The First Finding of Lichen Solorina Saccata at an Algific Talus Slope in Korea

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Park et al.

1 RESEARCH ARTICLE

3 The first finding of lichen Solorina saccata at an algific talus slope in Korea

5 Jung Shin Park1, Kwang-Hyung Kim2, Dong-Kap Kim1, Chang Sun Kim1, Sook-Young Park3*,

6 Soon-Ok Oh1*

8 1 Korea National Arboretum, Pocheon 11186, Korea, 2 APEC Climate Center, Busan 48058,

9 Korea, 3 Department of Plant Medicine, Sunchon National University, Suncheon 57922, Korea

13 Running title: Solorina saccata at an algific talus slope in Korea

17 *CONTACT

18 Sook-Young Park and Soon-Ok Oh

19 [email protected] and [email protected]

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22 Abstract

23 An algific talus slope is composed of broken rocks with vents connected to an ice cave,

24 releasing cool air in summer and relatively warmer air in winter to maintain a more stable

25 microclimate all year round. Such geological features create a very unusual and delicate

26 ecosystem. Although there are around 25 algific talus slopes in Korea, lichen ecology of these

27 areas had not been investigated to date. In this study, we report the first exploration of lichen

28 ecology at an algific talus slope, Jangyeol-ri, in Korea. A total of 37 specimens were collected

29 over 2017-2018. Morphological and sequencing analysis revealed 27 species belonging to 18

30 genera present in the area. Of particular interest among these species was Solorina saccata, as

31 it has previously not been reported in Korea and most members of genus Solorina are known

32 to inhabit alpine regions of the Northern Hemisphere. We provide here a taxonomic key for S.

33 saccata alongside molecular phylogenetic analyses and prediction of potential habitats in South

34 Korea. Sequences were generated from all S. saccata specimens collected in this study,

35 together with three S. saccata specimens from China for comparison. Phylogenetic analysis

36 based on nuclear small subunit (nuSSU) showed that all the S. saccata specimens are tightly

37 grouped into one clade with high support values (P=0.99) and showed close relatedness to S.

38 spongiosa than S. crocea. Additional analyses were carried out using concatenated sequences:

39 65 sequences of mitochondrial small subunit (mtSSU) combined with nuLSU and 66 sequences

40 of RPB1 with nuLSU. In these analyses, genus Peltigera was found to be the closest genus to

41 S. saccata. Furthermore, regions in South Korea potentially suitable for Solorina spp. were

42 predicted based on climatic features of known habitats around the globe. Our results showed

43 that the suitable areas are mostly at high altitudes in mountainous areas where the annual

44 temperature range doesn’t exceed 26.6˚C. Further survey of other environmental conditions

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45 determining the suitability of Solorina spp. should lead to a more precise prediction of suitable

46 habitats and trace the origin of Solorina spp. in Korea.

47 Keywords: Algific talus slope, Phylogenic analysis, Solorina saccata, Taxonomy

51 Introduction

52 Algific talus slopes are geological features in high altitude areas, composed of broken rocks

53 with vents connected to an ice cave. The air flow through these vents create a meteorologically

54 unique area, where cold air blows out of the vents or “wind-holes” in summer and relatively

55 warmer air is released in winter [1].

56 During the last ice age in the Pleistocene epoch of the Cenozoic Era, vegetation in the

57 Northern Hemisphere spread southwards. It was reported that due to the special microclimate

58 created by the temperature-stabilizing effect of algific talus slopes, they sheltered various

59 migrating animals and vegetation during interglacial periods [1-3]. Plants, mite-like insects or

60 snails typically inhabiting higher latitudes were found at algific slopes [4, 5], suggesting that

61 these geological features provide a distinctive environment from the surrounding area.

