First records of Enchytraeidae (Annelida, Clitellata) from the Three Parallel Rivers region Author Chen, Jing Author Jiang, Wanxiang Author Xie, Zhicai text Zootaxa 2016 4093 2 275 284 journal article 10.11646/zootaxa.4093.2.8 b448cb2d-ac08-4032-8a36-9e64f9ec6ce6 1175-5326 261808 6F6078C8-1245-46B7-BE63-8DEE720A35D4 Mesenchytraeus laojunensis sp. nov. ( Figures 2 , 3 , Tables 1 , 2 ) Type material. Holotype . Fully mature, whole-mounted specimen, stained, YNO201400001. Coniferous forest, Mt. Laojun, Yunnan Province (99° 43.185E , 26° 37.956N , 3958 m above sea level), under snow, in dark, sandy soil, under roots of Abies and moss, coll. J. Chen and W. X. Jiang, 27 April 2014 . Paratypes . YNO201200002–10, 4 dissected and 5 whole-mounted adult specimens, type locality, collection data as for holotype . Other examined material. About 10 submature and 15 immature specimens, also from type locality, March 2012 , October 2013 and 2014, coll. J. Chen and W.X. Jiang. Etymology. The new species is named after the type locality of Laojun Mountain in the Southwestern region of China . FIGURE 2. Mesenchytraeus laojunensis sp. nov. (A–I): A . Ventral chaetae in II. B . Ventral chaetae in V. C . Ventral chaetae in VII. D . Sperm funnel (in vivo). E . Pattern of clitellar glands. F . Coelomocyte. G . Sperm bundle. H . Spermatheca. I . Male organ in clitellar region. Scale bars: A, B, C, E, F, G 25 Μm; D, I 100 Μm; H, 50 Μm. Abbreviations: acg, accessory copulatory glands; at, atrium; bsf, body of sperm funnel; dt, diverticulum; ecd, spermathecal ectal duct; gr, granulocyte; hs, heads of spermatozoa; hy, hyalocyte; s, spermatozoa; sa, spermathecal ampulla; sp, spermathecal pore; vd, vas deferens. Description . Worms stout, grey or yellowish in vivo but intensely white in some preclitellar regions due to accumulations of coelomocytes. Body length 18–28 mm in vivo and 17.5–23 mm after fixation ( holotype : 22 mm ), body width 0.7–0.8 mm at V, 0.8–1.0 mm at clitellum ( Fig. 3 A). Segment number 64–72 ( holotype : 72). Head pore near the apex of prostomium, longitudinal slit ( Fig. 3 B). Epidermal gland cells conspicuous in I–III (especially on peristomium, with irregular distribution and accumulation at level of chaetae) but underdeveloped in remaining segments. Chaetal formula: 3,4 – 3,4: 4–6 – 4,5. Chaetae sigmoid and nodulated, distally thinner and simplepointed. Chaetae shortest in II ( Fig. 2 A), reaching maximum length in VII ( Fig. 2 B–C), and gradually reducing posteriorly ( Table 1 ). Lateral chaetae 90–120 Μm in length, 6–8 Μm in width. Ventral chaetae 95–152 Μm in length, 6–11 Μm in width, maximum width in VII ( Table 1 ). Chaetae of XII lacking in mature specimens. Clitellum elevated conspicuously, in XII–XIII (1/ 3XI –XIII in some specimens). Granulocytes and hyalocytes irregularly arranged, the former often in contact with each other, and with larger proportional distribution region than the latter ( Figs. 2 E, 3E). Two separate male pores ventro-laterally in the middle of XII. Paired spermathecal pores at 4/5 of each lateral line. TABLE 1. Chaetal size and number in different segments of Mesenchytraeus laojunensis sp. nov.

