Taxonomic Study on the Lichen Genus Cetrelia (Lecanorales, Ascomycota) in South Korea

Abstract

Seventy-two lichen specimens of Cetrelia collected in South Korea since 2003 were examined by both phenotypic and phylogenetic analyses. The phenotypic analysis was based on morphological and chemical characters, and the phylogenetic analysis was based on nrDNA ITS sequences. The result suggested that the presence and absence of isidia, soredia, lobules and medullar reaction C+ or C- are the important characters in the taxonomy of this genus. Four species of Cetrelia, C. chicitae, C. braunsiana, C. japonica, and C. pseudolivetorum have been identified in this study. Description of each species is presented with morphological and chemical characters. A key to the Cetrelia species is also presented.

Cetrelia W.L. Culb. & C.F. Culb. belongs to the lichenized ascomycete family Parmeliaceae Zenker, cetrarioid genera. The name of Cetrelia is a fanciful concoction from Cetraria and Parmelia, in which most of the species had previously been placed. Culberson and Culberson (1968) combined some species from the old genus Cetraria and some species from Parmelia into the new genus Cetrelia based on morphological and chemical characters. The main characters of this genus are the broad lobe, the presence of laminal pseudocyphella, and the production of aromatic compounds such as orcinol-type depsides or depsidones. Although there were many reports on the study of Cetrelia (Beguinot 1982; Chen 1986; Culberson and Culberson 1968; Elix 1994; Harada 1996; Lai 2001), almost no expert study on Cetrelia had been conducted in Korea until the macrolichen flora of South Korea was published (Park, 1990). In her paper, 6 species of Cetrelia were reported with brief description of each species and a key to the genus. However, there are still some problems such as ambiguous characters which made it difficult to differentiate species and to do the corresponding taxonomic work. According to the most newly published checklist of Korean lichens (Hur et al., 2005), there were 8 species of Cetrelia recorded in Korean peninsula. The aim of this study was to evaluate the importance of different taxonomic characters which have not been carefully examined by phenotypic and phylogenetic analyses so far.

Materials and Methods

Phenotypic analysis

Seventy-two lichen specimens of Cetrelia from South Korea were examined and are deposited in KoLRI (Korean Lichen Research Institute). The gross morphology and anatomy of the specimens were examined by the dissecting microscope (Nikon SMZ 1500) and compound microscope (Olympus BX50). The chemical characters were examined by medullar color reaction and thin layer chromatography (Culberson, 1972; White and James, 1985). Nineteen morphological and chemical characters were chosen for the phenotypic analysis (Table 1, 2). Maximum parsimony analysis was performed by PAUP version 4.0b10 (Swofford, 2002), with Cetraria islandica as the out group.

Table 1.

Nineteen phenotypic characters chosen for phenotypic analysis of Cetrelia genus in this study

Table 2.

Matrix form of 19 phenotypic characters used to phenotypic analysis

Note: '1' indicates positive, '0' indicates negative, '?' indicates unknown.

DNA extraction and nrDNA amplification

Twenty lichen thalli were fractioned with cryo-tissue-crasher (SK200, Tokken, Japan) (Table 3). Total DNA was extracted directly from whole thalli according to Ekman (1999) with DNeasy Plant Mini Kit (QIAGEN, Germany), then purified by PCRquick-spin™ PCR Product Purification Kit (iNtRON Biotechnology, INC.). The nrDNA ITS region (ITS1-5.8S-ITS2) was amplified by PCR. Primers for amplification were: ITS1F (5'-CTTGGTCATTTACAGGAAGTAA-3'; Gardes and Bruns, 1993) and ITS4A (5'-ATTTGAGCTCTTCCCGCTTCA-3'; White el al., 1990). Previously described conditions by Arup (2002) have been used for PCR amplification and cycle sequencing.

Table 3.

Twenty specimens used in phylogenetic analysis

Sequencing and phylogenetic analysis

PCR products were sequenced by ABI 3700 automated DNA Sequencer in NICEM at Seoul National University. The phylogenetic analysis was executed by the Software Mega2 (Kumar et al., 2001). Kimura 2-parameter model was taken, and gaps were retained initially while being excluded in the pairwise distance estimation. The neighbor joining (NJ) (Saitou and Nei, 1987) method was used in constructing the phylogenetic tree and the reliability of the inferred tree was tested by 1,000 bootstrap relications. Cetralia islandica (Genbank accession no.: EF373567) was used as the outgroup.