62 Algific talus slopes are located at latitudes of 35-45° north [6-11]. They are found in the

63 Northern Hemisphere in Europe, East Asia and the USA. In the USA alone, more than 400

64 locations have been reported in Minnesota, Iowa, Wisconsin, Illinois and Pennsylvania. In

65 Europe, several algific talus areas are distributed in mountainous regions around the Alps [12].

66 In central Honshu, Japan, there are more than 80 algific talus areas. Although a number of

67 comprehensive studies on climate, topography, surface geology and vegetation of algific talus

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68 areas have been carried out [13-16], only a few studies exist on lichen ecology and distribution

69 in these areas.

70 In a survey of lichen at White Pine Hollow State Park, Iowa, USA, 71 of 117 samples

71 were collected from algific talus areas and 4 out of 13 newly discovered species were found

72 in these areas. Species such as Peltigera ponojensis, Peltigera membranacea, and Physconia

73 muscigena were found at higher altitude than in Iowa, but were found in the field [17]. In

74 another study, a survey of lichens was conducted in the Spruce Creek Ice Caves, Huntingdon

75 County, Pennsylvania, USA. Many of the lichens identified including Arctoparmelia

76 centrifuga, Cladonia coccifera, Cladonia rangiferina, Porpidia tuberculosa, Protothelenella

77 corrosa, Rhizocarpon subgeminatum, Stereocaulon glaucescens, Vulpicida pinastri are

78 typically native of the northern alpine habitats [7].

79 In South Korea, 25 algific talus slopes are distributed from Jeju Island to Gangwon

80 province (Fig. 1) [2]. Among these areas, vegetation of Jangyeol-ri algific talus slope, in

81 Jeongseon, Kangwon province, was dominated by plant species Astilboides tabularis and

82 Pedicularis resupinata, which are known to be highly vulnerable to climate change, suggesting

83 that the biota typical of the northern regions are specifically present in the area [2].

84 The lichen genus Solorina is known to be distributed in bipolar, boreal, and arctic-

85 alpine environments [5]. The morphological characteristics of the genus Solorina Ach. are said

86 to include terricolous lichens with a foliose thallus and ascoma impressed in the upper surface

87 [18]. Genus Solorina belongs to the family Peltigeraceae, as is genus Peltigera, and is also

88 closely related to genus Nephroma. The relationship between the two genera is supported by

89 similar ascomatal ontogeny and ascus morphology [7, 8]. Unlike genus Peltigera, genus

90 Solorina have laminal apothecia, pigmented and verrucose spores [9].

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91 In a molecular phylogenetic analysis, genus Solorina was used as outgroups to assess

92 the specificity of lichen-forming fungi and genus Nostoc in the genus Peltigera section

93 Polydactylon [10]. The genera of Peltigeraceae including Solorina spp. have been used to

94 identify the phylogenetic location of lichen symbiotic members and to improve the

95 classification and phylogeny of Lecanoromycetes [11, 12]. In addition, genus Solorina has been

96 classified as a member of family Solorinaceae, and the relationship between species is

97 paraphyletic and monophyletic close to Peltigeraceae in a study to identify the morphogenic

98 phylogenetic location of Peltigeraceae or Pertigerales [19]. Another study on Peltigeraceae

99 claimed that genus Solorina belongs to the monophyletic family Peltigeraceae, and closely

100 related to the sister genus Peltigera [9].

101 Unfortunately, genus Solorina has been largely disregarded in molecular phylogenetic

102 studies on Peltigeraceae over the last decade have only been referred to as a sister genus to

103 Peltigera. Genus Solorina consists of about 10 species distributed throughout the world, with

104 4 species in Japan and 5 species in China [14, 15], but had so far not been found in Korea.

105 The objectives of this study were (i) to survey lichen species in Jangyeol-ri algific talus

106 slope, (ii) to identify the collected lichen species, and (iii) to identify lichen genus Solorina at

107 morphological and molecular levels. In this study, we catalog the variety of lichen species

108 present in Jangyeol-ri algific talus slope and also report the first finding of Solorina saccata, a

109 northern lichen species, in Korea.

