Chimaeradasys polytubulatus, Kieneke & Todaro, 2021

Kieneke, Alexander & Todaro, M. Antonio, 2021, Discovery of two ‘ chimeric’ Gastrotricha and their systematic placement based on an integrative approach, Zoological Journal of the Linnean Society 192 : -

CHIMAERADASYS POLYTUBULATUS SP. NOV.

( FIGS 3, 4)

Zoobank registration: urn:lsid:zoobank.org:act:DD1807A1-ED8C-4D76-9BE6-435BAFAAC278

Ty p e l o c a l i t y: I t a l y, S a r d i n i a, C o s t a Pa r a d i s o (41°3’8.84’’N, 8°56’15.71’’E); collection made on 4 September 2010 by scuba-diving at 35 m depth.

Type specimen: The single specimen that was documented microscopically and described herein is the holotype, fixed by monotypy (Article 73.1.2. of the International Code of Zoological Nomenclature, ICZN, 1999). The holotype specimen does not exist any longer (Article 73.1.4. of the International Code of Zoological Nomenclature, ICZN, 1999). Declarations according to recommendations 73G–73J ( ICZN, 2017): the living, anaesthetized specimen was extensively investigated microscopically in order to gain optimal, taxonomyrelevant data. The morphological distinctness and uniqueness is the reason for naming this new taxon now. The specimen was recovered from the microscopic slide and stored in pure ethanol until dissolving it in order to extract genomic DNA for genetic analyses. The remaining genomic DNA was stored at –20 °C. None of the remaining sediment samples yielded further specimens of the new species. Several gastrotrich taxonomists have been consulted prior to this taxonomic act. Following recommendations regarding taxonomic processing of soft-bodied meiofauna ( Garraffoni et al., 2019) were applied: the specimen was observed and documented using highresolution objectives and DIC, series of high-resolution digital images were deposited in MorphDBase (https:// www.morphdbase.de/?A_Kieneke_20200503-S-2.1 respectively https://doi.org/10.20363/mdb.mjky-s935).

Material examined: Only the single holotype specimen was surveyed using light microscopy with DIC. Furthermore, the genomic DNA was extracted from the single specimen and used as template for amplification of nuclear, as well as mitochondrial, gene fragments, although amplification has failed so far .

Ecology: Sediment was made up of coarse, well-sorted sand (mean grain size = –0.02 phi, sorting = 0.48). Temperature and salinity of the pore water at the time of tha samplings were 13 °C and 38 PSU, respectively.

Diagnosis: A Chimaeradasys of 391 µm in total length and 74 µm in maximum width. Anterior adhesive tubes, two per side, in a row just posterior to the mouth; TbV and TbD, absent; TbL, one per side at U86; TbVL, ten on the left side and 12 on the right side; the first tube of each side in the anterior pharyngeal region at U13 and the others unevenly distributed along the anterior two-thirds of the trunk; TbP, three per side, two at the end of each peduncular branch and one along the medial side, inserting ventrally near to the others. Mouth wide, (up to 60 µm in breadth), leading to a shallow, funnel-shaped buccal cavity and surmounted dorsally by a scalloped oral hood. Pharynx 92 µm in length, with pores at U28; PhIJ at U32. Intestine straight, slightly wider at midbody; anal opening at U78. Hermaphrodites; single testis on right body side, beginning just posterior to the PhIJ at U34; caudal organ glandomuscular; frontal organ sack-like, both with spermatozoa inside; ovary unpaired, large egg dorsal to intestine centered at U51.

Etymology: The species epithet is derived from Greek Π ολύ, much, and Latin tubulus, tubule, referring to the high number of ventrolateral adhesive tubes.

Description: The body measures 391 µm in total length ( Figs 3A, B, 4A, B). The habitus shows a clear widening in the region of the mouth opening (U0 to U8) that is surmounted by a scalloped oral hood and an expanded mouth rim. The mouth measures 60 µm in diameter ( Figs 3B, 4B, C). The region of the pharynx (U8 to U32) is slightly narrowed followed by the slightly widened trunk until approximately U52. The rear trunk continuously narrows until U89 and, between U89 and U96, it is just a narrow peduncle that splits into a pair of short appendages at U96 ( Figs 3A, B, 4A). Body width is 61/50/49/74/60/46/30/11 µm at U5/10/20/52/70/80/85/90, respectively. The trunk is dorsoventrally flattened with a flat venter and a moderately arched dorsum.

