Bdelloid rotifers (Rotifera, Bdelloidea) of China: diversity and new records

Abstract Bdelloid rotifers are a group of microscopic invertebrates known for their obligate parthenogenesis and exceptional resistance to extreme environments. Their diversity and distributions are poorly studied in Asia, especially in China. In order to better understand the species distribution and diversity of bdelloid rotifers in China, a scientific surveys of habitats was conducted with 61 samples (both terrestrial and aquatic habitats) from 11 provinces and regions of China, ranging from tropics to subtropics with a specific focus on poorly sampled areas (Oriental) during September 2017 to October 2018. A total of 59 morphospecies (including subspecies) were found, of which, thirty-nine morphospecies (including one genus) are new records for China, almost doubling the number of previous records. Four rare morphospecies (Adineta cf. acuticornis Haigh, A. beysunae Örstan, Habrotrocha ligula loxoglotta De Koning and H. serpens Donner) are depicted and redescribed, and an updated checklist of Chinese bdelloids with their location and ecological information is presented. This study provides new data from a large region of China, enriching the knowledge of bdelloid biodiversity, and their global biogeography.


Introduction
Bdelloid rotifers are microscopic invertebrates that constitute a subclass Bdelloidea of the phylum Rotifera, known for their peculiar obligate parthenogenesis (Welch and Meselson 2000;Welch et al. 2004) and outstanding ability to withstand harsh periods through anhydrobiosis (Ricci 1998;Gladyshev and Meselson 2008). The minute size of bdelloids (from less than 160 to 500-600 µm) allows their long-distance dispersal by wind, water, and animals to access to almost all possible habitats (Bohonak and Jenkins 2003;Fenchel 2004;Kellogg and Griffin 2006). They inhabit both aquatic (mainly freshwater lakes, ponds, and streams) and terrestrial habitats (e.g., mosses, lichens, tree barks, soil and litter) (Donner 1965). Rarely, bdelloids are found in marine and brackish waters Demirkalp et al. 2010;Song 2014).
Analysis of Bdelloidea taxonomy characteristics is problematic because only observation of living and active specimens allows appropriate identification of species. That is why it has not been widely carried out. Furthermore, there are no readily available reagents that can be used to anesthetize them and preserve their bodies fully extended (Örstan and Plewka 2017). Untill recently, only about 460 bdelloid species have been described worldwidely (Segers 2007), but there is ample evidence that the total number of bdelloid species is at least several times greater than the current one (Fontaneto et al. 2011;Robeson et al. 2011). In addition, the intensity of taxonomic researches on bdelloid species in different regions of the world was extremely uneven, thus the species diversity varies greatly from region to region. For instance, over 300 species are known from Europe (Fontaneto et al. 2007), while only about 50 species are found in the Oriental region (Segers 2007).
In China, only 48 bdelloid morphospecies have been reported Koste and Zhuge 1998;Yin and Xu 2016) (Table 1). The first study on the Chinese bdelloid rotifers was reported by Thorpe (1893), who found four species of Rotaria in Yangtze River area in Wuhu city, Anhui Province. After that, few fragmental reports from fresh waters and terrestrial environments in a large region of China were presented (Daday 1906;Stewart 1908;Gee 1927;Wang 1961;Bartoš 1963;Wang 1974;Gong 1983;Zhuge 1996, 1998;Zhuge et al. 1998;Yin and Xu 2016). Up to now, this taxon has not been actively studied in China comparing to Europe or even to Antarctica (Segers 2007), and the biogeography of bdelloids in South Asia is unclear, and their habitat preferences are incomplete. This study aimed to conduct a taxonomic work and evaluate the diversity of bdelloid rotifers in China, especially the poorly investigated tropical zones of the Oriental biogeographic region.