110

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111 Materials and Methods

112 Morphological examination

113 All lichen specimens collected in this study were deposited at the Korean National Arboretum

114 (KNA). Air-dried samples were observed using a stereomicroscope (Olympus SZX7) and a

115 compound microscope (Olympus CX22LED). Water mounts were hand sectioned with a razor

116 blade and microscopic features (ascomatal structure) were observed in water. Color reactions

117 were conducted as described [20].

118

119 DNA extraction and PCR amplifications

120 Four representative Solorina saccata specimens were selected and used for further molecular

121 analyses. Lichen thalli with apothecial discs were mainly used for DNA extraction. Samples

122 were ground and extracted with DNeasy plant mini kit (Qiagen, Valencia, CA, USA) according

123 to the manufacturer’s instructions. PCR amplifications were conducted using Amplitaq DNA

124 polymerase (ThermoFisher, Massachusetts, Watham, USA). The following primers were used

125 for PCR amplifications: mtSSU1 and mtSSU3R for mtSSU [21]; NS17UCB, NS20UCB for

126 nuSSU [22]; LIC24R [23] and LR7 [24] for nuLSU; fRPB2-7cf and fRPB2-11aR for RPB2

127 [25]. PCR conditions for nuSSU are as described in a previous study [26]. The following

128 program was used for amplification of nuLSU: initial denaturation for 4 min at 94℃, followed

129 by 30 cycles of 94℃ for 40 s, 52℃ for 40 s, 72℃ for 50 s and then a final extension step at

130 72°C for 8 min. Amplified DNA was concentrated and purified using a PCR quick-spin PCR

131 Product Purification Kit (INTRON Biotechnology, Inc. Sungnam City, Korea) for sequencing

132 analysis.

133

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134 Sequence alignments and phylogenetic analysis

135 Obtained sequences were aligned with Clustal W ver. 1.83 [27] and edited using the Bioedit

136 program. Based on the sequences, we selected commonly present sequence regions of

137 homology, and excluded uncommonly detected sequences. We utilized Gblocks 0.91b server

138 [28] for deleting ambiguously aligned regions in the concatenated alignment of nuSSU, nuLSU

139 and RPB1. We prepared a three-locus concatenated dataset containing nuSSU, mtSSU+nuLSU

140 and nuLSU+RPB1.

141 Phylogenetic analyses were conducted with MEGA 7.0 and MrBayes v. 3.2.6. Bayesian

142 analysis was carried out on the data set using the Metropolis-coupled Makov chain Monte Carlo

143 Method (MCMCMC) in MrBayes v. 3.2.6 [29, 30]. Best fit substitution models were estimated

144 using in Akaike-information as implemented in jModelTest v 2.15. [31]. The TVM+I model

145 in nuSSU, TIM3+I+G model in mtSSU+nuLSU, and JC+G model in nuLSU+RPB1 were

146 selected. Each MCMCMC run was performed with four chains and 10 million generations.

147 Trees were generated 1000 times and the first 25% was discarded. The remaining trees were

148 determined by calculating a majority-rule consensus tree with posterior probabilities (PP).

149 In addition, we also performed Maximum likelihood (ML) analysis using MEGA 7.0 with

150 1000 ML bootstrap values (BP) and GTR+G model was applied. We selected outgroup for

151 phylogenetic analysis according to previous research [32-34]. Phylogenetic trees were drawn

152 using Figtree v. 1.4.3 [35] with Treeview X v. 0.5.0 and posterior probabilities above 0.80 (PP)

153 were used near the bold branches.

