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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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.
Park et al.
1 RESEARCH ARTICLE
2
3 The first finding of lichen Solorina saccata at an algific talus slope in Korea
4
5 Jung Shin Park1, Kwang-Hyung Kim2, Dong-Kap Kim1, Chang Sun Kim1, Sook-Young Park3*,
6 Soon-Ok Oh1*
7
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
10
11
12
13 Running title: Solorina saccata at an algific talus slope in Korea
14
15
16
17 *CONTACT
18 Sook-Young Park and Soon-Ok Oh
19 [email protected] and [email protected]
20
21
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Park et al.
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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Park et al.
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
48
49
50
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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Park et al.
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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Park et al.
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.
Park et al.
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.
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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
21 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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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.