The various substrates of Usnea aurantiaco-atra and its algal sources in the Fildes Peninsula, Antarctica

CAO Shunan, ZHENG Hongyuan, LIU Chuanpeng, TIAN Huimin, ZHOU Qiming,5* & ZHANG Fang*

1 SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, China;

2 College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China;

3 School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, China;

4 Medical Faculty of Chifeng University, Chifeng 024000, China;

5 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

1 Introduction

Lichen is a typical symbiotic association, comprising the lichenized fungus (mycobiont) and its photosynthetic partner(alga or cyanobacterium, photobiont). In this symbiosis,the photobiont provides carbon sources, such as polybasic alcohol (green algae) or glucose (cyanobacteria) by photosynthesis activity[1–3], and the mycobiont protects its photosynthetic partner from strong radiation and desiccation by enveloping the algae cells. In the lichen thallus, the sexual reproduction of the photobiont is inhibited[4]or suppressed[5].However, the mycobiont has various ways of reproducing,such as by vegetative propagation (soredia or segment of the thallus), by a sexual procedure (the ascospores), or by an asexual method (the conidiospores). The ascospores of fungi must meet and recognize their compatible photobiont partner before they form a stabilized relationship developing into lichen thalli. This process is known as “l(fā)ichenization”.The sources of lichenized algae have attracted considerable attention because free photobionts are very rare in nature[6].

About 17500 lichenized fungi have been identified[7],but only 200 photosynthetic partners have been reported based on morphological studies (100 green algae and 100 cyanobacteria)[8]. This means that many different lichens must harbor the same photobiont. Conversely, some lichenized fungi may incorporate different algae as their photobionts[9-10],and it is believed that the mycobionts are able to adapt to various environments in this way[11-12].

There are two ぼowering plants, 104 mosses and about 427 lichens in Antarctica[13]. In the Fildes Peninsula, where the Great Wall Station is located, about 120 lichens have been reported (http://www.aari.aq/kgi/Vegetation/lst_lichens.html), of which the most dominant species is the fruticose lichenUsnea aurantiaco-atra(Jacq.) Bory. Most individuals of this species grow on rock, have erect and strong thalli,and apothecia; the minority grow with mosses, have thin and flattened thalli, and without apothecia.Umbilicaria antarcticaFrey & I. M. Lamb, whose diameter could reach 20 cm, was the most abundant foliose lichen in this area.Lichen substrates provide the micro-environment for their survival and have some specificity[14]. For example, all species in the genusUmbilicariaHoffm. were found to grow on rocks exceptU. yunnana(Nyl.) Hue; besides, wood is rarely also the substrate for someUmbilicarialichens under harsh environmental conditions[15-16].

Antarctica is an ideal area for the study of the recognition and association between mycobionts and photobionts. During the 27th and 28th Chinese National Antarctic Research expeditions (CHINARE), a fewUsnea aurantiaco-atraindividuals were found growing on the lichenUmbilicaria antarcticaand on wood. This provides a new insight in understanding the lichenization process.

ITS rDNA is one of the most widely used molecular markers in taxonomy, systematics and phylogenetics[17–19], and has been used as a DNA barcodes marker[20]. ITS rDNA was used to identify and analyze the phylogenetics of symbionts fromUmbilicaria antarcticaandUsnea aurantiaco-atrain Fildes Peninsula, Antarctica. Our study clarified the algal source in lichenization, and revealed the ecological function of the substratum preference.

2 Materials and Methods

2.1 Materials

2.1.1 Sampling

During the 27th and 28th CHINAREs, seven typicalUsnea aurantiaco-atraindividuals (two growing on rock, two on wood, two with moss and one onUmbilicaria antarctica)and sevenUmbilicaria antarcticaindividuals (Table 1)were collected in the Fildes Peninsula and Ardley Island,Antarctica. Fragments of thalli (200–500 mg) from 280 (on rock) and 104 (with moss) individuals ofUsnea aurantiacoatrawere gathered for molecular phylogenetic analysis. The sampling sites were marked using Google Earth 7.1.2.2041(Google Inc., USA) (Figure 1).

2.2 Methods

2.2.1 Morphology

Typical individuals ofUsnea aurantiaco-atra(with wellgrown thalli and clear features such as apothecia or soredia)and the special individuals (growing on other substrates such as woods or other lichens), together withUmbilicaria antarcticaindividuals, were photographed and collected. The morphological features were investigated using a SMZ–168 stereo zoom microscope (Motic China Group Co., LTD.) in the Scientific Research Building of the Great Wall Station.

