Two new species of genus Limnias from Thailand, with keys to congeners (Rotifera, Gnesiotrocha)

Abstract Two new species and two morphological variant taxa of sessile rotifers found during a survey of Thai rotifers are reported upon. Living specimens were collected and identified from twelve sampling sites. The specimens were photographed, and prepared for SEMs of dorsal plates and trophi. Two new species of genus Limnias are recognized. Limniaslenissp. n. possesses a corona with a relatively shallow ventral sinus, and a dorsal plate without any projection, whereas Limniasnovemcerassp. n. carries a corona with a deep and wide ventral sinus, and a dorsal plate with nine projections. Two morphological variants of Limniasmelicerta are discussed, which are designated as L.melicerta SH and L.melicerta LH on the basis of the length of the horns on their dorsal plates. Moreover, both a dichotomous key and a formula key are provide including all Limnias species known to date.


Introduction
Limnias Schrank, 1803 is a cosmopolitan genus of sessile rotifers (Segers 2007).This taxon is recognized by a combination of four characteristics: (1) a corona with two lobed and a wide dorsal gap, (2) a pair of distinct ventral antenna, (3) presence of a stiff dorsal plate, and (4) a firm tube that the adults inhabit (see figure 1 of Wallace et al. 2018).At present, six valid names of congeners are known: L. ceratophylli Schrank, 1803, L. melicerta Weisse, 1848, L. myriophylli (Tatem, 1868), L. shiawasseensis Kellicott, 1888, L. cornuella Rousselet, 1889and L. nymphaea Stenroos, 1898 (Jersabek et al. 2015;Wallace et al. 2018).In Thailand, two species of Limnias had to date been identified, L. melicerta and L. ceratophylli by Koste (1975) and Meksuwan et al. (2011).One easily recognized characteristic that separates species in the genus is the architecture of tube.For example, in L. melicerta the tube is composed of a series of rings stacked vertically forming a tube (Wright, 1954).The tube of L. ceratophylli lacks rings.Moreover, besides the ringed tube, L. melicerta possesses a deep ventral sinus corona and dorsal plate with seven projections, while L. ceratophylli has a corona with a shallow ventral sinus and a dorsal plate without projection.Based on our survey of diversity of sessile rotifers in Thailand started in 2011, we recognized two taxa belonging to Limnias which have distinct characters that do not fit any of the known members.They are here described as new species.We also update the dichotomous and formula keys to all species of genus Limnias by Wallace et al. (2018).

Collecting sessile rotifers
Specimens of Limnias species were collected from different localities in Thailand (Supplementary material 1).Collecting method for sessile rotifers was described by Edmondson (1944), Wallace (1977), and Meksuwan et al. (2011).Briefly, it includes moving live, aquatic plants into a container filled with water from the sampling site to a container without adding any anesthetics or other chemicals for preservation.In laboratory, plants were dissected into convenient sizes for manipulation and examination for presence of sessile rotifers.These were identified alive.We suggest filtering some source water through a 60-μm, mesh plankton net and adding this to the containers to provide phytoplankton and organic particles as food source for the rotifers.By using this mesh size, larger zooplankton that may hinder examination are removed.Samples may be held for several days by providing suitable conditions.For examination plasticine was used to form small supports at the four corners of a coverslip to prevent compression of specimens.

Scanning electron microscopy (SEM)
Dorsal plate.Each fixed, contracted specimen in 95% ethyl alcohol is extracted from its tube, and placed into a small drop of distilled water on a piece of cover glass.The specimen is oriented dorsally and left until the water is completely evaporated.Dried specimens on cover glasses are coated with gold, followed by examination under SEM.
Trophi.A sorted specimen is placed into a drop of commercial bleach (7% NaO-Cl).The remaining trophi is picked up and rinsed several times in drops of distilled water on a piece of cover glass.Then, the trophi is air dried, coated with gold and examined under SEM.SEM photographs were processed by a FEI Quanta 400 SEM.

Descriptions of new species
Classification of genus Limnias follows Segers (2002).We adhere to this view as it is supported by a number of molecular analysis that support Eurotatoria, as taxon consisting of subclass Bdelloidea and subclass Monogononta (e.g., Melone et al. 1998, Mark Welch 2000).Subclass Monogononta comprises superorder Pseudotrocha and superorder Gnesiotrocha.Genus Limnias is located in the Gnesiotrocha where the members lack a toe.Etymology.The species name is an adjective, derived from the Latin "lenis", meaning "soft, smooth, gentle" and refers to the smooth surface of its dorsal plate.
Distribution.The species is known only from its type locality.Etymology.The species name is a substantive, and refers to the number of projections (nine, fromthe Latin novem, and horn, from the Greek ceras) on the dorsal plate.
Diagnosis.Limnias novemceras sp.n. is easily recognized by its dorsal corona gap being much wider than the tube diameter, by its long ventral antennae that reach beyond the fully extended corona, and by its dorsal plate carrying nine projections.In addition, this species never raises its corona far beyond the tube opening (Figs 1D-F   more than twice as wide as trunk, ventral sinus depth between one third and one half of corona height, dorsal gap nearly 1.5 times as wide as trunk width (Figs 1D, 4B).Two long ventral antennae extending beyond the extended corona.Dorsal plate stiff, with nine projections, upper row a pair of short and broad projections, middle row with a central, distally bifid projection, two lateral rounded projections and, slightly more distally, two low and broad, ridges, lower row a pair of oblique, rounded, low projections, these rounded triangular with straight inner margin in SEM preparation.Dorsal antenna aperture situated at one fifth of the dorsal plate length from the anterior margin (Figure 7B).Trophi malleoramate, symmetrical.Left and right proximal unci teeth: 3/3, distal teeth: 16-17/16-17 (Figure 8B).Rami apophyses equal.Manubria crescent-shaped with three chambers.Corona width: 114-126, corona height:  Distribution.The species is known only from its type locality.

Keys to species of genus Limnias
We constructed dichotomous and formula keys of all known Limnias species based on investigation of four species, including the two new species, observed in Thailand.In addition to the four species, we reevaluate the identity of L. cornuella, L. myriophylli, L. nymphaea and L. shiawasseensis, based on original publications, the most recent revision of the group by Wallace et al. (2018), and illustrations available in the Rotifer World Catalog (Jersabek and Leitner 2013).We recognize that number of dorsal plate projections, length of ventral antennae, and tube structure are useful characters for species identification in genus Limnias.