The Trichoderma koningii aggregate species

Abstract

The morphological concept of Trichoderma koningii is found to include several species that differ from each other in details of phenotype (including conidium morphology, growth rate) and biogeography. Phylogenetic analysis utilizing partial sequences of the translation-elongation factor 1 alpha (tef1), as well as fragments of actin and calmodulin genes, indicate that phenotypic characters typical of T. koningii evolved independently in three well-separated main lineages. Combined molecular and phenotype data lead to the development of a taxonomy with the recognition of twelve taxonomic species and one variety within the three lineages. These lineages include: (1) T. koningii and T. ovalisporum and the new species T. caribbaeum var. caribbaeum, T. caribbaeum var. aequatoriale, T. dorotheae, T. dingleyae, T. intricatum, T. koningiopsis, T. petersenii and T. taiwanense; (2) the new species T. rogersonii and T. austrokoningii, and (3) the new anamorph T. stilbohypoxyli.Trichoderma koningii s. str. is an uncommon species restricted to Europe and eastern North America; T. caribbaeum var. aequatoriale, T. koningiopsis, and T. ovalisporum were isolated as endophytes of trunks of Theobroma species in tropical America, and T. ovalisporum from the woody liana Banisteropsis caapi in Ecuador; T. koningiopsis is common in tropical America but was isolated also from natural substrata in East Africa, Europe and Canada, and from ascospores in eastern North America, and as an endophyte in Theobroma species; T. stilbohypoxyli, originally described as a parasite of Stilbohypoxylon species in Puerto Rico, is found to be more common in the tropics, besides an endophytic isolate from Fagus in U.K. The additional new species are known almost exclusively from their teleomorphs. Isolates of T. ovalisporum and T. koningiopsis may have biological control potential. A morphophenetic key and a set of tools for molecular species identification were developed.

Fig. 1.

Bayesian radial tree showing position of T. koningii aggregate species on Hypocrea/Trichoderma phylogeny based on partial rpb2 sequences. Arrows indicate branches leading to currently recognized clades within the genus in sense of Chaverri & Samuels (2003), Druzhinina et al. (2005) and the present work. Circles at nodes indicate the posterior probability coefficients higher 0.95 as they were obtained after 3 million generations. All sequences except T. koningii CBS 979.70 DQ641671 and H. novaezelandiae G.J.S. 81-264 DQ 641672 were retrieved from NCBI GenBank as follows: H. voglmayrii CBS 117710 DQ086151; T. viride VD G.J.S. 89-127 AF545521; T. pubescens CBS 345.93 AF545552; T. hamatum CBS 102160.93 AF545548; T. strigosum CBS 348.93 AF545556; H. minutispora CBS 901.72 AY481588; H. pachybasioides CBS 820.68 DQ087238; H. pilulifera CBS 814.68 AF545519; H. citrina CBS 894.85 AF545561; H. pulvinata G.J.S. 98-104 AF545559; H. melanomagna CBS 114236 AY391926; H. lutea G.J.S. 89-129 AF545517; T. oblongisporum CBS 344.98 AF545551; T. fertile CBS 339.93 AF545546; H. chlorospora CBS 114231 AY391903; H. sinuosa CBS 114247 AY391942; H. aureoviridis CBS 245.63 AF545509; H. strictipilosa G.J.S 98-91 AF545538; T. spirale CBS 346.93 AF545553; T. aggressivum CBS 100525 AF545541; H. tawa CBS 114233 AY391956; H. lixii CBS 226.95 AF545549; H. catoptron CBS 114232 AY391900; T. tomentosum DAOM 178713a AF545557; H. gelatinosa CBS 114246 AY391924; T. helicum DAOM 230021 DQ087239; T. rossicum TUB F-718 DQ087240; H. jecorina TUB F-430 DQ087241; T. longibrachiatum CBS 816.68 DQ087242; T. saturnisporum CBS 330.70 DQ087243; H. schweinitzii G.J.S. 01-364 AF545565.

Fig. 2.