Table 1 Samples used in this study

Figure 1 Map of sampling locations.: Usnea aurantiaco-atra thalli fragments;: U. aurantiaco-atra on rock;: U. aurantiaco-atra on moss;: U. aurantiaco-atra on wood;: U. aurantiaco-atra on thallus of Umbilicaria antarctica;: Umbilicaria antarctica.

2.2.2 Extraction of total DNA

Total DNA was extracted using a modified CTAB Method[21]from the 14 morphologically inspected samples (sevenUmbilicaria antarcticaand sevenUsnea aurantiaco-atra)and 384U. aurantiaco-atracollected with a small amount of their thalli

2.2.3 ITS rDNA Amplification

The primer pairs ITS5 (5′–GGAAGTAAAAGTCGTAACAA GG–3′)/ITS4 (5′–TCCTCCGCTTAT TGATATGC–3′)[22]and nrSSU–1780–5′(5′–CTGCGGAAGGATCATTGATTC–3′) /nr LSU–0012–3′ (5′–AGTTCAGCGGGTGGTCTTG–3′)[4]were used to amplify ITS rDNA regions from the mycobiont and photobiont, respectively. PCR reactions were performed in a 50 μL reaction volume (100 ng of DNA template, 200 nM of each primer, 400 nMdNTP, 1×buffer,1 U of rTaq) as follows: an initial denaturation at 95°C for 5 min, followed by 33 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for 2 min, and completed with an extra extension at 72°C for 10 min.

2.2.4 Gel electrophoresis

The PCR product of each sample was detected in 1.2%agarose gel electrophoresis with DL2000 DNA Marker(Takara Biotechnology Co., Ltd., Dalian, China) as the marker.

2.2.5 Sequencing of the PCR product

PCR products were purified using E.Z.N.A.?Gel Extraction Kit (Omega Bio-tek Inc., USA). The products for those listed in Table 1 were bi-directionally sequenced using ABI3730XL. The PCR products from those samples collected in small amounts were digested with Sau3AI and detected by electrophoresis. Based on the electrophoresis results,products from 27 mycobionts and 24 photobionts were selected arbitrarily and sequenced.

2.2.6 Alignment and phylogenetic analysis

Sequences were assembled with Lasergene SeqMan Pro(DNASTAR, Inc., USA) and corrected manually, and then aligned using ClustalW in Mega 5.10[23-24]. The phylogenetic analysis was executed with Mega 5.10 software, and the Kimura-2 parameter was selected as the nucleotide substitution model. The maximum likelihood (ML)method was used to construct the phylogenetic tree and the reliability of the inferred tree was tested by 1000 bootstrap replications[25].

3 Results

3.1 Morphology and attachment

Usnea aurantiaco-atraindividuals growing on rock(Figure 2a), with moss (Figures 2b, 2c), on wood (Figure 2d)or onUmbilicaria antarctica(Figure 2e–2f) were inspected,and specimens were stored in the Resource-sharing Platform of Polar Samples (BIRDS) (Table 1). In general, apothecia were observed on the thalli of those attached to rocks(Figure 2a). Rare apothecia could be observed from those associated with mosses, both those growing on spalls beneath mosses (Figure 2b), and the others with moss but without any attachment (Figure 2c). Specially, there were no apothecia on thoseUsnea aurantiaco-atraindividuals growing on wood (Figure 2d) or on the lichenUmbilicaria antarctica(Figure 2e–2f).

Figure 2Usnea aurantiaco-atra on different substrates. a, on rock; b, with moss, growing on spall; c, with moss, no attachment; d, on wood; e–f, on Umbilicaria antarctica thallus.

3.2 ITS Sequences analysis

The ITS rDNA region of sevenUsnea aurantiaco-atraand sevenUmbilicaria antarcticawas sequenced and submitted to GenBank (Table 1). PCR-RFLP (restriction fragment length polymorphism) was used to analyze the genotypes for 384Usnea aurantiaco-atraindividuals (280 on rock,104 with moss) distributed around the Fildes Peninsula.The electrophoresis result showed that the genotypes for mycobionts or photobionts fromUsnea aurantiacoatrawere identical (result not shown here). Therefore, 27 mycobiont PCR products (F01-F27, GenBank Accession Nos. KR053321–KR053347) and 24 photobiont PCR products (A01–A24, GenBank Accession Nos. KR053362–KR053385) were selected randomly to be sequenced.