Sampling area, collection procedures and sample processing
A total of 61 samples was collected during the period from September 2017 to October 2018 in 11 provinces and regions of China across its subtropical and tropical zones at altitudes from 0-2850 m above sea level from four types of terrestrial habitat (soil ,  Table 1. Checklist of bdelloid rotifers recorded from China before 2015.
Samples from terrestrial habitats were placed into firmly closed paper envelopes, then dried at room temperature and stored in the envelopes for several weeks or months. Planktonic samples were obtained by filtering 1 to 5 liters of water through a plankton net with a mesh size 30 µm. Benthic ones were collected by scraping the bottom of water bodies with a 500 ml plastic bottle. Periphytic rotifers were obtained by shaking or scraping aquatic plants, then preserved in plastic bottles.
Samples from aquatic habitats were concentrated by a nylon net of 30 µm mesh size, then examined in lab immediately without fixating or anesthetization. Rotifers from mosses, lichens and leaf litter were extracted by washing the substrate with distilled water following the method of Peters (1993). Soil rotifers were extracted by the method of wet-sieving and centrifugation in a sugar gradient (Freckman 1993).

Light microscopy procedures
Rotifers isolated from waters were transferred into a Petri dish and sorted under a dark field dissecting microscope (SZX10, Olympus, Japan) with a magnification of 64×. Selected specimens were placed onto glass slides by using micropipettes, then

Species identification
Species were identified by both external morphology and anatomy using the keys of Donner (1965) and the original descriptions and redescriptions of specific species (Murray 1906;Song and Kim 2000;Yakovenko 2000aYakovenko , 2000bKutikova 2005;Bielańska-Grajner 2013;Song 2014Song , 2015Song and Min 2015;Song and Lee 2017). Drawings of some rare morphospecies were made with Adobe Illustrator CC 2018 and Photoshop CC 2017. All rotifers were measured from screenshots of digital videos after Iakovenko et al. (2013Iakovenko et al. ( , 2015 and Örstan (2018). Total length (TL) in the case of adinetid rotifers is the distance between the middle of the anterior rim of the head excluding rostrum, and the posterior rim of the spur pseudosegment; head length (HL) is the distance between the anterior edge of the head (posterior to the rostrum) and the anterior rim of the antennal pseudosegment, i.e., TL and HL do not include the rostrum, because it was usually bent under the head (Iakovenko et al. 2015). The head length in A. beysunae is the distance between the anterior edge of the head and an imaginary line passing through the innermost denticles of the rakes to better compare it with the original description (Örstan 2018). The number of denticles on each rake is represented formulaically using an 'en dash' (Örstan 2018). We counted the distal foot with the toes as a pseudosegment separate from the one carrying the spurs as Bryce (1894), Donner (1965) and Iakovenko et al. (2013Iakovenko et al. ( , 2015 did.

Species diversity
Fifty-nine morphospecies (including three subspecies) were identified in this survey (Table 3), and the bdelloids that were unidentifiable to the species level were not included in the list. Of them, thirty-nine taxa (including one genus) are new records for China, and thirty-eight species are new records for the Oriental region. The species list of Chinese bdelloid fauna has been increased from 48 to 87. Detailed information about their distribution and ecological information is reported in Tables 2, 3.
During our survey, five collected bdelloids have a general resemblance to known species, but also showed some dissimilar traits from previously described taxa, and they were qualified with 'cf.' and await further analysis. One of these doubtful species, reported as H. cf. spicula Bryce, which showed a upturned dorsal protrusion. Philodina cf. indica Murray, P. cf. proterva Milne and P. cf. parvicalcar showed relative wide range of variations in their head proportion, which need further analyses.
Among these new records, some species are very rare, and few were first found out of their type localities or habitats, e.g., Adineta beysunae Örstan and Habrotrocha ligula loxoglotta De Koning; some new morphological characteristics were observed and need to be added to the original descriptions, e.g., Adineta cf. acuticornis Haigh and Habrotrocha serpens Donner, which are redescribed and illustrated in the next section.
Species richness of bdelliods recorded between 1908 and 2018 in different provinces of China is presented in Figure 1, showing that sampling intensity greatly influenced the species diversity in different regions of China. For instance, the provinces of Guangdong, Yunnan and Hunan were the subject of 26, eight, and nine studies, which recorded 33, 18, and 16 morphospecies, respectively, whereas the provinces of Jiangsu, Zhejiang, Guizhou, Sichuan, Shanghai, Qinghai, Ningxia, and Gansu have no more than four investigations, which only recorded up to six morphospecies for each.  Table 3 Material. Eight specimens found in mosses and two specimens found in lichens, from tropical (GD 6) and subtropical (YN 5-6, SC 2) zones (Table 2).

