The Collothecidae (Rotifera, Collothecacea) of Thailand, with the description of a new species and an illustrated key to the Southeast Asian fauna

Abstract Following previous reports indicating a remarkable high diversity of sessile rotifers in Southeast Asian freshwaters, we report on an extensive study of the diversity of Collothecidae rotifers from fifteen freshwater habitats in Thailand. A total of 13 species, including two additional infraspecific variants, of Collothecidae are recorded, one of which is described as a new species of Collotheca. We further add taxonomic remarks on some of the taxa on record and illustrate the uncinate trophi of several representatives by scanning electron microscopic images. Finally, we provide illustrated identification keys to the Collothecidae recorded to date from Southeast Asia.

introduction Family Collothecidae is one of two families of the rotifer Order Collothecacea. The order is diagnosed by the presence of uncinate trophi (Segers 2002, Wallace et al. 2006) and a peri-buccal region expanded into a wide infundibulum, while family Collothecidae is further characterized by having a modified corona ciliata (short: corona) consisting of differentiated cilia implanted along the margin of, or grouped on knoblike, lobate or tentacle-like extensions of the infundibulum. The family contains two genera, Collotheca Harring and Stephanoceros Ehrenberg, and these respectively contain 45 and one valid species (Koste 1978, Segers 2007. Collothecid rotifers are essentially ambush predators. Their expanded and elongated corona lobes and cilia lobes form a fyke-like structure by which mobile prey, either zoo-or phytoplankton, are directed towards an enlarged funnel-shaped infundibulum. Once there, prey is trapped by contraction of infundibular sphincter muscles and swallowed through the pumping action of a membrane supported by the rod-shaped trophi. This specialized feeding strategy and its phylogenetic consequence have received considerable attention by rotifer research (e.g., Kutikova andMarkevich 1993, Sørensen andGiribet 2006), although large gaps remain in our knowledge of the diversity and evolution of the group.
To date, comparatively little is known on the distribution and diversity of sessile rotifers in general and of Collothecidae in particular, which is due to the fact that these animals require life observation for identification and study. This knowledge gap is especially evident regarding sessile rotifers from tropical regions. These animals are mostly dealt with on an ad hoc basis, and much of what little information that exists is contained in more general inventories of rotifers, in which the sessile taxa are represented as chance occurrences (e.g., Chittapun et al. 2007, Sanoamuang and Savatenalinton 2001, Segers and Chittapun 2001, Segers and Sanoamuang 2007. Some recent relevant studies on Southeast Asian sessile rotifers (Koste 1975, Meksuwan et al. 2011, Segers et al. 2010) report a remarkable diversity of the group, including several species of outstanding taxonomical and/or biogeographically interest, which sparked a more comprehensive study on this particular taxon of rotifers. Here we report on the diversity and taxonomy of Collothecidae found during our extensive study of the sessile rotifers of Thailand. Finally, realizing that the only available, relatively recent identification work dealing with Collothecidae is in German (Koste 1978), we present a key to the identification of the Collothecidae recorded from Southeast Asia, to facilitate and promote future studies on these remarkable animals.

Material and methods
We explored 15 freshwater habitats in 12 provinces of Thailand for Collothecidae during the present study (Fig. 1). Submerged parts of different species of aquatic plant were collected qualitatively to search for sessile rotifers. Collecting and observation methods are detailed in Meksuwan et al. (2011). Searching and identifying rotifers was performed under an Olympus SZ 51 stereo microscope and an Olympus CX 21 compound microscope. Drawings are based on photographs and observations of living animals. Trophi were prepared for scanning electron microscopy (SEM) following the method of Segers (1993), SEM photographs were taken using a FEI Quanta 400 SEM at the Scientific Equipment Center, Prince of Songkla University, Hatyai campus.

Diversity of family Collothecidae in Thailand
The samples examined contained 13 species and two infraspecific variants of Collothecidae (Table 1). This corresponds with ca. 28% of the world fauna of Collotheca species and all Stephanoceros species known to date (Segers 2007). Two of the species identified could not be ascribed to any known species and we conclude that the specimens pertain to new species, one of which is described below. Of the second possibly new species we opine that insufficient material is at present available to warrant a full description, hence we only provide a brief illustration to enable future recognition. One more species, C. ferox (Penard) is new to the Oriental region and C. ornata f. cornuta (Dobie) is new to Thailand. These results indicate a relatively diverse Collothecidae fauna in the studied region of Thailand, and the record of one, and possibly two new species leads us to surmise that an even higher and incompletely documented diversity can be expected to occur in Southeast Asia.