The adhesive tubes occur as anterior (TbA), lateral (TbL), ventrolateral (TbVL) and posterior (TbP) groups ( Figs 3A, B, 4A–C). There are two pairs of TbA. At U11, these two pairs of TbA are placed close to the ventral (posterior) mouth rim. Especially the inner pair may be masked by the withdrawn ventral, lip-like mouth rim ( Figs 3B, 4C). The distance between the insertions of both inner tubes is 16 µm and between the outer TbA it is 39 µm. The outer TbA are about 9 µm long, while the inner TbA are shorter (6–7 µm) and much more delicate. A single pair of TbL inserts at U86 ( Figs 3A, B, 4A); TbL are about 23 µm long and project laterally and towards posterior at an angle of 38° against the longitudinal axis of the animal. The TbVL occur in different numbers on left and right sides, mostly in the middle-third of the body ( Figs 3B, 4B). In total, there are ten pairs of TbVL inserting at U14, between U32 and U37, at U41, U46, U52, U58, U65 and U68, respectively. The most anterior TbVL are just 8–9 µm long, while all other TbVL measure between 15 and 19 µm in length. All TbVL project laterally and towards the posterior at an angle between 29° and 55° against the longitudinal axis of the animal. In addition to these paired TbVL, the examined specimen shows two further tubes on the right side, between U59 and U61. In total, there are six bilaterally arranged TbP, all confined to the peduncular branches ( Figs 3B, 4A). Each branch shows two tubes at the distal end, arranged in a V-shaped manner, and a third tube along the medial margin, inserting ventrally near the other two tubes. Distal tubes measure 10 µm in length, the medial tube is 12 µm long.

The ventral locomotory cilia start at U10 shortly posterior to the ventral mouth rim and end posterior to the anus at about U85. The cilia cover the entire ventral surface and are distinctly arranged in transverse rows ( Figs 3B, 4C). The field of locomotor cilia has a more or less uniform width of about 42 µm, but the density of cilia decreases slightly towards the posterior. Length of ventral locomotor cilia varies between 10 and 15 µm. At least four presumptively sensory, up to 12 µm long and pairwise arranged cilia insert on the dorsal surface of the oral hood. Further putative sensory cilia insert dorsolaterally along the trunk, arranged roughly pairwise ( Figs 3A, 4A, D). Some of these cilia are associated with epidermal glands. Along the dorsal (anterior) and lateral parts of the lip-like mouth rim, there are 13 presumptively sensory papillae, each one measures about 4 µm in height ( Figs 3A, B, 4C, D). The seven most anterior sensory papillae are evenly spaced from each other, while the distance between papillae decreases towards the lateral rims of the mouth opening. There is a c. 14-µm long cilium inserting on the tip of each papilla.

The surveyed specimen has seven pairs of more or less bilaterally arranged epidermal glands at about U8/18/25/33/62/71/79, respectively ( Figs 3A, 4A, D). The irregular, globular or slightly pear-shaped glands have a diameter of 9 to 11 µm and appear with granular content. At least for some glands, we were able to observe putative associated sensory cilia. In the area of the trunk, between U35 and U60, there are further epidermal glands in a roughly pairwise arrangement. However, these glands are much smaller and measure just 4 to 6 µm in diameter. Regarding the epidermal gland system, there is a certain degree of asymmetry, i.e. there are also some unpaired glands.

The digestive tract starts anteriorly with the ample mouth opening that continues in a funnelshaped buccal cavity. Fold-like structures at the dorsal epithelium of the buccal cavity and along the lip-like mouth rim indicate a certain contractile property of the whole oral hood ( Figs 3B, 4C). The pharynx is 91 µm in length with pharyngeal pores that open ventrolaterally at U28. The pharyngeointestinal junction is at U32 ( Fig. 3A). The width of the musculoglandular pharynx is 48/18/19/23/51/25 µm at U6/11/20/25/28/32, respectively. The intestine spans from U32 to U82, where it opens externally with an inconspicuous ventral anus. It has a more or less constant width of about 20 to 25 µm in its anterior half and tapers gradually toward the anus. Widths are 25/22/20/15/11 µm at U40/50/60/70/80, respectively. Only an inconspicuous regionalization of the intestine is recognizable with a higher density of refractive, presumptively enzymatic vesicles in the anterior twothirds and bigger, vacuole-like digestive vesicles in the posterior-third ( Fig. 4B). At low magnification, the middle-third of the intestine shows up in a greenish to brownish colour. However, the nature of the gut content was not ascertainable.