Bayesian radial phylogram showing the structure of the 'Viride' Clade as it was inferred based on sequences of two introns of tef1. Grey colour is used to separate specimens which are not discussed in this study but whose sequences were used to produce the multiple sequence alignment. Arrows indicate branches leading to species recognized within T. koningii aggregate species. In the highlighted part of the tree, grey filled circles at nodes indicate posterior probability coefficients higher than 0.90 as they were obtained after 5 million generations; black filled circles at nodes show support higher than 0.95. Font colours correspond to regions of sampling on the schematic map. Clades identified as “PS A–F” in the lower half of the tree represent undescribed phylogenetic species (see Table 1).

Fig. 3.

The concordance between two Bayesian phylograms as inferred based on partial act and cal gene sequences. Black circles at nodes indicate the posterior probability coefficients higher than 0.95 as they were obtained after 3 million generations. Grey circles in the cal tree indicate differences in topology when compared to the same isolates in the act tree. Taxon “PS A” indicates an undescribed phylogenetic species (see Table 1).

Fig. 4.

Principal Components Analysis, scatter plot of Eigen vectors. See Table 2 for statistics.

Fig. 5.

Growth rate curves after 72 h in darkness for the taxa considered. Dashed line = SNA; solid line = PDA. Standard error bars are indicated.

Figs 6–14.

Trichoderma cultures grown on PDA in 9-cm-diam Petri dishes for 96 h at 25 °C under 12 h darkness/12 h cool white fluorescent light. 6: T. austrokoningii G.J.S. 99-146. 7. T. austrokoningii (New Zealand) G.J.S. 99-116. 8. T. austrokoningii (Russia) G.J.S. 00-73. 9. T. caribbaeum var. caribbaeum G.J.S. 97-3. 10. T. dingleyae G.J.S. 99-105. 11. T. dorotheae G.J.S. 99-97. 12–13. T. intricatum (12: G.J.S. 96-13, 13: G.J.S. 97-88). 14. T. koningii G.J.S. 96-119.

Figs 15–23.

Trichoderma cultures grown on PDA in 9-cm-diam Petri dishes for 96 h at 25 °C under 12 h darkness/12 h cool white fluorescent light. 15–16. T. koningiopsis (15: G.J.S. 01-07, 16: G.J.S. 91-6). 17. T. ovalisporum DIS 172h. 18. T. petersenii DAOM 165782. 19–20. T. rogersonii (19: G.J.S. 90-125; 20: G.J.S. 92-116). 21–22. T. stilbohypoxyli (21: G.J.S. 02-143, in darkness; 22: G.J.S. 03-103, 7 d). 23. T. taiwanense G.J.S. 95-93.

Figs 24–35.

Hypocrea teleomorphs of Trichoderma species. 24–27. T. austrokoningii. 24–25 from type, 26 from Florida (C.T.R. 85-57), 27 from Russia (G.J.S. 00-73). 28–29. T. caribbaeum var. caribbaeum on stroma of?Penzigia sp. (G.J.S. 97-3). 30–31. T. dingleyae immature (30: G.J.S. 02-50) and mature (31: G.J.S. 99-105) stromata. 32–33. T. dorotheae mature and immature (arrow) stromata (32: G.J.S. 99-97, 33: G.J.S. 99-194). 34–35. T. intricatum immature (34: G.J.S. 96-13, Puerto Rico) and mature (35: G.J.S. 97-88, Thailand) stromata. Microscopy: all stereo. Bars: 24–25, 27–28, 30–34 = 1 mm; 26, 29, 35 = 0.5 mm.

Figs 36–50.

Hypocrea teleomorphs of Trichoderma species. 36–38. T. koningii (36, 38: G.J.S. 89-122, 37: G.J.S. 00-156). 39–40. T. koningiopsis (G.J.S. 93-20, type). 41–43. T. petersenii (41–42: G.J.S. 98-139; 43 G.J.S. 04-355 France), immature stromata shown in 41. 44–46. T. rogersonii. Immature stroma (arrow) in 44 (44: G.J.S. 95-217; 45: G.J.S. 90-79; 46: G.J.S. 90-125). 47–48. T. stilbohypoxyli (47: G.J.S. 96-43, 48: G.J.S. 03-103, immature). 49–50. T. taiwanense, overmature stromata (G.J.S. 95-93, type). Microscopy: all stereo. Bars: 36 = 2 mm; 37–39, 41, 44, 46, 47–49 = 1 mm; 40, 42–43, 45, 50 = 0.5 mm.

Figs 51–59.