Segments	II III	V	VI	VII	IX	XI
Length of lateral chaetae (Μm)	90–95 91–96	102–112	114–120	120–126	113–123	115–120
Width of lateral chaetae (Μm)	6–7.5 7.5	7.5–8	7.5	7–8	7–8	7–8
Number per bundle	3 3, 4	4	4	4	4	4
Length of ventral chaetae (Μm)	95–100 105–107	125–127	141–152	141–150	118–133	105–122
Width of ventral chaetae (Μm)	6–7.5 7.5–8	7.5–9	9–10	10–11	7.5–8	7–8
Number per bundle	4 4	5,6	5,6	4,5	6	6

FIGURE 3. Mesenchytraeus laojunensis sp. nov. (A–E): A . Dorsal view of anterior segments. B . Brain and head pore. C . Dorsal view of segments XI–XIII. D . A sperm bundle. E . Pattern of clitellar glands. Scale bars: A,C, 200 Μm; B, 100 Μm; D, 50 Μm. Abbreviations: bsf, body of sperm funnel; gr, granulocyte; hd, head pore; hy, hyalocyte; phg, pharyngeal glands; s, spermatozoa; sa, spermathecal ampulla; sp, spermathecal pore; sphg, secondary pharyngeal glands; vd, vas deferens. Brain ( Fig. 3 B) in I–II, trapezoidal, deeply concave anteriorly and weakly incised posteriorly, 150–250 µm wide and 200–300 µm long. Dorsal vessel arising from intestinal blood sinus in XIII, anterior bifurcation beneath front of brain, circumesophageal connectives merging ventrally in IV. Two pairs of circumesophageal (lateral) commissures connecting dorsal and ventral vessel, one in III (above pharynx) and another in IV. Blood colorless. Two pairs of primary pharyngeal glands in 4/5 and 5/6, both without dorsal connection and attached to septa ( Fig. 3 A). Four pairs of secondary pharyngeal glands in VI–IX ( Fig. 3 A). Gradual transition between oesophagus and intestine. No esophageal appendage. No intestinal diverticula. Chloragogen cells brownish, dense, granulated, beginning from V backwards. Coelomocytes ( Fig. 2 F) lemon-shaped, densely distributed, more concentrated in anterior segments, dark in transmitted light due to numerous refractile droplets. Its live size from 40 to 55 Μm in length by 19 to 25 Μm in width. Nephridia from 6/7 backwards, five pairs in preclitellar region. Anteseptale containing funnel only, about 50 µm long, postseptale bilobed, with folded canal and little interstital tissue, 120– 150 µm long and 50 µm wide, with efferent duct arising anteroventrally between the two lobes, close to septum. Sperm funnel much developed, long, cylindrical, occupying segments XI–XII; fixed dimensions: length 900– 2250 µm, width 200–300 µm ( Figs. 2 D, 3C); collar as wide as or slightly wider than funnel body. Heads of spermatozoa ca. 22–33 µm long. Vas deferens with ciliated canal, ca. 25–38 µm wide, wound in irregular spirals in coelom of XII–XIV, and entering subterminally into the atrium. Atrium in XII, distinct, cylindrical ( Fig. 2 I), ca. 100–130 µm wide in vivo, length 300–400 µm, connecting with penial bulbs centrally in XII. Atrial glands absent. Penial bulbs ventral, compact, consisting of masses of glandular cells and muscle strands, ca. 160–200 µm in diameter. Several accessory copulatory glands extending in different directions around the male pore ( Fig. 2 I). Testes in XI, compact. One pair of well-developed and asymmetrical sperm sacs originating from XII and extending backward into XVII–XXII, containing numerous flame-shaped sperm bundles ( Figs. 2 G, 3D). Spermatozoal heads in the wider part of the bundle and tails in the narrower one; length of a sperm bundle ca. 500– 600 µm, width ca. 50–70 µm. Egg sac present, extending into XIX. One or two eggs mature at a time. Spermathecae confined to V, external openings at midline of 4/5 ( Fig. 2 H). No ectal gland visible. Ectal duct short, 30–50 µm in length and 60–80 µm in maximal width, thick-walled, both epidermis and muscle layers welldeveloped. Ectal duct expanding abruptly into onion-shaped ampulla (ca. 200 µm in diameter); ampulla continued into the narrowing ental duct. Each ampulla with one diverticulum. Ampullar diverticulum elongate and cylindrical, thick walled, 250–320 µm in length and 130–180 µm in width. Ental ducts ca. 70–90 µm wide and 48– 49 µm long, thin-walled, connecting with oesophagus separately