There is a single, 165-µm long right testis spanning from U35 to U76, including the posteriorly directed vas deferens that starts at about U60 ( Figs 3A, 4F). However, the actual termination of the sperm duct could not be determined with certainty (see below). The maximum width of the single testis is 16 µm at U50. Apart from the most anterior portion that is filled with vesicular content (approximately U35 to U40), the whole testis, including the vas deferens, is stuffed with filiform spermatozoa with clearly spiralled sperm heads ( Fig. 4F). Lateral to the posterior (distal) portion of the vas deferens, between U68 and U76, there is a thick-walled, muscular, hollow structure, presumptively the caudal organ ( Figs 3A, 4F, H). It is roughly club-shaped and narrows posteriorly (maximum width is about 8 µm at the muscular section at U74) into a kind of outlet duct that curves to the left and ventrally under the intestine. The anterior (proximal) portion seems to be folded by almost 180° and reclines parallel and medial to the muscular section of the caudal organ ( Figs 3A, 4F, H). Dorsal to the intestine in a median position, between U67 and U75, there is a sac-like structure measuring roughly 10 by 31 µm, the frontal organ ( Figs 3A, 4G). In addition to the testis, there are filiform spermatozoa with clearly spiralled heads inside the frontal and caudal organs ( Fig. 4F–H). While sperm lies in dense, more or less parallel bundles inside the testis and the caudal organ, there are only few, single and coiled spermatozoa inside the frontal organ. Slightly dorsal and lateral to the right side of the intestine there is an unpaired ovary spanning between U40 and U67 ( Figs 3A, 4B, E–H). Between approximately U60 and U67 there are a few early oocytes (10–20 µm in diameter) that mature in a cephalic direction. Between U40 and U60 there is a large, roughly drop-shaped egg (26 by 80 µm) densely filled with refractive vesicles and containing a large nucleus (almost 19 µm in diameter) with distinct nucleolus ( Fig. 4E).

Remarks on external anatomy and reproductive structures: The observed specimen is characterized by an interesting asymmetry, which concerns the adhesive tubes of the ventrolateral series. More specifically, it shows ten TbVL on the left side and 12 TbVL on the right side. Phenomena of asymmetry regarding tubes of the ventral and ventrolateral series has previously been reported several times within marine Gastrotricha and appear to be particularly frequent in taxa of the subfamily Thaumastodermatinae , e.g. Ptychostomella orientalis Lee & Chang, 2003 , Tetranchyroderma inequitubulatus Todaro, Balsamo & Tongiorgi, 2002 and T. weissi Todaro, 2002 (Todaro, 2002; Todaro et al., 2002; Lee & Chang, 2003).

Similar to the specimen of C. oligotubulatus , this specimen also has some uncertainties concerning the three reproductive structures: testis, the frontal and the caudal organs. We were not able to unambiguously detect external and/or internal pores. However, the spermatozoa inside the frontal organ are in closest proximity to the mature or maturing egg, respectively. Furthermore, the connectivity between the three organs could not be clarified with certainty. But as in the other new species, our incomplete data indicate accordance of function of these reproductive organs with that of phylogenetically allied species of the taxon Thaumastodermatinae . Hence, the vas deferens and caudal organ much likely open independently on the ventral side or throughout the anus and there probably is a connection between the frontal and the caudal organ lumina. On the left side of the intestine, more or less in the same region as the frontal organ, around U70, there is an accumulation of droplets and globular material about 7 µm wide ( Fig. 4F, H). We were not able to clarify whether this is just vesicular mesodermal tissue, or secretory material of a new type of gland.

Variability and remarks: Information on character variability among individuals of the new species is currently not available.

TAXONOMIC AFFINITIES

Based on general appearance, we initially regarded the discoveredspecimenseitherasbelongingto Dendrodasys (fam. Dactylopodolidae ), due to the branched caudal peduncle, or to the Thaumastodermatidae , perhaps close to Ptychostomella in virtue of the wide mouth and the bare cuticular covering. Undeniably, at first sight both new species appear like ‘chimeras’ of both mentioned genera, being ‘compound’ from the anterior two-thirds of a Ptychostomella and the posterior-third of a Dendrodasys ( Fig. 5A–C).