T. austrokoningii, anamorph (Queensland, including type; all from CMD). 51–52. Conidial pustules. 53–58. Conidiophores and phialides. Intercalary phialide shown in 58 (arrow). 59. Conidia. Figs 51, 53–55, 59 from G.J.S. 99-147; 52, 56–58 from G.J.S. 99-146. Microscopy: 51–52 = stereo; 53–56 = PC; 57–59 = DIC. Bars: 51 = 1 mm; 52 = 0.5 mm; 53–58 = 20 μm; 59 = 10 μm.

Figs 60–70.

T. austrokoningii, Hypocrea teleomorph (Queensland, including type). 60. Median longitudinal section of a mature perithecium. 61. Surface view of stroma showing two ostiolar openings (arrow) and small cells that form hairs. 62–63. Hairs arising from stroma surface (arrows). 64. Section of stroma surface showing hairs (arrows) and pigmented outer region. 65. Median longitudinal section through an ostiolar canal. 66. Cells of stroma interior below perithecia. 67–68. Asci. Apical ring visible in 68–70. Discharged ascospores. Spores shown in optical section in 69, surface view showing ornamentation in 70. Figs 60, 63–64, 66–67, 69–70 from G.J.S. 99-147; 61–62, 65, 68 from G.J.S. 99-146. Microscopy: 60 = BF; all others DIC. Bars: 60 = 100 μm; 61–68 = 20 μm; 69–70 = 10 μm.

Figs 71–79.

Trichoderma austrokoningii from Russia (G.J.S. 00-73) on CMD. 71–72. Conidial pustules. Note long, entirely fertile conidiophores in 72 (examples marked with arrows). 73–78. Conidiophores and phialides. Intercalary phialides indicated by arrows in 78. 79. Conidia. Microscopy: 71–72 = stereo; 73–76, 78 = PC; 77, 79 = DIC. Bars: 71 = 1 mm; 72 = 0.5 mm; 73–78 = 20 μm; 79 = 10 μm.

Figs 80–88.

Trichoderma austrokoningii from New Zealand on CMD. 80. Aggregated conidiophores. 81–87. Conidiophores. 88. Conidia. Figs 80–84, 86–87 from G.J.S. 99-116; 85, 88 from CBS 243.63. Microscopy: 80–81 = stereo; 82–85 = PC; 86–88 = DIC. Bars: 80 = 1 mm; 81 = 0.5 mm; 82–87 = 20 μm; 88 = 10 μm.

Figs 89–101.

Trichoderma austrokoningii from Florida and Taiwan on CMD. 89–91. Conidial pustules. 92–99. Conidiophores. 100–101. Conidia. Figs 89, 91, 97–99, 101 from G.J.S. 96-163; 90, 92-96, 100 from C.T.R. 85-57. Microscopy: 89–91 = stereo; 92–99 = PC; 100–101 = DIC. Bars: 89–90 = 1 mm; 91 = 0.5 mm; 92–99 = 20 μm; 100–101 = 10 μm.

Figs 103–112.

Trichoderma caribbaeum var. caribbaeum, anamorph from CMD. 103–104. Conidial pustules. 105–110. Conidiophores. 111–112. Conidia. Figs 103, 107, 109–111 from G.J.S. 98-43; 104–106, 108, 112 from G.J.S. 97-3. Microscopy: 103–104 = stereo; 105–106, 109 = PC; 107–112 = DIC. Bars: 103 = 1 mm; 104 = 0.5 mm; 105–110 = 20 μm; 111–112 = 10 μm.

Figs 113–121.

Trichoderma caribbaeum var. caribbaeum, Hypocrea teleomorph. 113. Stromata of Hypocrea (light coloured) growing on xylariaceous host. 114. Face view of stroma surface. 115. Section through a single stroma. 116, 118. Median longitudinal sections through mature perithecia. 117. Internal tissue of the stroma below perithecia; the lower wall of a perithecium seen on the right. 119. Section through ostiolar canal. 120. Stroma surface. 121. Ascus with ascospores (in 1 % aq. phloxine). Figs 113, 115–121 from G.J.S. 97-3; 114 from G.J.S. 98-43. Microscopy: 113 = stereo, 114, 116–120 DIC; 115, 121 = BF. Bars: 113 = 1 mm; 114, 121 = 10 μm; 115 = 200 μm; 116–120 = 20 μm.

Fig. 102.