154

155 Estimation of optimum temperature for Solorina spp.

156 Although Solorina species are native to polar and alpine regions, our specimens were collected

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157 at a relatively low altitude of 400-450 m above sea level. To determine climatic requirements

158 for Solorina spp., we first identified all from collection sites of Solorina spp. from previously

159 published literature and relevant reports, and then collected 30 years’ climate data on minimum

160 and maximum air temperature for the identified habitats

161 A few criteria were applied to filter out unusable data. Since the climate in the Southern

162 Hemisphere has different seasonal variations, data from the Southern Hemisphere were

163 excluded. Sites with uncertain sampling location or unspecific records above district level were

164 also excluded. Multiple sites located within 1 km radius were combined and considered as a

165 single site, as we used a 1 km resolution grid dataset to extract corresponding climate data.

166 As a resut, the number of sites was narrowed down from 73 to 63 sites for subsequent

167 climatological analysis. Coordinates (latitude and longitude) of the chosen sites were first

168 estimated based on available site information and using the Google Maps

169 (https://www.google.com/maps). Then monthly minimum and maximum temperature

170 information at the identified coordinates were extracted from the WorldClim, a monthly

171 climatology dataset provided at 1 km resolution (https://www.worldclim.org/). Average

172 monthly minimum and maximum temperatures were calculated and plotted together with all

173 the temperature ranges of the 63 sites (Fig. 7).

174 Site suitability for Solorina spp. in South Korea was determined based on the temperature

175 data of 63 sites. The temperature range for each month was defined by the minimum and

176 maximum values among the 63 sites. The temperature range values were used to identify

177 suitable areas with similar monthly temperature variations in South Korea. If the local

178 temperature of an area satisfies the above defined temperature range profile, it was deemed

179 “Suitable”. Other areas were considered “Unsuitable”. Local temperature profiles to 1 km

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180 resolution of South Korea were obtained from a historical climate dataset produced by the

181 Korean Meteorological Administration (KMA). High resolution KMA temperature datasets

182 were produced from 75 KMA Automated Synoptic Observing System (ASOS) stations and

183 462 KMA Automated Weather Stations (AWS) using PRIsm-based Downscaling Estimation

184 (PRIDE) model [36], and included daily weather data from 2001–2010.

185

186

187

188 Results and Discussion

189 Survey of lichen species in Jangyeol-ri algific talus slope

190 Jangyeol-ri algific talus slope is located at 200-450 m above sea level in Jeongseon, Kangwon

191 province (37˚27'06.14"N, 128˚41'04.18"E). It is 200 m in breadth and sloped at 40 degrees

192 (Fig. 2A-E). The geological features were consistent with the Paleozoic Ordovician strata, most

193 of which were limestone and dark red forest soils [2].

194 A total of 37 lichen specimens were collected from 2017 to 2018. From these samples, 27

195 species belonging to 18 genera were identified by morphological examination and sequencing

196 analysis: genus Amandinea (A. punctate); Caloplaca (C. flavovirescens); Candelaria (C.

197 concolor); Cladonia (C. furcata subsp. furcata and C. pyxidata); Collema (C. japonicum and

198 C. leptaleum var. biliosum); Everniastrum sp.; Flavoparmelia (F. caperata); Graphis (G.

199 scripta); Heterodermia (H. diademata and H. hypoleuca); Lecanora (L. argentata and L.

200 strobilina); Leptogium (L. cyanescens); Myelochroa (M. aurulenta); Ochrolechia (O.

201 akagiensis); Peltigera (P. elisabethae, P. horizontalis, and P. rufescens); Phaeophyscia (P.

202 primaria); Protoblastenia (P. rupestris); Ramalina sp.; Solorina (S. saccata) (Table 1).

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203 In this survey, we could not find any lichen species overlapping with those found in

204 previous studies on lichen inhabiting algific talus slopes in White Pine Hollow, Iowa, USA

205 [17] and Spruce Creek Ice Cave, Pennsylvania, USA [7]. Interestingly, the most predominant

206 lichen species we found in Jangyeol-ri algific talus belonged to genus Solorina (10.8% of all

207 specimens collected), which are mainly observed in circumpolar, arctic alpine and boreal areas

208 including Europe, Asia and North America [37]. Although there is no overlap between lichen

209 species found in Jangyeol-ri and Spruce Creek Ice Cave, the species collected at these two

210 different algific talus slopes were species common to northern alpine areas, indicating that the

211 vegetation at algific talus slopes are very different from the surrounding areas at an equivalent

212 latitude.

