Linking morphology and molecules: integrative taxonomy of spathidiids (Protista: Ciliophora: Litostomatea) from Korea Author Jang, Seok Won Author Vďačný, Peter Author Shazib, Shahed Uddin Ahmed Author Shin, Mann Kyoon text Journal of Natural History 2017 2017-05-15 51 17 - 18 939 974 http://dx.doi.org/10.1080/00222933.2017.1319520 journal article 10.1080/00222933.2017.1319520 1464-5262 5181143 Spathidium papilliferum Kahl, 1930 ( Figures 5 (a–k), 6(a–j) and 7(a–i); Table 2 ) Spathidium papilliferum Kahl, 1930: 164 , fig. S. 164, 7 Epispathidium papilliferum – Foissner, 1984: 84 , Abb. 43 (combining author, description of Austrian population) Epispathidium papilliferum – Strüder-Kypke et al., 2006: 262 (18S rRNA gene sequences) Description of Korean populations Two populations of this species were studied: one from the Jungjok Mts. (pop. 1) and the other from the surroundings of the Ssanggyesa temple (pop. 2). The populations were very similar; therefore, the description combines all in vivo observations. However, morphometric data were obtained only from the second population. Body size 150–190 × 35–50 µm, usually near 175 × 45 µm in vivo and 105–185 × 25– 65 µm, usually near 140 × 40 µm after protargol impregnation; not contractile but flexible ( Table 2 ). Shape narrowly spatulate with a length:width ratio of 3.5–4.3: 1 in vivo and 2.7– 4.8:1 after protargol impregnation; oral region very conspicuous because oral bulge with two or usually three papillae and about as wide as broadest posterior body portion; neck region rather distinct and flattened laterally; posterior end rounded ( Figures 5 (a,e–k), 6(a,b, e–h) and 7(a,b,f–i)). Nuclear apparatus scattered throughout body, except for the anteriorly flattened cell portion. Number of macronuclear nodules highly variable, ranging from 26 to 79, usually about 50; individual nodules globular, ovate to oblong and about 7.5 × 4.5 µm in size after protargol impregnation; nucleoli small- to medium-sized and rounded. On average 16 micronuclei distributed among macronuclear nodules, globular and about 2.0 µm in diameter after protargol impregnation ( Figures 5 (d–f,i) and 7(a–c,e–h); Table 2 ). A single contractile vacuole at posterior body end ( Figures 5 (a,e,f), 6(a,e) and 7(a,b)). Extrusomes densely packed in oral bulge papillae and scattered throughout cytoplasm, impregnate with the protargol method used; oral extrusomes rod-like, almost straight or slightly curved, about 13 µm long in vivo ; developmental cytoplasmic stages either rod-like or composed of a rod-like anterior part and a fusiform or asymmetric posterior part ( Figures 5 (c,g,h,j), 6(b,d,f–i) and 7(d–g,i)). Cortex very flexible, contains several rows of colourless granules between adjacent ciliary rows. Cytoplasm colourless, contains macronuclear nodules, extrusomes, and few to many lipid globules and food vacuoles ( Figure 6 (a,b,e)). Movement conspicuously slow and worm-like. Figure 4. Spathidium ascendens from life (a–h) and after protargol impregnation (i–m). (a, i) Right side view of representative individuals; (b) detail of posterior body portion showing contractile vacuole; (c) resting cyst; (d, l) detail of anterior body portion showing extrusomes attached to oral bulge; (e) dorsal brush bristles; (f) extrusomes; (g) cortical granulation; (h) epibiotic bacteria; (j, k) right side and dorsolateral views of ciliary pattern in anterior body portion; (m) optical section showing filiform macronucleus and mature rod-like as well as developing fusiform cytoplasmic extrusomes. CK, circumoral kinety; CV, contractile vacuole; DB, dorsal brush; EX, extrusomes; MA, macronucleus; SK, somatic kinety/somatic kineties. Scale bars: a, i = 100 µm; b–d, j–m = 10 µm; e– h = 5 µm. Figure 6. Spathidium papilliferum from life, Jungjok Mts. population (a–d) and Ssanggyesa temple population (e–j). (a, e) Right side view of representative individuals; (b, f–h) detail of anterior body portion showing oral bulge papillae (triangles) studded with extrusomes; (c) dorsal brush bristles; (d, i) extrusomes; (j) resting cyst. CV, contractile vacuole; DB, dorsal brush; EX, extrusomes; LD, lipid droplets. Scale bars: a, e = 100 µm; c = 5 µm; b, d, f–j = 10 µm. Figure 5. Spathidium papilliferum , Ssanggyesa temple population from life (a–c, g, h) and after protargol impregnation (d–f, i–k). (a) Right side view of a representative individual; (b) resting cyst, 45 µm in diameter; (c) mature and developing extrusomes, about 13 µm long; (d) nuclear apparatus in mid-body; (e, f) variability of body shape and size as well as of nuclear apparatus; (g, h) variability of oral bulge papillae; (i, j) dorsolateral view of ciliary and nuclear pattern in anterior body portion; (k) right side