Growth curves of variants of T. austrokoningii. Solid line = PDA, broken line = SNA. The following isolates were included: Australia (G.J.S. 99-146, G.J.S. 99-174), New Zealand (G.J.S. 99-116, CBS 243.63), Russia (G.J.S. 00-73), U.S.A./Taiwan (C.T.R. 85-57, G.J.S. 96-163). Standard error bars are shown.

Figs 122–133.

Trichoderma caribbaeum var. aequatoriale from SNA (DIS 320c). 122–125. Conidiophores formed in the aerial mycelium. 126–127. Conidial pustules. 128–132. Conidiophores produced from pustules. 133. Conidia. Microscopy: 122, 126–127 = stereo, 123 = BF; 124–125, 131–133 = DIC; 128 = FL; 129–130 = PC. Bars: 122, 127 = ¼ mm; 126 = 1 mm; 123, 125, 128–130 = 20 μm; 124, 131–133 = 10 μm.

Figs 134–142.

Trichoderma dingleyae, anamorph from CMD. 134–135. Conidial pustules. 136–141. Conidiophores. 142. Conidia. Figs 134–135 from G.J.S. 02-50; 136, 139, 141 from G.J.S. 99-203; 137–138, 140 from G.J.S. 02-50; 142 from G.J.S. 99-203. Microscopy: 134–135 = Stereo, 136, 139, 141–142 = DIC, 137 = PC; 138, 140 = FL. Bars: 134–135 = 1 mm; 136–138, 140 = 20 μm; 139, 141–142 = 10 μm.

Figs 143–152.

Trichoderma dingleyae, Hypocrea teleomorph. 143. Young stroma, note velutinous surface and poorly visible ostiolar openings (one shown at arrow with exuded ascospores). 144. Face view of a stroma. 145. Section through stroma with immature perithecia. 146–147. Section through stroma surface; note loosely hyphal nature of stroma surface and hairs (arrows) that give a velutinous aspect. 148. Cells of interior of stroma below perithecia. 149. Section through ostiolar canal. 150–152. Asci and ascospores; note ornamented ascospores in 151. Figs 143, 146–148, 150, 152 from G.J.S. 02-05; 144–145, 149 from G.J.S. 99-105; 151 from G.J.S. 99-194. Microscopy: 143 = stereo; 144–152 = DIC. Bars: 143 = 1 mm; 144, 146, 148–149 = 20 μm; 145 = 100 μm; 147 = 20 μm; 150–152 = 10 μm.

Figs 153–164.

Trichoderma dorotheae, anamorph from CMD. 153–154. Conidial pustules. Note in Fig. 154 that individual conidiophores are not visible in the pustule. 155–162. Conidiophores and phialides; arrows in Fig. 158 indicate percurrently proliferated phialides. 163. Conidia. 164. Chlamydospore. Figs 153–154, 159, 162, 164 from G.J.S. 99-97; 155–158, 160–161 from G.J.S. 99-202; 163 from G.J.S. 99-194. Microscopy: 153–154 = stereo; 155, 157 = PC; 156, 158, 160–161, 163 = DIC; 159, 162 = FL; 164 = BF. Bars: 153 = 1 mm; 154 = 0.5 mm; 155–157, 159, 162, 164 = 20 μm; 158, 160–161, 163 = 10 μm.

Figs 165–173.

Trichoderma dorotheae, Hypocrea teleomorph. 165. Section through an immature perithecium. 166. Face view of stroma. 167–169. Hairs (arrows) formed on stroma surface in face view (167) and in section (168–169). 168–170. Section through stroma surface and ostiolar region (170). 171–173. Asci and ascospores. Note ornamented ascospores in 173. Figs 165–166, 168–170 from G.J.S. 99-202, 167, 172–173 from G.J.S. 99-194, 171 from G.J.S. 99-97. Microscopy: all from DIC. Bars: 165, 170 = 20 μm; 166–169, 171–173 = 10 μm.

Figs 174–185.

Trichoderma intricatum, anamorph from CMD. 174–176. Conidial pustules; individual plumose conidiophores can be seen in the pustule (176, arrows). 177–184. Conidiophores and phialides. 185. Conidia. Figs 174, 177–179, 181, 183 from G.J.S. 97-88; 175–176, 180, 182, 184–185 from G.J.S. 96-13. Microscopy: 174–176 = stereo; 177–179, 181 = PC, 180, 182, 184 = FL; 183, 185 = DIC. Bars: 174–175 = 1 mm; 176 = 0.5 mm; 177–182, 184 = 20 μm; 183, 185 = 10 μm.