The peculiar posterior body region, ending with the narrow peduncle that splits into two short appendages, whose tips are equipped with adhesive tubes, resembles the anatomic condition of Dendrodasys (see, for example, the recently described D. rubomarinus Hummon, 2011 ; see also Fig. 5A). The two noticeable and posteriorly directed lateral adhesive tubes shown by the new species amplify the overall similarity of their posterior body region with that of Dendrodasys because a pair of similar tubes is present also in members of the latter taxon (e.g. D. duplus Lee, 2012 and D. gracilis Wilke, 1954 ; see also Fig.5A). However, there is an important difference regarding this character: while in Dendrodasys these tubes insert on the elongate posterior peduncle (less often at its base), in Chimaeradasys they are clearly inserted anterior to it. The situation concerning the gonads is somewhat ambiguous. In general, Dendrodasys possesses paired testes and paired ovaries (e.g. Wilke, 1954; Schmidt, 1974; Valbonesi & Luporini, 1984; Hummon, 2011), but at least one species, Dendrodasys affinis Wilke, 1954 ( Fig. 5A) possesses a single right testis and a single ovary (see: Hummon et al., 1998), therefore matching the new species concerning these characters. Another species, D. duplus , shows a certain reduction of the right testis ( Lee, 2012) pointing to a phenomenon that is recurrent in Gastrotricha (e.g. in Urodasys ; see: Schoepfer-Sterrer, 1974; Todaro et al., 2019a). The reported instances indicate that reduction and/or complete loss of testes, respectively, in Dendrodasys happened at least twice independently, while such a character loss occurred only once within the stem lineage of the Thaumastodermatinae (Todaro et al., 2011; Kieneke & Schmidt-Rhaesa, 2015). But there are even more striking dissimilarities between both new species and Dendrodasys : first, the anterior adhesive tubes of Dendrodasys (always a single pair) are borne on extensible fleshy bases; second, none of the hitherto known species of Dendrodasys possesses lateral or ventrolateral adhesive tubes ( Fig. 5A); and, third, there are only two adhesive tubes per posterior appendage instead of three like in both new species of the new genus ( Fig. 5A, B).

Species of the new genus are also strikingly similar to members of Thaumastodermatidae Remane, 1927 . Species of this family possess an ample mouth surmounted by an oral hood, which is diagnostic of the family. Importantly, the new species also share additional characteristics with members of the subfamily Thaumastodermatinae Remane, 1927 , including the following: (1) anterior adhesive tubes originating directly from the body surface, (2) presence of ventrolateral adhesive tubes, (3) paired posterior ‘adhesive pedicles’, (4) small posterior pharyngeal pores, (5) unpaired right testis and (6) single ovary with gametes maturing in a caudocephalic direction ( Fig. 5B–E; Ruppert, 1978; see also: Kieneke & Schmidt-Rhaesa, 2015). Among the Thaumastodermatinae , the new species are most comparable with species of Ptychostomella with which they share also the arrangement of the anterior adhesive tubes and the unarmored (bare/smooth) cuticle ( Fig. 5B, C). However, due to the extremely exceptional shape of their posterior region, we regard both new species as part of an evolutionary lineage distinct from that of Ptychostomella , supporting, therefore, the erection of a new genus that we name Chimaeradasys here. The results of the molecular phylogenetic analyses (see below) provide further support for this proposal.

Several morphological differences between the two discovered specimens support the idea of two species within the new genus. These are a smaller total body length, less anterior and ventrolateral adhesive tubes, less sensory papillae and a lower number of epidermal glands in Chimaeradasys oligotubulatus compared with C. polytubulatus . Furthermore, C. oligotubulatus possesses a pair of ventral adhesive tubes that are absent in C. polytubulatus . Also, the geographic distance between the two type localities may account for the erection of two new species. Of course, we cannot completely rule out that the aforementioned differences just represent the range of character variability of one widespread species, because we only were able to investigate a single specimen per location. However, the presence of two separate species, one occurring at the Azores and another at Sardinia, is the most likely hypothesis that has to be tested once further data are available.