213

214 Phylogenetic analysis of Solorina saccata using nuSSU, nuLSU and RPB1

215 We generated nuSSU, mtSSU, nuLSU, ITS, RPB1 and RPB2 sequences from the four S.

216 saccata specimens in this study (Supplemental Table 1). Additionally, we also generated

217 sequences from three S. saccata specimens previously collected in China for comparison. We

218 successfully obtained sequences from all above specimens, except one Korean S. saccata

219 specimen, Oh KL17-0241. The five ribosomal gene and ITS region sequences obtained from

220 our specimens were over 99% identical to the S. saccata sequences in GenBank (data not

221 shown).

222 For phylogenetic analysis, we retrieved reference sequences of family Peltigerales

223 (Dendriscocaulon, Nephroma, Leptochidium, Lobaria, Lobariella, Peltigera,

224 Pseudocyphellaria, Solorina and Sticta) from GenBank (Supplemental Table 1). A

225 phylogenetic tree was generated using 26 nuSSU sequences (Fig. 3). All the sequences from

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226 our putative S. saccata specimens used in this analysis were tightly grouped into one clade with

227 high support values (P=0.99). In addition, phylogenetic analysis revealed that S. saccata is

228 more closely related to S. spongiosa than S. crocea. This is in correlation with the color of the

229 lower surface, which is white for S. saccta and S. spongiosa and orange for S. crocea [38].

230 Furthermore, where nuSSU sequences and either mtSSU or RPB1 sequences were available,

231 we performed phylogenetic analysis using 65 concatenated mtSSU+nuLSU sequences (Fig. 4)

232 and 66 nuLSU+RPB1 sequences (Fig. 5). The two phylogenetic trees in Fig. 4 and Fig. 5, more

233 convincingly showed that S. saccata was clearly different from other genera in the family.

234 Within the family, genus Peltigera was closest to Solorina in both the trees from concatenated

235 sequences. These results are in accordance with a previous analysis based on nuSSU sequences

236 showing that Solorina is located in the Hydrothyria-Peltigera clade and is a sister-clade to

237 Peltigera with 100% bootstrap support [23]. Also, when using mtSSU and nuLSU sequences,

238 S. crocea and Massalongia calrosa were closely related to Peltigera with very high support

239 values [33].

240

241 Comparision of average temperatures at Jangyeol-ri algific talus slope and the

242 surrounding area, Jeongseon-gun

243 To investigate whether Jangyeol-ri algific talus indeed has a distinct temperature profile from

244 the surrounding Jeongseon-gun region, we examined the monthly temperature variation (Fig.

245 6). Because the temperature measurements in Jangyeol-ri algific talus slope were only available

246 from April to November 2017, the data were compared with those of Jeongseon-gun in 2017

247 (Fig. 6).

248 The maximal difference in average temperature was observed in August 2017, when

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249 Jangyeol-ri was 15.7℃, which was 7.3℃ lower than the average temperature of 23.0℃ in

250 Jeongseon-gun. In comparison, in November 2017, the average temperature of Jangyeol-ri

251 algific talus slope was 2.93℃, which was 0.87℃ lower than the average temperature (3.8℃) in

252 Jeongseon-gun. These results indicate that the greatest differences in temperature between

253 Jangyeol-ri algific talus slope and the rest of Jeongsun-gun is in the summer months. It is

254 presumed that the cooler temperatures in summer may contribute to the continued existence of

255 Solorina spp. in Jangyeol-ri.

256

257 Prediction of suitable areas for Solorina spp. in South Korea

258 Suitable areas for Solorina spp. in South Korea were predicted using the monthly temperature

259 profiles of 63 sites across the globe where the Solorina spp. were found, assuming that monthly

260 temperature variations are a major factor determining suitability as a habitat. Our results

261 showed that the highest monthly maximum recorded was 26.6˚C in August and the lowest

262 monthly minimum temperature was -43.9 ˚C in January (Fig. 7). The minimum temperature of

263 -43.9˚C indicates that Solorina spp. may be able to endure the extreme cold temperatures in a

264 state of dormancy like other lichens in polar regions.