Results and Discussion

Phenotypic analysis

Phenotypic analysis was performed by morphological and chemical characters (Table 1 and 2). Maximum parsimony analysis showed that Cetrelia was divided into two sections (I and II), according to the presence or absence of lobules, indicating that this character is the most important character to distinguish the species (Fig. 1)

Fig. 1.

UPGMA tree of 4 species of Cetrelia in South Korea, and Cetraria islandica as outgroup. Data matrix has 5 taxa, 19 characters. All characters are of type 'unord', all characters have equal weight, among which 15 variable characters are parsimony-uninformative, number of parsimony-informative characters = 4. Tree length = 19, consistency index (CI) = 1, homoplasy index (HI) = 0, CI excluding uninformative characters = 1, HI excluding uninformative characters = 0, retention index (RI) = ,1, rescaled consistency index (RC) = 0.

The results also indicated that either morphological characters or chemical characters were not enough to distinguish the species in Cetrelia and thus they should be considered together. For example, in section I, both C. pseudolivetorum (Asahina) W.L. Culb. & C.F. Culb. and C. japonica (Zahlbr.) W.L. Culb. & C.F. Culb. are lobulate and that is why they are easily wrong in brief morphological identification. Medullar color reaction of C is further significant to distinguish them. Olivetoric acid (C+ reaction) was only present in C. pseudolivetorum but was absent in C. japonica (Fig. 3). In section II, C. chicitae (W.L. Culb.) W.L. Culb. & C.F. Culb. and C. braunsiana (Müll. Arg.) W.L. Culb. & C.F. Culb. produce almost the same chemical compounds of alectoronic acid and α-collatolic acid. It is difficult to distinguish them by medullar color reaction or TLC, but they are morphologically very different, C. chicitae is marginal soridate while C. braunsiana is isidate.

Fig. 3.

1. Isidia-like lobules of C. pseudolivetorum 030784; 2. Isidia of C. braunsiana 040425; 3. Lobules of C. japonica 050267: a. pycnidia; 4. Soredia of C. chicitae 060317; 5. Pseudocyphella on upper surface of C. japonica 050267; 6. Acervulus of C. japonica 050314.

Phylogenetic analysis

The NJ consensus tree constructed by Mega2 is shown in Fig. 2. According to the tree, each species finely assembled together and this proved that the taxonomy of Cetrelia based on morphological and chemical characters is reliable.

Fig. 2.

NJ consensus tree based on nrDNA ITS sequences. Nucleotide: Kimura 2-parameter, pairwise deletion, bootstrap = 1000. The numbers in each node represents bootstrap support value, and the numbers lower than 50 were not shown.

Besides, all tested specimens were divided into two sections (I and II) according to the presence or absence of lobules, indicating that this character is very important in the taxonomy of Cetrelia, which is accordant with the phenotypic analysis (Fig. 1).

In conclusion, according to the comprehensive analysis of phenotypic and phylogenetic analysis, four characters are thought to be important in the taxonomy of Cetrelia and they are listed as follows: presence or absence of lobules, presence or absence of soredia, presence or absence of isidia, medullar reaction C- or C+. Among these four species, C. japonica and C. pseudolivetorum have closer relationship whereas C. chicitae and C. braunsiana are closely related.

In this paper, only ITS region was involved in phylogenetic anlysis, so, not all characters could be well evaluated. The amount of examined specimens is limited and only 4 species had been identified from South Korean materials. Some characters which are not considered to distinguish these four species might be significant to distinguish them from other species. Therefore, more corresponding work needs to be done in the future.

Taxonomic treatment of the genus

According to the above comprehensive analysis, a key to the genus is presented with morphological and chemical characters. Only representative specimens were cited when the number is large.