view of ciliary pattern in anterior body portion. CK, circumoral kinety; CV, contractile vacuole; DB, dorsal brush; EX, extrusomes; MA, macronuclear nodules/macronucleus; MI, micronuclei/micronucleus; OB, oral bulge. Scale bars: a, e, f = 100 µm; d, i–k = 10 µm. Figure 7. Spathidium papilliferum after protargol impregnation, Ssanggyesa temple population. (a, b) Right side view of representative individuals; (c) nuclear apparatus in mid-body; (d, e) developing and mature cytoplasmic extrusomes; (f) dorsolateral view showing three-rowed dorsal brush and oral bulge papillae studded with extrusomes; (g, h) right and left side views of ciliary pattern in anterior body portion; (i) detail of anterior body portion showing oral bulge papillae with extrusomes. CV, contractile vacuole; CK, circumoral kinety; DB, dorsal brush; EX, extrusomes; MA, macronuclear nodules/macronucleus; MI, micronuclei/micronucleus; SK, somatic kinety/somatic kineties; triangles in (a, b, h, i) denote oral bulge papillae. Scale bars: a, b = 100 µm; c–i = 10 µm. Cilia about 8 µm long in vivo , spacing loose to ordinary except for about 10 densely spaced cilia in curved anterior kinety portion. About 26 and 27 equidistant, ordinarily spaced meridional ciliary rows forming an Epispathidium -like pattern (i.e. anterior portion of right side rows curved dorsally and left side rows curved ventrally to parallel the circumoral kinety; Figures 5 (i,k) and 7(g,h); Table 2 ). Dorsal brush inconspicuous because bristles stump-like and only up to 2.5 µm long in vivo ; dikinetidal; three-rowed; isostichad – that is, all rows of similar length ( Figures 5 (i,j), 6(c) and 7(f)). Oral bulge conspicuous because carrying two or usually three papillae; extends over inclined anterior body end ( Figures 5 (a,e–k), 6(a,b,e–h) and 7(a,b,f–i)). Circumoral kinety at base of oral bulge; continuous and distinctly separated from ciliary rows; composed of narrowly spaced and perpendicularly oriented dikinetids ( Figures 5 (i,k) and 7(g,h)). Nematodesmata not recognisable in vivo or after protargol impregnation. Resting cysts of Ssanggyesa temple population Cysts colourless, about 40–45 µm across in vivo ; without escape apparatus. Cyst wall inconspicuous because smooth and only about 1 µm thick. Cytoplasm finely granulated, less densely in cyst periphery. Cyst centre studded with globules and macronuclear nodules. Contractile vacuole still present in young cysts. Extrusomes, cortical granules and cilia not recognisable ( Figures 5 (b) and 6(j); Table 2 ). Comparison with original description and remarks Korean populations match Kahl’ s (1930) description and Foissner’ s (1984) re-description in their narrowly spatulate body, prominent oral bulge papillae armoured by batteries of rod-shaped extrusomes, several scattered macronuclear nodules, and inconspicuous dorsal brush. However, Korean specimens are smaller than Kahl’ s individuals (150– 190 µm vs 170–260 µm), but their size falls well within the range (100–200 µm) reported by Foissner (1984) . As concerns the number of macronuclear nodules, there are 7–10 nodules according to Kahl (1930) , while Korean individuals possessed 26–79 nodules and Austrian cells about 100 nodules. Foissner (1984) assumed that the macronucleus was not fully fragmented in Kahl’ s specimens, because many minute nodules were scattered among a few larger ones in Austrian cells. On the basis of our observations, we agree with Foissner (1984) that the difference in number of macronuclear nodules can be attributed to life-cycle stage. The length of extrusomes is very similar among all populations: about 13 µm in both Kahl’ s and Korean specimens and 10–12 µm in Foissner’ s individuals. Likewise, the number of ciliary rows seems to be similar between Korean and type specimens when Kahl’ s drawing (fig. S. 164, 7 ) is considered. However, Foissner’ s specimens possessed slightly more ciliary rows than the Korean cells (32–36 vs 26–27). Our comparison of S. papilliferum populations indicates that this species is either rather variable in body size and number of ciliary rows, or consists of several cryptic species. The second scenario is favoured by molecular analyses (see below). However, the matter is more complex, because Korean populations are not monophyletic and none of them group with the Austrian isolates investigated by Strüder-Kypke et al. (2006) . It is not possible to solve this taxonomic problem given our present knowledge; this problem is also discussed in the phylogeny section. Because the genus Epispathidium is polyphyletic, we tentatively prefer Kahl’ s (1930) original classification of papilliferum in the genus Spathidium . Further, we consider S. papilliferum to be a collective group.