in posterior of V. DNA sequences. Fragments of the 16S rDNA (Genbank Acc. No. KU360271 ), 28S rDNA (Acc. No. KU360272 ) and 12S rDNA (Acc. No. KU360273 ) of three non-type specimens of M. laojunensis sp. nov. obtained from the type locality were sequenced and deposited in GenBank. Remarks. Mesenchytraeus laojunensis sp. nov. is differentiated from all its congeners by the combination of the following characters: 1) secondary pharyngeal glands extending to IX; 2) coelomocytes with distinct refractile vesicles; 3) much swollen spermathecal ampulla associated with one diverticulum, with ental duct communicating dorsally with oesophagus in V; 4) sperm funnel cylindrical and very long (up to 2000 µm in length); 5) spermatozoa forming multiple flame-shaped sperm bundles in sperm sacs with spermatozoal heads clumped at one end; 6) vas deferens extending backwards to XIV and communicating subterminally with enlarged atrium; 7) sperm sacs extending backward into XVII–XXII; 8) well-developed accessory glands around the male pores ( Table 2 ). Among these traits, both the extra long sperm funnel and the subterminal entering of vas deferens into the atrium are exceptional traits in Mesenchytraeus . The new species belongs morphologically to a group of Mesenchytraeus species that is characterized by the absence of enlarged chaetae, spermathecae communicating with oesophagus and each ampulla bearing one diverticulum (Christensen & Dózsa–Farkas 1999; Healy & Timm 2000; Schmelz & Collado 2010, 2012). Five species have thus far been reported in this group. Among them, M. celticus Southern 1909 , described from moist soil in Ireland and Scotland , is most similar to the new species by its large body size, the origin of the dorsal vessel (XIII), the backward extension of the pharyngeal glands, and the presence of several accessory glands around the male pore. M. celticus differs from the new species by a higher number of chaetae per bundle (up to 13), a subcylindrical and thin-walled ampulla with smaller diverticulum, pear-shaped sperm funnel (length: width ratio ca. 2:1) associated with a short sperm duct, and an indistinct atrium (Southern 1909; Schmelz & Collado 2010) ( Table 2 ). TABLE 2. Cοmparisοn οf Mesenchytraeus laojunensis sp. nov. with similar species

Species/	M. celticus	M. kuril	M. ogloblini	M. torbeni	M. viivi	M. laojunensis sp.n.
character	Sοuthern, 1909	Healy & Τimm, 2000	Cernοsvitοv, 1928	Christensen & Dοzsa-Farkas,	Τimm, 1978
1999
Length/mm	12-25	4-5	10-15	5- 9	10-14	18-28
Segment number	unknοwn	32-40	54-62	25-38	59-61	64-73
Chaetal fοrmula	5-7-?∶	4-7(8,9) - 2-5 ∶	2-3(4) - 5,6(7) ∶	1-4 - 2-5(6,7) ∶	3-5 - 5-8 ∶	3,4 - 3,4 ∶
10-11-?	4-7(8,9) - 2-5	5-7 - 6-7	2-5 - 2-5	6-9 - 6-9(10)	4-6 - 4,5
Pharyngeal glands	in Iv-X	3 pairs primary glands in	secοndary glands in	2 pairs primary glands in	2 pairs primary glands in	2 pairs primary glands in
3/4-5/6, nο secοndary	v-vII	4/5-5/6, secοndary glands in	4/5-5/6, secοndary glands in	4/5-5/6, secοndary glands
glands	v-vII	Iv-vI (vII)	in vII-IX
Preclitellar	?	οne preclitellar pair in	?	frοm 6/7	6/7-10/11	6/7-10/11
nephridia	7/8-8/9 οr 9/10
Spermathecae	in v, 1	in v, 1 diverticulum	in v, 1 elοngate	in v, 1 diverticulum	in v, 1-2 diverticula	in v, 1 elοngate
diverticulum	diverticulum	diverticulum
Width ampulla ∶	2∶1	1∶ 2	3∶1	2∶1	1∶1	1∶1
diverticulum
Width ampulla ∶	1•8∶1	1∶1	3∶1	ca• 1•4∶1	2∶1	6-6•5∶1
ectal duct
Dοrsal vessel οrigin	XIII?	XII	XIv-Xv	XII-XIv	XvI-XvII	XIII
Sperm funnel	1•5-2∶1	3-4∶1	1•5∶1• vas deferens	1•5∶1	1•5∶1, vas deferens shοrt	lοng cylindrical, ca•6-10∶1•
length ∶ width	shοrt	vas deferens extending tο
XIv•
Sperm sacs	?	paired, in IX-XIII (XIv)	?	absent οr small in XII, XIII	1 οr 2, in XII	XIX
Egg sacs	?	paired, extending intο	?	?	1, extending intο XvII	extending intο XvIII-XXII
XIII-XIv
Cοelοmοcytes	dark, nucleus	?	dark	?	dark, nucleus invisible
clear
Atrium	indistinct	withοut atrium	present, withοut	present, withοut prοstate	indistinct	cylindrical, distinct
enlargement	prοstate
Penial bulb	several	absent	2 large	2 large	several	several