Genus Collotheca Harring
Collotheca ferox (Penard) http://species-id.net/wiki/Collotheca_ferox Remarks. The morphological characters of our specimens agree closely with the description of the species by Penard (1914): the corona of the specimens is more than twice as broad as its trunk and bears five broad lobes ( Fig. 2A, B, 4J). The dorsal lobe tip is relatively large and rounded anteriorly; the lateral lobes are intermediate in size whereas the triangular ventral lobes are relatively small and are set close together. The features of the ventral lobe are unique to this species and prevent confusion with other five-lobed species of the genus. Our photographs of living specimens and trophi of C. ferox confirm, in particular, the unique features of the ventral corona lobes illustrated by Penard (1914). Differential diagnosis. The presence of a five-lobed corona separates the new species from most of the known members of genus Collotheca. In comparison with other Collotheca species having a five-lobed corona (C. algicola (Hudson), C. ambigua (Hudson), C. annulata (Hood), C. bilfingeri Bērziņš, C. ferox and C. campanulata (Dobie)), C. orchidacea sp. n. can be distinguished by its uniquely well-developed thumb-shaped lateral and semi-circular ventral corona lobes. It has a relatively broad infundibulum, and short foot and trunk, similar only to C. ambigua and C. ferox. In addition, C. orchidacea sp. n. and C. ferox hold their infundibulum and corona towards the substratum, whereas most other species including C. ambigua and C. campanulata normally hold their body and corona upright.

Collotheca orchidacea
Description. Habitus ( Fig. 2C-F): infundibulum funnel-shaped, trunk and corona opening held horizontally. Infundibulum and proventriculus about twice as long as the trunk. Infundibulum large, more than twice as wide as trunk. Foot short, length Stephanoceros fimbriatus (Goldfusz, 1820) Stephanoceros millsii (Kellicott, 1885) (* = new to Oriental region and Thailand; ** = new to Thailand). 1 recorded by Koste (1975), not seen during this study  about half of trunk, contractile, with a short peduncle. Corona five-lobed: single dorsal, and a pair of well-developed lateral and of ventral lobes. Infundibulum with a weak line running parallel to the edge of the corona, and at least four ring-shaped structures (circular muscles?). Dorsal lobe large, elongate, basally with straight and converging lateral margins; parallel-sided medially, with smoothly curved antero-lateral corners. Tip of dorsal lobe transversally sinuate. Lateral lobes relatively the smallest, thumb-shaped, about half as wide as the dorsal lobe. Ventral lobes broadest, smoothly Trophi (Fig. 5E)  Etymology. The species name -orchidacea is a noun in apposition, and refers to the shape of the new species' corona, which is reminiscent of the flower of certain orchid species. As such, the name of the species also refers to the biodiversity of Thailand, characterized by an abundance of orchid species.
Distribution. The species is known from its type locality only.

Collotheca ornata f. cornuta (Dobie)
Note. This taxon (Figs 4G, H) is differentiated from the nominal form by the corona bearing an elongate projection dorsally to the dorsal lobe. The presence/absence of this projection has classically been interpreted as of infrasubspecific relevance only (see Edmondson 1940, Koste 1978. In the absence of additional data (morphological, molecular or behavioural), we prefer to be cautious and record the taxon separately. Specimens were found in Khlong Lam Chan Non-Hunting Area, Trang province ( Fig. 1: S3); the present is the first Thai record of the taxon.

Collotheca sp.?
Remarks. We found a single specimen of a species that we could not identify (Fig.  3D). Its corona consists of two lobes, one large dorsal lobe and one minute ventral lobe, which is similar to Collotheca calva (Hudson). The specimen, however, exhibits a unique cluster of long setae dorsally on the tip of the dorsal lobe and, in addition, shows two ring-shaped structures in the infundibulum. The presence of an egg in its gelatinous case indicates that the specimen was mature and not some incompletely developed juvenile. We believe that it represents an undescribed species but refrain from describing and naming it due to the lack of a sufficient number of specimens. The animal occurred in Khlong Lam Chan Non-Hunting Area, Trang province ( Fig. 1: S3).

Remarks. Genus
Stephanoceros is diagnosed (Koste 1978) by having extraordinarily long extensions of the corona (tentacles) bearing transversally implanted rows of medium-long cilia, in addition to short mobile cilia. Following this diagnosis he suggests that C. stephanochaeta (Edmondson), which has short corona lobes bearing similarly inserted rows of cilia, might be better placed in Stephanoceros rather than Collotheca, while he discards the relevance of the absence of such transverse rows of cilia in Stephanoceros millsii (Kellicott) by considering the latter a mere infrasubspecific ecotype of S. fimbriatus (Goldfusz).
We believe that the diagnosis of Stephanoceros is questionable, considering that neither the presence of long corona lobes (also in Collotheca judayi Edmondson and C. tenuilobata (Anderson)) nor the presence of transverse rows of cilia on the corona lobes (present in Collotheca stephanochaeta, absent in Stephanoceros millsii, see below) can serve as synapomorphic diagnostic feature for the genus. To the contrary, we hypothesize that the two species now attributed to Stephanoceros are merely species in which the prolongation of corona lobes already present in many species of Collotheca has evolved to its greatest extent. We look forward to a more complete phylogenetic analysis of the taxa, knowing that a molecular phylogenetic study of the group is ongoing. A synonymy between Collotheca and Stephanoceros would have to result in the reallocation of all taxa of the junior Collotheca to the senior Stephanoceros.