265 On the contrary, warmer temperatures may not be suitable to sustain Solorina spp., as

266 previously reported areas are confined in bipolar, boreal, and arctic-alpine environments [18,

267 37, 39-43]. With an assumption that the Solorina spp. in Korea would share the same

268 temperature requirements as those found at other sites around the world, we used the annual

269 temperature ranges of other reported sites to predict potential areas in South Korea that

270 Solorina spp. may exist.

271 Our result showed that the suitable areas are mostly at high altitudes in mountainous areas,

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272 where annual temperature does not exceed 26.6˚C. The fact that Solorina spp. in Korea were

273 found in the algific talus area, where the temperature is lower than surrounding areas in the

274 summer months, supports our prediction results. It is highly probable that the Solorina spp.

275 found in the algific talus area may not result from a migration from mountainous areas all the

276 way down to the lower altitudes (Fig. 8). Rather, we speculate that Solorina spp. have been

277 inhabiting the algific talus area likely since thousands of years ago when the algific talus area

278 was at a high altitude before going through diastrophism.

279 Other algific talus areas in Korea also show similar temperature variations to the predicted

280 areas suitable for Solorina spp. Based on our preliminary analysis using temperature records

281 of those sites over a few years, the highest temperatures recorded are generally 1-2˚C less than

282 26.6˚C, which is the highest monthly maximum temperature of the global sites of Solorina spp.

283 Although it is possible that Solorina spp. indeed exist in the mountainous areas as predicted in

284 our study, temperature alone may not be sufficient as an indicator of suitability for the habitat

285 of Solorina spp.; other environmental factors such as relative humidity and rainfall may also

286 be necessary determinants. However, it is very difficult to find out these environmental

287 conditions with very limited data available from algific talus areas in Korea. Further survey on

288 other environmental factors and their effect on Solorina spp. should result in more reliable

289 prediction of their habitats in Korea and other global areas.

290

291

292

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293 Key to differentiate between the three related genera, Peltigera, Nephroma and Solorina

294 1. Photobiont in thallus mainly green (chlorococcoid), thallus lobes rounded, with a single

295 urceolate apothecia immersed in the centre of the thallus or if absent, lower surface orange and

296 without distinct veins...... Solorina

297 1. Photobiont in thallus mainly blue-green (cyanobacterial, especially Solorina simensis) ...... 2

298 2. Lower surface naked or sparingly tomentose, apothecia (if present) on upper surface of lobe

299 ends ...... Nephroma

300 2. Lower surface with a well-developed coarse network of white or brown veins, apothecia (if

301 present) on short marginal protections ...... Peltigera

302

303 Key to identify known species of Solorina

304 1. Photobiont in thallus mainly blue-green (cyanobacterial), apothecia mainly plane, 4-spored

305 in ascus ...... S. simensis

306 1. Photobiont in thallus mainly green ...... 2

307 2. Lower side of thallus vivid orange ...... S. crocea

308 2. Lower side of thallus white or pale brownish ...... 3

309 3. 1-2 spores in each ascus ...... 4

310 3. 4-8 spores in each ascus ...... 8

311 4. 1 spore in each ascus ...... 5

312 4. 2 spores in each ascus ...... 6

313 5. Spores 2-septate, spore size 90-128(-161) × 30-44 μm ...... S. monospora

314 5. Spores 2-3 septate, spore length 85-120 μm ...... S. embolina

315 6. Cephalodia immersed in thallus ...... S. bispora var. subspongiosa

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316 6. Cephalodia not immersed in thallus ...... 7

317 7. Spores > 90 μm long ...... S. bispora var. macrospora

318 7. Spores < 90 μm long ...... S. bispora var. bispora

319 8. 4 spores in each ascus ...... 9

320 8. 6-8 spores in each ascus ...... 10

321 9. Apothecia urcelate, distinctly sunken in depression in the upper surface ...... S. saccata

322 9. Apothecia thinly depressed when immature but becoming level when mature ......