accessοry glands
Distributiοn	Ireland ,	Ζοlοtοϳ Klϳuc stream,	river sediment,	sοil, N Υamal Peninsula,	lake shοres in Kοla peninsula,	frοzen sοil, Υunnan, China
Scοtland, mοist	Kamcatka, Russia	Carpathians, Balcan	Russia	Russia
sοils	peninsula
Reference	Sοuthern 1909;	Healy & Τimm 2000	Cernοsvitοv 1928;	Christensen & Dοzsa-Farkas	Τimm & Pοpchenkο 1978;	this study
Schmelz &	Schmelz & Cοlladο	1999	Schmelz & Cοlladο 2010
Cοlladο 2010	2010

Characters of the remaining four species that differ from those of the new species are as follows. Mesenchytraeus kuril Healy & Timm, 2000 , described from a small river in Kamčatka, Russia , has three pairs of primary glands in 3/4–5/6, only one pair of preclitellar nephridia and 4–5 pairs in the postclitellar region at XIV– XVIII–XX, an indistinct spermathecal ampulla, a sperm funnel 3–4 times as long as wide, and no atrium and accessory copulatory glands (Healy & Timm 2000). Mesenchytraeus ogloblini Černosvitov, 1928 , another riverdwelling species described from the Eastern Carpathians in South-East Europe, has secondary pharyngeal glands in V–VII as large as or larger than the primary glands, smaller coelomocytes (length ca.15 µm), a shorter vas deferens entering terminally into a somewhat swollen atrium, penial bulb with only two large and stalked accessory glands, and a smaller and trumpet-shaped sperm funnel (length: width ratio ca. 1.5: 1) (Černosvitov 1928; Schmelz & Collado 2010). Mesenchytraeus viivi Timm, 1978 , described from lake sediments of the Kola Peninsula, Russia , differs from M. laojunensis in a higher number of chaetae (up to 9 per bundle), a more posterior origin of the dorsal vessel (XVI–XVII), a lower number of secondary pharyngeal glands (V–VI/VII), a smaller sperm funnel (1.5: 1) associated with a shorter sperm duct, a slightly swollen spermathecal ampulla (twice as wide as ectal duct); it further has occasionally two ampullar diverticula and possibly one small ectal gland at the spermathecal orifice (Timm & Popchenko 1978; Schmelz & Collado 2010). Mesenchytraeus torbeni Christensen & Dózsa-Farkas, 1999 is a genuine terrestrial enchytraeid species collected in the North Yamal Peninsula of the Siberian tundra. It differs from the new species in: three lobed secondary pharyngeal glands in V–VII, sperm sacs absent or small (confined to XII or XIII), sperm funnel smaller, 1.2 times as long as wide, sperm duct shorter, only two large accessory glands around each male pore, an undeveloped spermathecal ampulla (ca. 1.4: 1 as wide as ectal duct), and a smaller ampullar diverticulum (Christensen & Dózsa–Farkas 1999) ( Table 2 ). To date, only three species of Mesenchytraeus are known that possess the specific trait of regular sperm bundles in the sperm sacs, M. gigachaetus Xie, 2012 (= M. megachaetus Shen et al. , 2011 ), M. anisodiverticulatus Shen et al. , 2012 and M. monodiverticulus Shen et al. , 2012 (Shen et al. 2011, 2012a,b; Xie 2012). All of them were described from China . They diifer from M. laojunensis sp. nov. in enlarged ventral chaetae in some preclitellar bundles (Shen et al. 2011, 2012a,b; Xie 2012). In particular, the new species otherwise resembles M. monodiverticulus by the single diverticulum in each spermatheca. However, the new species differs from M. monodiverticulus in many traits, such as larger body size, different patterns of clitellar granulocytes and hyalocytes and sperm bundles, and more developed sperm sacs. Geographic distribution and habitat requirements. The worms were only found at the snowpack of the top of Laojun Mountain (ca. 3800–4000 m asl) in Yunnan Province of southwestern China , where the dominant plants are Abies spp., Rhododendron lapponicum and mosses. These worms prefer to live beneath the moss and above the frozen soil, where the soil moisture is ca. 18.3% and the soil temperature ca. -2–4 °C. Mesenchytraeus laojunensis sp. nov. was usually dominant in the enchytraeid assemblages and co-occurred with Ch. ozensis during the sampling seasons.