Redescriptions of some rare morphospecies
Description. Body transparent and colorless, with smooth skin. No eyespots. Rostrum rather long when animal creeps and stretches out, distal rostral pseudosegment semi-circular and flattened. Rostral lamella divided into two broad sickles-like lobes, immobile, laterally elongated, no trace of cilia under the present microscope image. Small oval head, HW 63-90% of HL and 11-16% of TL, HL 15-18% of TL. Five rectangular denticles in each rake.
Neck width not distinct from head and trunk. The width of the first two pseudosegments of neck approximately equal to HW, the second neck pseudosegment much wider and swollen than the first one. Antenna of two pseudosegments, with length 56-64% of the bearing pseudosegment width. Trunk oval, BW 15-22% of TL. Rump conical, TrL 54-67% of TL. The stomach lumen very narrow and Z-shaped Table 3. Bdelloid rotifers found in this study with their updated biogeographic distribution after Segers (2007  ( Fig. 2D). Oviparous; egg oval and smooth, one knob at each pole (Fig. 2C); Vitellarium large with eight nuclei. Foot slim and short, of four pseudosegments. Spurs long, the inner edge of the spurs almost parallel to the straight outer edge for two-thirds of its length, then a small bulge followed by a contraction and tapers to a sharp point (Fig. 2E). SL 4-8% of TL, and 143-193% of SSW. Three long and unsegmented toes. Dorsal toe longer than two ventral toes. Trophi small, round. Dental formula 2/2.
Measurements. The detailed measurements are summarized in Table 4 with a comparison of the original data from Haigh (1967).
Remarks. Adineta acuticornis has not been found since its original description by Haigh (1967) and was considered as an endemic morphospecies of New Zealand (Shiel and Green 1996). It was found in China for the firstly time also in the Oriental biogeographic region recorded in two provinces of China in 2017 or 2018. It was recorded in damp mosses on soil face in the type locality, whereas in this study, numerous specimens were recorded in both dry and damp mosses, and two specimens in lichens on soil surface.
A distinct characteristic differentiating this morphospecies from Adineta vaga Davis is its wide and rostral lamellae which are slightly wider than the anterior head, while the rostral lamellae of A. vaga are narrower than the anterior head. It differs from Adineta glauca Wulfert by its spur shape, which is short and has a flat base, while A. glauca spur with a swollen base. This morphospecies differs from Adineta longicornis Murray by its spur shape which has bulge, while A. longicornis spur is slender and acute (Murray 1906: 5a, 5b).
The general morphology of the Chinese specimen conforms to the description of the New Zealand population, except the position of the spur contraction is closer to the tip (the contraction is in the middle of the spur in Haigh's description) and the stomach lumen do not have distinct two loops as Haigh's description. A comparison with Haigh's (1967) body dimensions showed a similar body proportion (Table 4). Since there was no genetic evidence to prove it actual systematic status, we assigned 'cf.' (resembling original description) as the status of this find. Besides, we observed three new morphological features missed by Haigh (1967): each rake with five denticles, a larger vitellarium with eight nuclei and egg with one knob on each pole. Figure 3; Table 3 Material. Numerous specimens found in leaf litter from three provinces (GD13-14, 25-26, YN7, JS1) across tropical and subtropical zones. One specimen found in dew on leaves from Southwest of China (YN8) ( Table 2).
Neck distinct from head, the first two pseudosegments of neck narrower than HW. Trunk oval. Posterior end of the first rump pseudosegment with a pair of lateral angular knobs.
Foot of five pseudosegments with two pairs of lateral knobs on its first two pseudosegments, FL 14-22% of TL. Spurs long and sturdy, with short interspace, SL 6-8% of TL, 172-284% of SSW. Three short unsegmented toes. Ventral toe longer than two dorsal toes. Dental formula 2/2.
Remarks. This is the second report of this morphospecies since its original description by Örstan (2018) in rainwater and plant debris from the United States. In the present study, A. beysunae was found in leaf litter and dew on leaves. And interestingly, it  was abundant in 60% of all leaf litter samples. Our study suggested A. beysunae might have a habitat preference for leaf litter and temporary waterbodies.
Description. Body slender and transparent, integument smooth. Rostrum long and strongly bent ventrally. Rostral lamellae divided into two semi-circular lobes and wider than the anterior rim of rostrum. Head similar to hexagon, HW 89% of HL. Corona slightly narrower than collar, with papillae clearly seen in the middle of each trochus, CW 97% of HW. Trochal discs separated by a narrow, V-shaped sulcus, in which a cylindrical ligula bends obliquely to the dorsal side (Fig. 4F, G). A slight contraction near the tip which then forms a small papilla on the tip of the ligula, attaining the level of the discs at the inner side (Fig. 4D, E). Upper lip a flat bow. Neck also bent ventrally when animal creeps. The first pseudosegment of neck slightly narrower than the head at the corners of the mouth, not distinct from head and trunk. A pair of lateral cuticular bulges on the dorsal antenna pseudosegment. Antenna with two segments, its length 30-40% of the bearing pseudosegment width.
Trunk slender and cylindrical, TrL 59-67% of TL. Rump conical, with both pseudosegments somewhat swollen and strong arched up dorsally and roofing the foot, the posterior rim of the second pseudosegment creased, RL 8-10% of TL.
Foot short with three pseudosegments, FL 6-8% of TL. Bulbous spurs short and triangular shape, with distinct tips and wide interspace, base swollen. The width of interspace 114% of SL, 97% of the swollen width. Three stout unsegmented toes of the same length. Trophi small, dental formula 3/3.
Remarks. Habrotrocha ligula loxoglotta was originally described from Holland (De Koning 1947), later reported from beech-oak needle-litter in Germany, from dry mosses in France (Donner 1951) and from mosses in Austria (Kutikova 2005). In this study, it was found for the first time in China (Yunnan Province) as well as in the Oriental region. Figure 5; Table 3 Materials. Five specimens found in dry mosses on bark from southern China (GD6) ( Table 2). Description. Body extremely slender (BW is only about 6% of TL), long and cylindrical, integument transparent and smooth. Rostrum rather long, with two pseudosegments. The first pseudosegment circular and slightly bigger than the second one which often contracted (Fig. 5B). One whole semi-circular lamella not divided into lobes, rather large, broader than the rostrum, covers the long and stiff tactile cilia. Head slender, HW 44% of HL, 22% of TL. Corona also slender, a little wider than the head, CW 107% of HW. Trochal pedicels grown together, central rounded papillae on each separated trochal discs, incline to the dorsal side. Upper lip low, narrow and without lobes, slightly arched, not covered by the incompletely extended rostrum. Lower lip spoon-shaped, strongly protrudes forward.