323 ...... S. platycarpa

324 10. 6 spores in each ascus, external cephalodia forming a dark spongy mat between and under

325 the apothecia ...... S. spongiosa

326 10. 8 spores in each ascus ...... S. octospora

327

328 Identification guide for Solorina saccata.

329 Solorina saccata (L.) Ach., K. Vetensk-Acad. Nya Handl. 29: 228 (1808) (Fig. 9)

330 Thallus well developed, spreading, forming round lobes with waxy margins, smooth, forms

331 rosettes, greenish gray when dry but green when wet, coarse white pruinose at least close to

332 the margin, thin spread; lower surface pale brown, rhizine sparsely present, scattered, 2-2.5

333 mm long, pale brown, indistinctly veined. Apothecia frequent, reddish brown, to 6.5 mm diam.,

334 deeply sunken in depression in the upper surface, with or without margin, margin 0.5 mm thick,

335 concolor with thallus. Asci numberous, 4-spored, epihymenium brown to dark brown, 25-32.5

336 μm, hymenium hyaline, 155-187.5 μm; ascospores 45-53 μm × 17.5 μm, pale brown to brown,

337 1-septate, ellipsoid-oblong. No lichen substance detected by TLC.

338

15 bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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339 Acknowledgements

340 The authors grateful to Bun-Yeon Park for assistance in DNA sequencing, Jeong-Jae Woo and

341 Young-Nam Kwag for helpful comments. This work was supported by a grant from the Korean

342 Forest Service Program through the Korea National Arboretum (KNA1-1-22, 17-2), and the

343 National Research Foundation of Korea (NRF-2017R1D1A1B04035888).

344

345 Jung Shin Park1, Kwang-Hyung Kim2, Dong-Kap Kim1, Chang Sun Kim1, Sook-Young Park3*,

346 Soon-Ok Oh1*

347

348

349 Author Contributions

350 Conceptualization: Jung Shin Park, Sook-Young Park, Soon-Ok Oh

351 Data analysis: Jung Shin Park, Kwang-Hyung Kim, Dong-Kap Kim, Sook-Young Park

352 Funding acquisition: Chang Sun Kim, Sook-Young Park, Soon-Ok Oh

353 Investigation: Jung Shin Park, Kwang-Hyung Kim, Sook-Young Park

354 Wring- original draft: Jung Shin Park, Kwang-Hyung Kim, Sook-Young Park, Soon-Ok Oh

355

356

357

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454 43. Gärtner G., Dablander A., Kofler W. Zur Taxonomie von Solorina bispora NYL. ssp. bispora 455 (Ascolichenes) nach Sporenmerkmalen. Ber. Naturwiss.-med. Ver. Innsb. 2011;97:27-33.

456

457

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458 Table 1. Summary of lichen species in Jangyeol-ri algific talus slope