Key to Cetrelia species in South Korea

1. Thallus sorediate or isidiate, not lobulate.

• 2. Thallus sorediate, medullar reaction C-...C. chicitae (Bunkalgujieu)

• 2. Thallus isidiate, medullar reaction C-...C. braunsiana (Otkalgujieu)

1. Thallus neither sorediate nor isidiate, but lobulate

• 3. Thallus with dorsiventral flat lobules, medullar reaction C-...C. japonica (Jageunipmaehwakalgujieu)

• 3. Thallus with isidioid thick lobules, medullar reaction C+ pink or red...C. psuedolivetorum (Deungunipmaehwakalgujeu)

Taxonomy

1. Cetrelia chicitae (W.L. Culb.) W.L. Culb. & C.F. Culb., Contrib. U.S. Natl. Herb. 34 (7): 504-505, 1968

External morphology

Thallus medium, about 8.5 cm broad, lobes 0.8~1.8 cm broad. Upper surface ashy olive-green, margins somewhat rolled and downward; sorediate along margins (Fig. 3-4), pseudocyphellate, pores small, punctiform to enlongate but rarely exceeding 1mm. Lower surface jet-black, margins brown or colored like upper surface, rhizines to 1 mm or less, black, tips white. Medulla white. Pycnidia and apothecia not present in Korean specimens.

Chemistry

Thallus medullar C-, K-, KC+ pink, P-, contains atranorin, alectoronic, α-collatolic acid, 4-O-methylphsodic, and physodic acid (Fig. 4).

Fig. 4.

TLC plate for compounds known in Cetrelia under UV light, developed with solvent B' system. Lane C refers to control (Artranorin and norstictic acid); Lane 1, 4, 5, 6, 7, 8, 9, 10, 13 and 14 are C. japonica; Lane 2, 11 and 15 are C. braunsiana; Lane 3 is C. chicitae; Lane 12 is C. pseudolivetorum. Spot A: atranorin, B: norstictic acid, a: α-collatolic acid, b: alectoronic acid, c: microphyllinic acid, d: 4-O-demethylmicrophyllinic acids, e: olivetoric acid.

Distribution

Mt. Jiri (Fig. 5).

Fig. 5.

Distribution of Cetrelia species in South Korean. Circles (•) and squares (▪) represent the localities previously (Park, 1990) and newly reported in this study, respectively. The previous records confirmed by newly collected specimens in this survey were indicated as stars (⋆).

Habitat and ecology

Alt. 1620 m; on trunk of Abies.

Remarks

This species is rare and easily distinguished from other Korean species by its marginal soredia. It might be morphologically mistaken with C. japonica having poorly developed lobules, but they can be separated from each other by chemical analysis.

One specimen examined

060317 Jae-Seoun Hur, June 17, 2006.

2. Cetrelia braunsiana (Müll. Arg.) W.L. Culb. & C.F. Culb. Contrib. U.S. Natl. Herb. 34 (7): 493-498, 1968

External morphology

Thallus medium to large, 5~17 cm broad, lobes 0.5~0.7 cm broad. Granular or coralloid isidia finely or poorly developed along the margins or on the upper surface (Fig. 3-2), upper surface ashy-green, tan or uniformly brownish in some old herbarium specimens. Margins ascendant or downward. Pseudocyphellate, pores small, punctiform to irregular but rarely exceeding 1mm. Lower surface black, margins brown or grayish like the color of upper surface, rhizines black, about 1 mm. Apothecia not seen, pycnidia present in some specimens, limited on the tips of isidia.

Chemistry

Thallus medullar C-, K-, KC+ pink, P-, contains atranorin, alectoronic, α-collatolic acid, 4-O-methylphsodic, and physodic acid (Fig. 4).

Distribution

Mt. Baekwoon, Mt. Halla, Mt. Jiri, Mt. Juhul, Mt. Odae, Mt. Sokri, Mt. Wolak (Fig. 5).

Habitat and ecology

Alt. 510~1700 m; on trunk of Abies, Betula, Pinus and Quercus; sometimes on rock.

Remarks

Morphologically C. braunsiana is similar to C. pseudolivetorum because of the isidia-like lobules of C. pseudolivetorum, but chemically they are quite different. Medullar color reaction of C. braunsiana is C-, whereas C. psuedolivetorum is C+.