Figs 186–195.

Trichoderma intricatum, Hypocrea teleomorph. 186. Section through a stroma with mature perithecia. 187–189. Stroma surface in face view (187) and in section (188–189); hairs shown at arrows; note the loose nature of the stroma surface that gives the stroma a velutinous aspect (188–189, 191). 190. Median longitudinal section through a mature perithecium. 191. Section through the ostiolar canal. 192. Cells of the stroma interior below perithecia. 193–194. Asci and ascospores. 195. Discharged part-ascospores. Figs 186, 189–192 from G.J.S. 97-88; 187–188, 193–195 from G.J.S. 96-13. Microscopy: 186 = BF, all others DIC. Bars: 186 = 200 ìm; 187–188, 194–195 = 10 μm; 189–193 = 20 μm.

Figs 196–207.

Trichoderma koningii, anamorph from CMD. 196–197. Conidial pustules; individual conidiophores can be seen in 197 (e.g. arrow). 198–205. Conidiophores and phialides; in 198 conidiophores as viewed with the stereo microscope at the periphery of a pustule can be seen; note the densely clustered phialides in 201–202 and 205. 206. Conidia. 207. Chlamydospores. Fig. 196 from G.J.S. 96-119; 197 from G.J.S. 90-18; 198, 202 from CBS 979.70; 199 from G.J.S. 92-18; 200–201 from G.J.S. 97-117; 203 from G.J.S. 00-156; 204–207 from ATCC 64262. Microscopy: 196–198 = stereo; 199–202 = FL; 203, 205 = PC; 204, 206 = DIC, 207 = BF. Bars: 196 = 1 mm; 197–198 = 0.5 mm; 199–202, 205, 207 = 20 μm; 203–204, 206 = 10 μm.

Figs 208–216.

Trichoderma koningiopsis, anamorph from CMD. 208–210. Conidial pustules; note individual conidiophores at the periphery of a pustule in 210 (e.g. arrow). 211–215. Conidiophores and phialides; note intercalary phialide in 215 (arrow); note “pachybasium”-like arrangement of phialides in 214. 216. Conidia. Figs 208, 210–211 from G.J.S. 91-6; 209, 212–215 from G.J.S. 01-09; 216 above from G.J.S. 97-273, 216 below from G.J.S. 91-7. Microscopy: 208–210 = stereo; 211–215 = FL, 216 = DIC. Bars: 208–209 = 1 mm; 210 = 0.5 mm; 211–215 = 20 μm; 216 = 10 μm.

Figs 217–225.

Trichoderma koningiopsis, Hypocrea teleomorph. 217. Section through a mature stroma. 218. Stroma surface in face view with hairs visible (e.g. arrows). 219. Median longitudinal section through a mature perithecium. 220–222. Section through stroma surface; the remains of the loose surface can be seen in 221 and a hair is visible in 222 (arrow). 223. Cells of the stroma interior below perithecia. 224–225. Asci and ascospores; 225 stained with 1 % aq. phloxine; the thickened ascus apex can be seen in 225. All from G.J.S. 93-20. Microscopy: 217 = BF, all others from DIC. Bars: 217 = 200 ìm; 218, 220–223 = 20 μm; 219 = 50 μm; 224–225 = 10 μm.

Figs 226–236.

Trichoderma ovalisporum, anamorph from CMD. 226. Conidial pustules. 227–234. Conidiophores and phialides; intercalary phialides visible in 228, 233–234 (arrows). 235–236. Conidia. Figs 226–230, 234–235 from DIS 172h; 231, 233, 236 from DIS 203c; 232 from DIS 70a. Microscopy: 226 = stereo, 227–228 = PC, 229–230, 232 = FL, 231, 233–236 = DIC. Bars: 226 = 1 mm; 227–230, 232–234 = 20 μm; 231, 235–236 = 10 μm.

Figs 237–247.