No. of Year of Species Family specimens collection Caloplaca flavovirescens Teloschistaceae 1 2017 Candelaria concolor Candelariaceae 1 2017 Cladonia furcata subsp. furcata Cladoniaceae 2 2017 Cladonia pyxidata Cladoniaceae 3 2017 Collema japonicum Collemataceae 1 2017 Collema leptaleum var. biliosum Collemataceae 1 2017 Everniastrum sp. Parmeliaceae 1 2017 Flavoparmelia caperata Parmeliaceae 1 2017 Heterodermia diademata Physciaceae 1 2017 Heterodermia hypoleuca Physciaceae 2 2017 Lecanora argentata Lecanoraceae 1 2017 Lecanora strobilina Lecanoraceae 1 2017 Leptogium cyanescens Collemataceae 1 2017 Myelochroa aurulenta Parmeliaceae 1 2017 Peltigera elisabethae Peltigeraceae 3 2017 Peltigera horizontalis Peltigeraceae 1 2017 Peltigera rufescens Peltigeraceae 2 2017 Phaeophyscia primaria Physciaceae 1 2017 Ramalina sp. Ramalinaceae 1 2017 Solorina saccata Peltigeraceae 1 2017 Amandinea punctata Caliciaceae 1 2018 Cladonia furcata subsp. Furcata Cladoniaceae 1 2018 Graphis scripta Graphidaceae 1 2018 Lecanora strobilina Lecanoraceae 2 2018 Ochrolechia akagiensis Ochrolechiaceae 1 2018 Protoblastenia rupestris Psoraceae 1 2018 Solorina saccata Peltigeraceae 3 2018 Total 37 459

460

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461 Figure legends

462 Fig 1. Location of 25 algific talus slopes in Korea. The black circle indicates Jangyeol-ri algific

463 talus slope. Other sites are indicated in grey circles.

464

465 Fig. 2. Photographs of Jangyeol-ri algific talus slope. (A) The collection area is marked by the

466 dashed-line circle and the exposed area of the talus slope is marked by dashed-line rectangle.

467 (B) The collection site of lichens in Jangyeol-ri algific talus slope. (C) Two vents. (D) Lichen

468 species Solorina saccata in front of the vent. (E) Exposed area. The smaller insert shows more

469 detail at the ground level.

470

471 Fig. 3. Phylogenetic relationship within family Pertigerales inferred from Bayesian analysis

472 using nuclear small subunit (nuSSU) sequences. Only posterior probability values higher than

473 80% are shown. Strongly supported nodes are indicated in bold.

474

475 Fig. 4. Phylogenetic relationship within family Pertigerales inferred from Bayesian analysis of

476 concatenated mitochondrial small subunit (mtSSU) and nuclear large subunit (nuLSU)

477 sequences. Only posterior probability values higher than 80% are shown. Strongly supported

478 nodes are indicated in bold.

479

480 Fig. 5. Phylogenetic relationship of Pertigerales inferred from Bayesian analysis of

481 concatenated nuLSU and RPB1 sequences. Only posterior probability values higher than 80%

482 are shown. Strongly supported nodes are indicated in bold.

483

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484 Fig. 6. Comparison of average temperatures at the Jangyeol-ri algific talus slope and the

485 surrounding area, Jeongsun, in 2017. X-axis: Temperature / C, y-axis: Month.

486

487 Fig. 7. Analysis of minimum and maximum air temperatures at Solorina spp. habitats

488 worldwide. (A) 63 sites where Solorina spp. has been reported, indicated by red dots. Monthly

489 minimum (B) and maximum (C) temperatures of the 63 sites were extracted from the

490 WorldClim dataset (https://www.worldclim.org/). Average (mean) monthly minimum (B) and

491 maximum (C) temperatures are indicated as solid black lines in each graph and temperature

492 range at the 63 sites are indicated in grey.

493

494 Fig. 8. Areas potentially suitable as habitats for Solorina spp. in South Korea, solely based on

495 monthly minimum and maximum temperatures in 1 km grid cells. Predicted habitats are

496 marked in red over the elevation map of South Korea.

497

498 Fig. 9. Morphology of Solorina saccata. (A) Solorina saccata KL17-241; (B) Cross section of

499 the ascomata (4-spored asci) (Scale bars: B=40 µm).

500

501

502

503 Supporting information

504 S1 Table 1. A total 126 taxa and the generated or retrieved nuSSU, nuLSU, mtSSU, RPB1,

505 RPB2, and ITS sequences from NCBI.

22 bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/710947; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.