Based on the phylogenetic analysis of the mycobiont ITS rDNA, there were minimum differences withinUsnea aurantiaco-atraorUmbilicaria antarctica. The ML tree based on mycobiont ITS rDNA sequences showed these two lichen species were supported well with high bootstrap values (96%forUsnea aurantiaco-atraand 99% forUmbilicaria antarctica)(Figure 3a).Usnea aurantiaco-atraindividuals were unable to form monophyletic groups based on their substrates. For the photobiont, all the samples were clustered withTrebouxia jamesii(Hildreth & Ahmadjian) G?rtner (Figure 3b) by a bootstrap value of 99%, which meant that all photobionts in our study wereT. jamesii. The results also demonstrated that some photobionts fromUsnea aurantiaco-atraandUmbilicaria antarcticacould share the same genotype. For example, ITS genotypes of AG282, AG236 and AG247 (fromUsnea aurantiaco-atra) and those of AG041 and AG035 (fromUmbilicaria antarctica) were identical (Figure 3b).

Figure 3 ML trees based on ITS rDNA sequences of mycobiont (a) and photobiont (b). The numbers in each node represent bootstrap support values. Only bootstrap values higher than 50% are indicated. : on rock; ■: on moss;▲: on wood; : on thallus of Umbilicaria antarctica. Italic font indicates the sequences obtained by the authors.

4 Discussion

As the dominant organism in extreme terrestrial environments,lichen is able to adapt to various harsh conditions[26–28]. The symbiotic relationship between mycobiont and photobiont plays an important role in lichen’s adaptability. Hence the key process allowing lichen to spread to novel habitats,especially for those dispersing by ascospores, is the obtaining by the mycobiont of its compatible photobiont.

Nearly 20%–40% of bare ground (not covered by permanent snow) in the Fildes Peninsula, Antarctica, is covered by lithophilousUsnea aurantiaco-atra[26], classified in theUsneasubgenusNeuropogon. During the investigation at the Great Wall Station, we found that some individuals ofU. aurantiaco-atracould grow on wood, and even on other lichen thalli. The species provided the ideal materials to reveal the sources of the photobiont in lichenization. Generally,the substrate ofU. aurantiaco-atrawas rock[27], but our researches suggested that this lichen species was not strictly substrate-dependent (Figures 2d–2f). Two growth forms were reported forU. aurantiaco-atra[29]: Individuals of form I grew on rock and had erect branches and apothecia (Figure 2a);those of form II grew with mosses and had prostrate branches,but noapothecia (Figure 2b–2c). The individuals growing with mosses were attached to spalls beneath the mosses in most cases, and those without connection to spalls(Figure 2c) were thought to have been split away from rocks.At the Great Wall Station, it was recorded that the annual mean wind speed was 7.3 m·s-1, there were 137 d with galeforce winds and the fastest wind speed reached 35 m·s-1[30].Individuals belonging to forms I or II would have had to adapt to the windy environment of Fildes Peninsula through attaching to either rocks or to mosses, to prevent them from being blown away to drift into the ocean.

TwoUsneaspecies had been reported from this region,U. aurantiaco-atrahaving apothecia but no soredia, andU.antarcticaDu Rietz having soredia but no apothecia[31-32].Recently, phylogenetic study suggested thatU. aurantiacoatraandU. antarcticacannot be distinguished at molecular level as they share the same ITS genotype, and thatU.antarcticashould be treated a synonym ofU. aurantiacoatra[33]. Therefore,U. aurantiaco-atraandU. antarcticawere not treated as separate species in present study.

PCR-RFLP was performed in our study in addition to morphological identification. The results showed that the difference for the ITS rDNA region among mycobionts or photobionts ofU. aurantiaco-atrawas very small, so only a minority of PCR products (27 for mycobionts and 24 for photobionts) were sequenced, representing 384 samples collected in a small amount. The sequencing results showed that there were 0–4 bps discrepancy among mycobionts and 0–10 polymorphism sites in photobiont sequences. The unique sequence suggested that randomly sequenced PCR products could have reぼected the local genetic background ofU. aurantiaco-atrain this region.