Habrotrocha serpens Donner, 1949
Neck slender. Throat very short, pharyngeal tube long, undulating before the mastax. Dorsal antenna slender, with two segments, its length 86% of the antennal pseudosegment width. Trunk slender, the two lateral sides of trunk almost parallel when animal fully extended, the last trunk segment often strongly contracted. Rump conical, with both pseudosegments swollen, arched up dorsally and roofing the foot, RL 12% of TL.
Foot very short, of four pseudosegments, FL 5% of TL. Spurs triangular and have swollen base, each with curved inner margins and a very small interspace. SL 63% of SSW. Three short unsegmented and of approximately equal length toes (Fig. 5C). Trophi large, dental formula 4/4.
Measurements. The detailed measurements are summarized in Table 5 with a comparison of the original data from Donner (1949;1970).  Donner (1949;1970 Remarks. The general morphology of our sample conforms with the description of the Austrian population except that the rostrum is not always fully expanded to/ exceeding the upper lip in a feeding position. It may because of the second pseudosegment of rostrum often contracted. Additionally, we observed three approximately equal-lengthed toes which were not clear in Donner's (1949) description. This morphospecies was first described from soil from Austria by Donner (1949), and then recorded in moss and soil from Austria and Czechoslovakia (Bartoš 1951); in needle litter, Calamagrosits turf, grasses and leaf litter from Austria, Czechoslovakia, Romania, and Spain (Donner 1965(Donner , 1970. It is new for China as well as for the Oriental region.

