Henlea rodionovae, Rota & Martinsson & Erséus, 2018

Rota, Emilia, Martinsson, Svante & Erséus, Christer, 2018, Two new bioluminescent Henlea from Siberia and lack of molecular support for Hepatogaster (Annelida, Clitellata, Enchytraeidae), Organisms Diversity & Evolution (New York, N. Y.) 18 (3), pp. 291-312 : 301-304

Henlea rodionovae sp. n. ( Figs. 6–7 )

Holotype MCZR OLIGOCHAETA 0214 , whole-mounted, mature specimen, fixed in Bouin Nov 2015.

Type locality Bolshie Koty (51° 54 ′23.5″ N, 105° 04′ 32.1″ E), a former gold-mining site now belonging to Pribaikalsky National Park , 70 km from Irkutsk, soil inside and immediately outside greenhouse (loc. 6) .

Paratypes All from the type locality (loc. 6). MCZR OLIGOCHAETA 0215–0216 , two posteriorly amputated specimens (vouchers CE27993 , CE27994) preserved in 85% ethanol, Nov 2015; posterior parts used for DNA extraction (COI Barcodes, Nuclear markers and GenBank nos. listed in Table 1). MCZR OLIGOCHAETA 0217 , one mature specimen, whole-mounted, unstained, Dec 2015. SMNH Type Collection 9079, one mature specimen, whole-mounted, Nov 2015. SMNH Type Collection 9080, one posteriorly amputated specimen (voucher CE31472 ) preserved in 85% ethanol, Apr 2017; posterior parts used for DNA extraction (COI Barcode, Nuclear markers and GenBank nos. listed in Table 1) .

Additional material One whole-mounted, submature specimen from forest soil by Kacha river (loc. 1), fixed in Bouin Jan 2002. Material not preserved from Listvyanka (51° 50′ 52.7″ N, 104° 52′ 43.0″ E), near the southern tip of Lake Bajkal, soil within and around greenhouse (loc. 5), Aug 2015. Numerous specimens from loc. 6 in first author’ s collection.

Etymology Named for Natalja S. Rodionova, researcher of the Russian Academy of Sciences, for her lifelong dedication and excellent accomplishments in the biochemical study of enchytraeid bioluminescence.

External Colour yellow with a shade of orange-brown, emitting flashes of bluish light upon stimulation. Body wall relatively soft, transparent, contractile. Live body size: length 20–24 mm, width at V 650–680 μm, at clitellum 750–850 μm, posterior half of body distinctly tapering caudad. Fixed dimensions: length 14 mm; width at V 650–720 μm, at clitellum 730– 1000 μm. Segments 60–67, average 64.6 (n = 8). Prostomium blunt conical, narrow at base, as long as wide, in vivo 200– 230 μm from head pore to tip, very mobile ( Fig. 6a, b, c, d, f, g ), on all sides incised by grooves, some of which transversal, longitudinal or radial; grooves vary in position between specimens; longitudinal grooves extend ventrally also all along peristomium ( Fig. 6b, f ); grooves visible also after fixation. Peristomium (segment I) in vivo 250–280 μm long, 380– 520 μm wide; after fixation 130–150 μm long, 450 μm wide. Head pore as a transversal slit at 0/1. Epidermal gland cells small, irregularly shaped, forming 3 complete rows ( Fig. 6d, e, f , arrows). Chaetae straight, arranged in lateral and ventral bundles, 2÷4(5) – 2÷7: 2÷4(6) – 2÷7, most numerous around segments XL; the outer chaetae in a bundle larger than the inner ones (Suppl. Fig. 2 ), length up to 130–150 μm (caudally), thickness up to 12 μm. Chaetae of XII missing in mature specimens. Clitellum over 1/4 XI–3/4 XIII, slightly elevated. Paired male pores ventral in XII, inside deep male bursae; clitellar gland cells absent between them. No copulatory glands. Paired spermathecal pores in lateral lines at 4/5.

