registration no. PY2010092801/1) was deposited in the Laboratory of Protozoology, Ocean University of China ( OUC). A paratype slide was deposited in the Natural History Museum, London (registration number NHMUK 2015.7.10.2) .

Etymology

The species-group name ovalis (Latin adjective; eggshaped, oval) refers to the oval body outline.

Description

Cell about 70 – 110 9 40 – 60 lm in vivo, oval in outline ( Figs 3A View Figure 3 , 4A – F), anterior end slightly narrowed and bends leftwards when cell changes direction during locomotion, posterior end more or less broadly rounded. Ratio of length to width about 5:3 – 2:1; dorsoventrally flattened about 2:1. Pellicle very flexible. Buccal field narrow, transparent, and conspicuous in vivo, occupying about 1/3 of body length. Cytoplasm colourless; whole body except buccal field usually packed with numerous dark-greenish algae (3 – 10 lm across; we are unsure whether they are endosymbionts or not) and several food vacuoles containing diatoms and small ciliates, rendering cell slightly grey-greenish ( Figs 3I View Figure 3 , 4A – G). Several bacterial plaques of varying size on dorsal surface, each composed of c. 1 – 2 9 0.5 lm bacilli ( Fig. 4H). Cortical granules absent. Contractile vacuole about 10 lm across, positioned left of midline in mid-body region ( Fig. 3A View Figure 3 ). Locomotion by slowly crawling on substrate; swimming cells not observed.

Cilia of anterior membranelles approximately 12 lm long in vivo. All ventral cirri about 8 – 10 lm long except frontal and transverse cirri, which have cilia about 12 lm long; transverse cirri do not project beyond posterior body margin ( Fig. 3A View Figure 3 ). Dorsal bristles about 3 lm long in vivo.

Infraciliature as shown in Figure 3B – I View Figure 3 . Adoral zone composed of 19 – 25 membranelles with about four membranelles in distal portion located apically. Bases of distal membranelles comprise three short equal-length rows of kinetosomes; bases of proximal membranelles comprise one short and three long rows of kinetosomes ( Fig. 3B, D, E View Figure 3 ). Paroral and endoral membranes equal in length, arranged in parallel, occupying only about half length of buccal field ( Fig. 3B, D, I View Figure 3 ). Consistently three enlarged frontal cirri located near anterior end of cell, arranged in a slightly oblique pseudorow ( Fig. 3B, D, G View Figure 3 ). Single enlarged buccal cirrus located right of anterior end of paroral membrane ( Fig. 3B, D, G – I View Figure 3 ). Amphisiellid median cirral row (ACR) usually composed of eight to 11 cirri, commencing behind rightmost frontal cirrus and terminating at about level of buccal vertex ( Fig. 3B, D, I View Figure 3 ). Three to seven (usually four) frontoventral cirri left of anterior portion of ACR ( Fig. 3B, D, G, H View Figure 3 ). Usually five transverse cirri arranged in J-shaped or U-shaped row. One to three, usually two, pretransverse ventral cirri positioned ahead of transverse cirri. Two marginal rows not reaching posterior end of body and thus separated posteriorly. Right marginal row commencing near anterior part of ACR, composed of 19 – 31 cirri; left marginal row starting about level with buccal vertex, composed of 16 – 24 cirri ( Fig. 3B, G – I View Figure 3 ). Invariably three dorsal kineties more or less shortened anteriorly; number of bristles per kinety increases from left to right ( Fig. 3C, F View Figure 3 ). Two ellipsoidal macronuclear segments slightly left of cell midline, transparent in vivo, conspicuously separated but connected to each other via a thread ( Figs 3A, C, E View Figure 3 , 4A, E); rear end of anterior macronuclear segment a little behind level of buccal vertex, rear end of posterior segment slightly anterior of transverse cirri when observed in vivo. Two spherical micronuclei, c. 2 lm in diameter, each closely associated with a macronuclear segment ( Fig. 3C, H View Figure 3 ).

CV, coefficient of variation in %; Max., maximum; Mean, arithmetic mean; Med., median value; Min., minimum; N, number of specimens examined; SD, standard deviation.

SSU RRNA GENE SEQUENCE AND PHYLOGENETIC ANALYSES

The SSU rRNA gene sequence of Pol. monilata sp. nov. (GenBank accession number KT192639 View Materials ) is 1698 bp long and has a G + C content of 45.11%; that of L. ovalis sp. nov. (GenBank accession number KP266625 View Materials ) is 1729 bp long and has a G + C content of 45.58%.

The topologies of the BI and ML trees inferred from SSU rRNA gene sequences were basically congruent with variable support values; therefore, only the ML topology (with nodal support from both methods) is shown ( Fig. 5 View Figure 5 ). The phylogenetic analyses showed that neither order Stichotrichida nor Sporadotrichida is monophyletic. Within these two orders, three families out of seven (i.e. Trachelostylidae , Gonostomatidae , and Spirofilidae ) and one

TWO NEW BRACKISH HYPOTRICHOUS CILIATES 483

G A E B C D F H

ACR, amphisiellid median cirral row; CV, coefficient of variation in %; Max., maximum; Mean, arithmetic mean; Med., median value; Min., minimum; N, number of specimens examined; SD, standard deviation.

subfamily (i.e. Stylonychinae) are monophyletic. Protogastrostyla pulchra and Protogastrostyla sterkii cluster in a clade that branches off at the base of the ingroup. Lamtostyla ovalis sp. nov. clusters with five stichotrichid species, i.e. Uroleptoides magnigranulosus , Bistichella cystiformans , Bistichella variabilis , Parabistichella variabilis , and Orthoamphisiella breviseries , near the base of the SSU rRNA gene trees forming a group that is sister to a large assemblage comprising most of the stichotrichids, sporadotrichids, and (core) urostyloids for which SSU rRNA gene sequence data are available. Polystichothrix monilata sp. nov. is sister to Pseudogastrostyla flava with high support (96% ML, 1.00 BI), and is next most closely related to Rubrioxytricha ferruginea , Rubrioxytricha haematoplasma , Ponturostyla enigmatica , and Pseudocyrtohymena koreana . Except for Pseudogastrostyla flava , all of these species have 18 frontal-ventral-transverse cirri and are members of the subfamily Oxytrichinae . In order to further investigate evolutionary relationships within the family Oxytrichidae , we mapped morphological characters onto the phylogenetic tree ( Fig. 5 View Figure 5 ). Based on cirral patterns, Oxytrichidae is divided into three groups, viz. oxytrichids with 18 frontal-ventral-transverse cirri (blue stars), oxytrichids with more than 18 cirri (red stars), and oxytrichids with fewer than 18 cirri (green stars). The Oxytrichidae can also be separated into two groups based on the presence or absence of dorsal kinety fragmentation, viz. those with dorsomarginal kineties but lacking dorsal kinety fragmentation (orange triangles), and those both with dorsomarginal kineties and dorsal kinety fragmentation (black triangles). However, grouping patterns based on either of these two morphological character states are not possible in the topology of the SSU rRNA gene trees ( Fig. 5 View Figure 5 ).