Trichoderma petersenii, anamorph from CMD. 237–238. Conidial pustules; individual conidiophores can be seen in 238 (e.g. arrow). 239–245. Conidophores and phialides; note densely clustered phialides in 245. 246–247. Conidia. Figs 237–239, 241, 243 from G.J.S. 05-351; 240 from G.J.S. 90-86; 242, 244, 247 from G.J.S. 04-355; 245 from G.J.S. 99-48; 246 from DAOM 165782. Microscopy: 237–238 = stereo; 239–243 = PC; 245 = FL; 244, 246–247 = DIC. Bars: 237 = 1 mm; 238 = 0.5 mm; 239–243 = 20 μm; 244, 246–247 = 10 μm; 245 = 20 μm.

Figs 248–259.

Trichoderma petersenii, Hypocrea teleomorph. 248. Stromata formed on bark. 249. Longitudinal section through a mature stroma. 250, 252. Face view of a stroma; hairs visible in 252 (arrow). 251, 255. Median longitudinal sections through perithecia. 253–254. Sections through stroma surface; hairs marked by arrows. 256. Cells of stroma interior below a perithecium. 257–258. Asci. 259. Discharged part-ascospores. Figs 248, 252–254, 256–259 from G.J.S. 91-99; 249, 255 from G.J.S. 99-48; 250 from G.J.S. 04-355. Microscopy: 248 = stereo; all others = DIC. Bars 248–249 = 0.5 mm; 250, 253, 255–257 = 20 μm; 251 = 250 μm; 252, 254, 258–259 = 10 μm.

Figs 260–268.

Trichoderma rogersonii, anamorph from CMD. 260–261. Conidial pustules; individual conidiophores can be seen protruding from the periphery of the immature pustule in 261. 262–267. Conidiophores and phialides; an intercalary phialide can be seen in 264 inset (arrow). 268. Conidia. Figs 260–261 from G.J.S. 90-78; 262 from G.J.S. 04-158; 263, 267 from G.J.S. 04-157; 264–265 from G.J.S. 90-108; 266, 268 above from G.J.S. 90-79; 268 below from G.J.S. 98-82. Microscopy: 260–261 = stereo; 262–266 = PC; 267–268 = DIC. Bars: 260 = 1 mm; 261 = 0.5 mm; 262–267 = 20 μm; 264 inset, 268 = 10 μm.

Figs 269–280.

Trichoderma rogersonii, Hypocrea teleomorph. 269. Stromata; immature stromata indicated by arrows. 270. Stroma surface in face view; ostiolar opening of a perithecium indicatd by “O”; note the mottled appearance resulting from small piles of cells and short hairs (e.g. arrows). 271–273. Cells and hairs (arrows) at stroma surface in face view (271–272) and in section (273). 274. Median longitudinal section through mature perithecia. 275. Section through stroma surface; note hairs (e.g. arrows); note the loose stroma surface in 274–275. 276. Section through the ostiolar canal and surrounding stroma tissue; hair indicated by arrow. 277. Tissue of stroma interior below a perithecium. 278–279. Asci and ascospores. 280. Discharged part-ascospores. Fig. 269 from G.J.S. 95-217; 270–271, 274 from G.J.S. 98-75; 272, 278–279 from G.J.S. 04-157; 273, 275–276 from G.J.S. 90-108; 277 from G.J.S. 92-116; 280 from G.J.S. 04-158. Microscopy: 269 = stereo, all others DIC. Bars: Fig. 269 = 1 mm; 270–278 = 20 μm; 279–280 = 10 μm.

Figs 281–289.

Trichoderma stilbohypoxyli, anamorph from CMD. 281. Conidial pustule. 282–288. Conidiophores and phialides. 289. Conidia. Figs 281, 283–284, 289 from G.J.S. 96-32; 282, 286 from G.J.S. 96-30; 285, 287–288 from G.J.S. 96-42a. Microscopy: 281 = stereo, all others DIC. Bars: 281 = 0.5 mm; 282–288 = 20 μm; 289 = 10 μm.

Figs 290–299.

Trichoderma taiwanense. 290–295. Anamorph from CMD. 290–294. Conidiophores and phialides; note enlarged and roughened conidiophore base (arrows). 295. Conidia. 296–299. Asci and ascospores. Figs 296, 298 stained in 1 % aq. phloxine. All from G.J.S. 95-93. Microscopy: 290–293 = PC; 294–299 = DIC. Bars: 290–293 = 20 μm; 294–299 = 10 μm.