Internal Brain ( Fig. 6g ) concave anteriorly, deeply indented posteriorly, longer than wide, ca. 140–160 μm wide and 190– 210 μm long. Septa 6/7–8/9 thickened. Oesophageal appendages adhering to gut wall, forming frilled ridges, giving off numerous (5–6 per side) branched extensions into coelom dorsally and ventrally in VI ( Fig. 7c, e, f , arrows). Intestinal diverticula as a multitubular apple-shaped structure surrounding gut and occupying much of the coelomic cavity in VIII, length 300–320 μm, maximum cross diameter at midlength, ca. 400–500 μm ( Fig. 7a, b, c, d ); mass of tubules symmetrically paired, arising from intestine near 8/9, extending forwards adhering to gut, midlaterally the anterior border is concave. The tubules end blindly against cephalic border of the organ. Chloragogen cells from IV, finely granular, forming a dense opaque orange-brown or chestnut-brown tissue from X ( Fig. 7a, c ). Dorsal vessel originating in IX ( Fig. 7b ), with heart-like expansions in IX, VIII and VII. Preclitellar nephridia 6 pairs, in 5/6–10/11, efferent duct arising anteroventrally ( Fig. 7g ). Coelomocytes abundant, with distinct nucleus and evenly fine-granulated cytoplasm, discoid, shorter than half the largest outer chaetae ( Fig. 7d, g ), in vivo 40–50 μm, at most 60 μm across; after fixation, 30–35 μm. Seminal vesicles X–XI. Sperm funnels elongate conical, narrowing entally to ectally, in vivo ca. 400 μm long and 110 μm wide; collar 20 μm high, wider than the body of funnel, reflected ( Fig. 7j ). Heads (i.e., nuclei) of spermatozoa about 50 μm long. Vasa deferentia confined to XII, long, forming many regularly loops, relatively thin (in vivo 13– 15 μm thick) ( Fig. 7j ). Penial bulbs simple, small, of lumbricilline-type. Spermathecae one pair, in V ( Fig. 7h ). Some accessory gland cells at each spermathecal pore; glands elongate, club-shaped, attached by their narrower ends to ectal pore, each gland 75 μm long, 40 μm wide at bottom. Ectal ducts stout, cylindrical, in vivo 200–300 μm long and 60 μm wide at pore, thick-walled (ca. 15 μm), with inner canal expanding markedly from pore to spermathecal midcourse (from 6 to ca. 30 μm). Ampullae oval, in vivo 100 μm wide, externally not clearly demarcated from ectal ducts. Walls of ampullae ca 15 μm thick. Ental ducts 280 μm long, spindle sac-like, merging into a common chamber communicating with oesophagus in posterior of V ( Fig. 7h ).

Taxonomic relationships Henlea rodionovae sp. n. belongs to the same group of Henlea as H. petushkovi sp. n., i.e., it possesses Hepatogaster -like gut diverticula in VIII consisting of masses of branched tubules (see Suppl. Table 1). Morphology and DNA (see below) indicate that the two species are very closely related, but differ from each other by the body wall strength, the structure of prostomium, brain shape, size of coelomocytes and structure of intestinal diverticula. The number of chaetae and body segments as well as the size of coelomocytes makes H. rodionovae most similar to H. tubulifera from Michigan, but the oeosophageal appendages, the sperm funnels and the spermathecae have different structure. The body colour and geographical location would instead suggest kinship to H. ochracea , described by Eisen (1878, 1879) from the Siberian Arctic. However, the latter species was originally described with a smaller size (15 mm, 52 segments) and with efferent duct arising from the posterior end of the nephridial body. Later, Welch (1919) gave a more extended description of Eisen’ s taxon based on material from the Northwest Territories of Canada, a description that confirmed the aberrant nephridial morphology.

Despite these morphological similarities with species known from other parts of the northern hemisphere, this study shows that H. rodionovae sp. n. is most closely related to H. petushkovi sp. n., and that these two taxa can even hybridize with each other.

Figures

Fig. 6 Henlea rodionovae sp. n. Morphology of prostomium and body wall in living worms. a–d, f, g Different views of prostomium (a, g dorsal. b, c ventral. d, f ventrolateral). e Body wall of segments III–V. Arrows point to rows of epidermal glands. Scale bars: a–g 200 μm

Fig. 7 Henlea rodionovae sp. n. Internal anatomy as seen in living worms. a, b Dorsal view of segments VI–X and close-up of gut diverticula in VIII. c, d Ventral view of segments VI–X and close-up of gut diverticula in VIII. e, f Dorsal and ventral views of oesophageal appendages and their branches (arrows) in VI. g Close-up of nephridium (black arrow, nephrostome; white arrow, efferent duct) and coelomocytes (cc). h Spermatheca. j Sperm funnel. Scale bars: a–j 200 μm

Fig. 2 Histological cross sections of the gut diverticula in Enchytraeus leptodera Vejdovský (a) and Enchytraeus ventriculosus d’Udekem (b), and in Archienchytraeus nasutus Eisen (c), as shown by Michaelsen (1886) and Michaelsen (1889), respectively