TheU. aurantiaco-atraindividuals growing on wood and on lichen thalli (Figure 2d–2f) were observed during the 27th and 28th CHINAREs at the Great Wall Station.No distinction was detected at molecular level among some individuals growing on lichenUmbilicaria antarctica(AG282), on wood (AG235, AG236) and on rock (AG251,AG297, F01-F27) according to the mycobiont ITS rDNA analysis (Figure 3a). The photobionts ofUsnea aurantiacoatraandUmbilicaria antarcticaall belonged to the same algal speciesT. jamesii(Figure 3b). BecauseUsnea aurantiaco-atrawas the dominant lichen in the Fildes Peninsula, the dominant photobiont species wasT. jamesiihere. The same photobiont genotype was observed forU. aurantiaco-atragrowing on different substrates; furthermore,U. aurantiaco-atraandUmbilicaria antarcticacould share the same photobiont genotype, which indicated that there was an algae pool from which lichenized fungi could obtain their photobionts.

The algae pool suggests one lichen could capture its photobiont from the thallus of another lichen. SomeUsnea aurantiaco-atrahas the same photobiont as those inUmbilicaria antarctica, which confirmed that these two lichen species shared the same algae pool. Molecular data showed that the ITS rDNA sequences of the mycobionts fromUmbilicaria antarctica(AG017, AG019, AG023, AG024,AG038 and AG041) were identical, and their photobionts were all from the same species,T. jamesii, although a few variance sites existed in the ITS rDNA region. The result was fully consistent with that of the latest pyrosequencing of Antarctic lichens[34]. It could be inferred that the photobiont ofUmbilicaria antarctica, on whose thallusUsnea aurantiacoatra(AG282) was growing, should also be the same as that of AG282. However, theUmbilicaria antarcticaindividual had died, and so information from its photobiont could not be obtained directly. Ascospores fromUsnea aurantiacoatrawere able to germinate on the thallus ofUmbilicaria antarctica, capture its photobiont and finally form a lichen thallus. Individuals ofUsnea aurantiaco-atragrowing on wood might undergo a similar progress. Free-living photobionts are very rare in nature, but the photobionts could be released from vegetation fragments and survive for a short time[35]. Our research implied that free-living photobiont was present in the Fildes Peninsula, especially on wood surfaces,because no other lichens were observed on the wood.

Meanwhile,Usnea aurantiaco-atragrowing with mosses, was the dominant organism in the Ardley Island.More attention should be paid to the mosses growing withU. aurantiaco-atrato elucidate the process of succession between these two organisms, and to examine whether there is a specific relationship between lichen and moss.

Our findings, especially the discovery ofU. aurantiacoatragrowing onUmbilicaria antarctica, provided evidence for photobionts transferring directly between lichens. TheUsnea aurantiaco-atrafound on wood also confirmed there was free-livingT. jamesiiin the Fildes Peninsula, because there were no other lichens on the wood. There is a possibility that the thalli ofU. aurantiaco-atraon wood or other lichens were from vegetable structures such as soredia, and this could not be excluded completely. However, the unique fungal ITS genotypes for AG238 (on wood) and AG282 (onUmbilicaria antarctica) strongly implied that they had passed through a process of sexual reproduction so that their fungal ITS sequences were not in accordance to those growing on rocks.This meant that ascospores had captured the algal partner on substrates other than rocks.

There are multiple algal species in the Fildes Peninsula,but only one species was found accompanying the dominant lichen speciesUsnea aurantiaco-atra. This indicates that this algal species has adapted to the micro-environment,becoming the preponderant lichenized algae. The various substrates ofU. aurantiaco-atraindicated that the sources of its photobiont were not unique; the alga in a new thallus could be obtained from the parental thallus, from other lichens or from the environment. Some works have demonstrated that the decrease in selectivity of the mycobiont to its photobiont may be helpful for lichen surviving in extreme environments because mycobionts could form a lichen thallus with a broad range of photobionts to survive[10,13,36]. However, the decreased selectivity ofU. aurantiaco-atrato the substrates is also a strategy to survive in harsh environments. For example, some lichens belonging to the genusUmbilicaria, found growing on rocks, can also inhabit wood[37].

In summary, morphology and molecular analysis demonstrated the available photobiont sources, and confirmed that there was an algae pool in this area. This provides real insight into the growth ofUsnea aurantiaco-atraon various substrates, which in turn will help us to understand the distribution of photobionts, photobiont transfer mechanisms and the process of lichenization.

AcknowledgementOur research was facilitated by the Resource-sharing Platform of Polar Samples (http://birds.chinare.org.cn/) where information on lichens and data are stored. We are grateful to the Chinese Arctic and Antarctic Administration for its help in carrying out the project in the Great Wall Station during the 27th and 28th CHINAREs. This research was supported by State Oceanic Administration, P. R. China (Grant nos. 10/11 GW06, 2011GW12016), and the National Natural Science Foundation of China (Grant nos. 31000010, 31270118, 41206189).

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