Taxonomy and diversity of bdelloid rotifers in China
Only 48 species were recorded in eleven studies conducted in China between 1908 and 2018 (Table 1), which implies that taxonomic and diversity researches on Chinese bdelloids are very limited. Moreover, only 65% (31 of 48) of the recoreded morphospecies were illustrated and described (e.g., Wang 1961;Bartoš 1963;Gong 1983), and many of the illustrations are inaccurate, not showing important details and the descriptions are not detailed enough to verify their identity. Besides, there are 17 morphospecies listed in the literature without any illustrations, photographs or descriptions (e.g., Koste and Zhuge 1996;Yin and Xu 2016), which need further verification. Also, some species were recorded out of their specific habitats (e.g., H. thienemanni Hauer and P. roseola Ehrenberg) and some recorded in unusual environments (e.g., Habrotrocha pulchra Murray, H. thienemanni Hauer, Mniobia tentans Donner, Macrotrachela bullata Murray, and Philodina vorax Janson were abundant in glacier over 5500 m a.s.l.) (Table 1). These ecological differences may hide potential cryptic taxa and need further studies combined with new techniques such as DNA taxonomy.
Due to a lack of insufficient taxonomic and diversity research in China, species richness is extremely uneven in different provinces of China. More morphospecies were recorded in the Tibetan Plateau (27 morphospecies) and Guangdong Province (22 morphospecies) with more samples collected (Stewart 1908;Bartoš 1963;Wang 1974;Gong 1983;Yin and Xu 2016). Four new morphospecies were reported in Guangdong, including Habrotrocha modesta Bartoš, H. flexicollis Bartoš, Pleuretra proxima Bartoš, and P. similis Bartoš. Unfortunately, they were never found again, and these are considered as disappeared 'endemic morphospecies' in latter researches. Research on different habitats of bdelloids were also uneven. Most studies were only focused on fragmented fresh-water bodies or mosses, but did not pay attention to other habitats such as brackish waters, soil and litter. Therefore, more studies are necessary to explore the taxonomy and diversity of bdelloid rotifers in China, especially with a focus on the areas and habitats that were not well studied.

Geographical distribution and ecological information of Chinese bdelloids
The high dispersal potential of bdelloids has supposedly led to their generally cosmopolitan distribution (Fenchel and Finlay 2004). The previous extensive sampling of bdelloids confirms that some species can be found in distant areas on different continents, but also some species can only be found in specific area (Donner 1965;Segers 2007). At present, studies of biogeography on these taxa are not comprehensive. For example, Adineta ricciae Segers and Shiel, previously considered as an Australia-endemic species, was observed in South China (the Oriental region); A. beysuanae has been described in a container filled with plant debris and rain water from the United States (Örstan 2018), and it was then found in similar or drier habitats (dew and leaf litter) from China. These findings imply that the currently described distribution of bdelloids is incomplete and may be strong influenced by the sampling effort, especially in the poorly investigated areas, such as South Asia.
With study extending to more ecological habitats, some morphospecies were found in a broader range of habitats. We observed five brackish water morphospecies: Rotaria rotatoria Pallas, R. laticeps Wulfert, R. tridens Montet, Philodina megalotrocha Ehrenberg and Macrotrachela hewitti Murray. They were found among aquatic plants or brackish temporary puddle with sediment in mangrove. Noticeably, R. rotatoria was abundant and dominated in Gracilaria lichenoides (a red alga) culture ponds, possibly because G. lichenoides could provide suitable habitats. These ecological differences seem to represent different ecological niches, which may hide some interesting phenomena of separated evolutionary lineages. For example, Adineta vaga, which occurs in the multiple types of habitats, has a large amount of cryptic diversity (Fontaneto et al. 2011).

More extensive surveys of bdelloids in Asia
More than half of the recorded morphospecies from this study (some presumed cosmopolitan) are new records for the Oriental region as well as for South Asia. As there are still considerable gaps in faunistic studies in the Oriental region, we do not yet have sufficient faunistic data to determine the true distributions of bdelloids. Our findings highlight the need for further taxonomic studies on bdelloids in Asia. Furthermore, asexual bdelloids have evolved independently in spite of being effectively sympatric, indicating that they may adapt to different ecological niches, thus the type of habitat is a key player for microscopic species diversity and evolution (Birky et al. 2005). Applications of molecular phylogeny for identification of bdelloid species would be invaluable in uncovering the actual systematic status of some euryoecious or variable morphospecies so that we may better understand the true distribution of bdelloid species.