Representatives of 14 specimens

040425 Jae-Seoun Hur, May 7, 2004.

3. Cetrelia japonica (Zahlbr.) W.L. Culb. & C.F. Culb. Contrib. U.S. Natl. Herb. 34 (7): 511-513, 1968

External morphology

Thallus medium to large, 6~16 cm broad, lobes 0.5~1 cm broad, tips pruinose occasionally, the margins densely fringed with multi-branched lobules (Fig. 3-3), lobules narrow or broadly expanded and lobe-like, flat. But in some specimens, lobules poorly developed, unbranched and flat. Upper surface greenishgray to yellowish-green, or tan in some old herbarium specimens. The margins ascendant or downward. Pseudocyphellae conspicuous, punctiform to elongate smaller or about 1 mm (Fig. 3-5). Lower surface black, margins brown or grayish like the color of upper surface, rhizines black, tips white sometimes, about 1 mm, but in some specimens, rhizine absent or few. Apothecia not seen, pycnidia black, about 0.1 mm broad, the ostiole prominent, terminal, or absent, acervulus present occasionally (Fig 3-6), laminal, about 1 mm broad. Conidia 1.25 × 5 µm, rod shaped, the ends somewhat enlarged.

Chemistry

Thallus medullar C-, K-, KC+ pink or KC-, P-, contains atranorin, microphyllinic, 4-O-methylolivetoric, and 4-O-demethylmicrophyllinic acids (Fig. 4).

Distribution

Mt. Baekwoon, Mt. Dukyoo, Mt. Halla, Mt. Jiri, Mt. Jumbong, Mt. Odae, Mt. Sobaek, Mt. Sorak, Mt. Taebaek (Fig. 5).

Habitat and ecology

Alt. 450~1900 m; on trunk of Abies, Acer and Quercus; occasionally on rock with mosses.

Remarks

It may be confused with C. pseudolivetorum without a chemical test if the lobules are not well developed. The lobules tend to be flatter and more dorsiventral in C. japonica than in C. pseudolivetorum. This species is the most common species in South Korea.

Representatives of 52 specimens

041594 Jae-Seoun Hur, October 12, 2004.

4. Cetrelia pseudolivetorum (Asahina) W.L. Culb. & C.F. Culb. Contrib. U.S. Natl. Herb. 34 (7): 519-521, 1968

External morphology

Thallus medium to large, 5~15 cm, lobes 0.5~1.5 cm broad, isidia-like dorsiventral dissected lobules present on margin of the lobes or on the upper surface (Fig. 3-1). Upper surface grayish or uniformly light brownish or tan in old herbarium specimens, smooth or becoming cracked, psuedocyphellate small to medium, about 0.5 mm broad, punctiform or slightly elongate. Lower surface black, margins brown or grayish like the color of upper surface, rhizines black. Apothecia not seen. Pycnidia present on the tips of lobules, few or abundant.

Chemistry

Thallus medullar C+ pink or red, K-, KC+ pink to red, P-, contains atranorin, olivetoric acid, anziaic acid as an accessory (Fig. 4).

Distribution

Mt. Dukyoo, Mt. Jiri, Mt. Sobaek, Mt. Taebaek (Fig. 5).

Habitat and ecology

Alt. 1300~1550 m; mostly on rock: sometimes on trunk of Quercus.

Remarks

C. pseudolivetorum is very close to C. japonica when it has well developed dorsiventral lobules, and close to C. braunsiana when it has isidia-like lobules, but their chemistry is very different.

Representatives of 5 specimens

030784 Jae-Seoun Hur, October 2, 2003.

The species not found in this time

Two more species of Cetrelia monachorum (Zahlbr.) W.L. Culb. & C.F. Culb. and C. olivetorum (Nyl.) W.L. Culb. & C.F. Culb. were previously recorded in South Korea (Park, 1990). These were rarely found even 20 years ago and all her collections were deposited in Duke University, USA. However, we have seen no corresponding specimens during our expeditions and, possibly, they are under threaten of extinction. C. monachorum is currently treated as synonymy of C. olivetorum. This species is easily distinguished from C. japonica and C. pseudolivetorum because of having soredia. It is not confused with C. chicitae and C. braunsiana because of C+ reaction due to the presence of olivetoric acid.

Cetrelia isidiata (Asahina) W.L. Culb. & C.F. Culb. and Cetrelia nuda (Hue) W.L. Culb. & C.F. Culb. were floristically reported in Korean peninsula without detailed description. These were not traceable due to lack of voucher specimens at this moment, so they are not included in this paper.