Stephanoceros fimbriatus (Goldfusz) versus Stephanoceros millsii (Kellicott) (revised status)
We found specimens matching the descriptions of two taxa in Stephanoceros, Stephanoceros fimbriatus (Fig. 3H) and S. millsii (Fig. 3G, 5F) (see Kellicott 1887, Koste 1978. S. fimbriatus has five very long, stout corona lobes carrying transverse rows of robust setae along their length, while S. millsii has five relatively slender corona lobes carrying longitudinal rows of long, fine setae. The corona lobes of S. fimbriatus are relatively shorter than those of S. millsii, when compared to their trunk length. Regarding trophi, the unci tips of S. millsii are acutely pointed whereas those of S. fimbriatus have arrow-shaped tips, and the unci are more strongly curved in S. millsii (compare Fig. 5F with Fig. 1B in Sørensen and Giribet 2006).
Because the morphological characters of these two taxa enable a reliable diagnosis and because the two have wide and overlapping distribution ranges, we argue that these two taxa are distinct species, in contrast to Koste (1978) who considered S. millsii an infrasubspecific variant ("Anscheinend Ökotyp") of S. fimbriatus. Ours are the first photographs of living animals and trophi of S. millsii.
S. millsii is common in Thailand whereas S. fimbriatus is quite rare in our survey. Both species are cosmopolitan (Koste 1978).

The uncinate trophi of Collothecidae
The uncinate trophi type is one of nine trophi types recognized in phylum Rotifera (Wallace et al. 2006). This trophi type is characterised by unci possessing few teeth and by weakly developed manubria and fulcrum (Koste 1978) and has hardly been considered in the taxonomic analysis of Collothecacea (Families Collothecidae and Atrochidae). We examined the uncinate trophi of 6 species of Collothecidae to evaluate whether morphological differences, which might be taxonomically relevant, exist.
We found that, in all species examined, the uncinate trophi are composed of two pairs of large and sturdy unci teeth, whereas manubria, rami and fulcrum are less developed components (Figs 5A-F). Of the unci, the distal tips can be gradually sharpened (5C-D), stout (5B), or with set-off tips (5A), and the tips may carry a terminal, median groove (e.g., 5E-F). The unci are mostly strongly curved, either more or less evenly (e.g., 5A, E) or in their proximal third (5B), or terminally (5C), and the terminal tips may be slightly incurved (5A), straight (5E), or outcurved (5B). The unci pairs can be relatively equal (5A, D-F) or strongly unequal (5B, C) in length. The unci teeth are quite sturdy, as they are not easily dissolved by low concentration of commercial bleach (lower than 5% final concentration). The manubria, rami and fulcrum, on the other hand, are very weak and dissolve easily in bleach making it particularly hard to reliably compare their morphology. Nevertheless, the rami scleropilli usually remain after treatment (5E-F).
As illustrated here, the uncinate trophi, in particular the unci, do exhibit features that might be useful for taxonomic analysis. We suggest that 1) shape of the head of the unci; 2) shape of the unci teeth; and 3) relative size of the two pairs of unci teeth might be registered in future studies of Collotheca rotifers. Of course, the inclusion of these features in taxonomic analysis requires addition of information on more species of Collotheca, and evaluation of the intraspecific variability by comparing different populations of Collotheca species.

Feeding in Collotheca
As mentioned above, Collothecidae species are essentially ambush predators. They remain immobile until a prey organism, guided by their long cilia and infundibulum that forms a fyke, and water current created by the beating of short cilia, comes in range of a sensory organ situated dorsally on the inner side of the infundibulum. When this organ is triggered, the cilia, corona lobes and infundibulum contract which restrains the prey organism within the infundibulum, and the prey is finally ingested whole. We observed that some species of Collotheca, and these appear to be species that have an enlarged funnel-shaped infundibulum, arrange their corona near the surface of the substrate they are attached to (e.g., Collotheca sp., Figs 3D; C. ferox, Fig. 2A, B -note that the specimen in Figs 2A, B was not in normal position; C. orchidacea sp. n., Figs 2C, D). Other species, mostly those that have a relatively smaller infundibulum but well-developed bands of cilia along the corona or on knobs, and a long foot, expose their expanded corona in the water column (e.g., C. campanulata f. longicaudata, Fig. 3B; C. ornata, Fig. 3C; C. tenuilobata, Fig. 3F). We hypothesize that the two groups may have different diets. The latter group probably feeds on free-swimming, planktonic/periphytic organisms, while species of the former group may target browsing animals, in a way that is strikingly similar to Cupelopagis vorax (Leidy, 1857) (Bevington et al. 1995).

Identification key to the Collothecidae of Southeast Asia
The keys presented here include all species recorded hitherto from Southeast Asia (Brunei, Cambodia, Indonesia, Malaysia, Myanmar, Philippines, Singapore, Thailand, Timor Leste, Vietnam), as included in De Ridder and Segers (1997) and more recent publications. To facilitate identification and discovery of species not included in the key, we provide both a dichotomous as well as a formula key to the Southeast Asian Collothecidae.

Dichotomous key
1 Length of corona lobe(s) shorter than trunk (Figs 3A-F Corona with a single dorsal lobe carrying one group of long cilia ....C. libera -Corona with five knob-shaped projections, the dorsal one on a triangular lobe; all bearing a group of long cilia (Fig. 3C, 4B) (3)