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Keywords:

  • liverwort and moss;
  • fungal endophytes;
  • polar ecosystem;
  • extremophiles;
  • culture- dependent method

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Reference

Endophytic fungi associated with three bryophyte species in the Fildes Region, King George Island, maritime Antarctica, that is, the liverwort Barbilophozia hatcheri, the mosses Chorisodontium aciphyllum and Sanionia uncinata, were studied by culture-dependent method. A total of 128 endophytic fungi were isolated from 1329 tissue segments of 14 samples. The colonization rate of endophytic fungi in three bryophytes species were 12.3%, 12.1%, and 8.7%, respectively. These isolates were identified to 21 taxa, with 15 Ascomycota, 5 Basidiomycota, and 1 unidentified fungus, based on morphological characteristics and sequence analyses of ITS region and D1/D2 domain. The dominant fungal endophyte was Hyaloscyphaceae sp. in B. hatcheri, Rhizoscyphus sp. in C. aciphyllum, and one unidentified fungus in S. uncinata; and their relative frequencies were 33.3%, 32.1%, and 80.0%, respectively. Furthermore, different Shannon–Weiner diversity indices (0.91–1.99) for endophytic fungi and low endophytic fungal composition similarities (0.19–0.40) were found in three bryophyte species. Growth temperature tests indicated that 21 taxa belong to psychrophiles (9), psychrotrophs (11), and mesophile (1). The results herein demonstrate that the Antarctic bryophytes are an interesting source of fungal endophytes and the endophytic fungal composition is different among the bryophyte species, and suggest that these fungal endophytes are adapted to cold stress in Antarctica.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Reference

Fungal endophytes are a diverse group of fungal species that inhabit living plants at some time during their life cycle without causing apparent symptoms of infection (Wilson, 1995). Most of them belong to the phylum Ascomycota, and others belong to Basidiomycota (Flor et al., 2011; Koukol et al., 2012; U'Ren et al., 2012) and Zygomycota (Gazis & Chaverri, 2010). The host and habitat range of these fungi is also diverse; they have been isolated from many different land plants and from all terrestrial ecosystems ranging from the tropics to polar regions (Arnold & Lutzoni, 2007; Rosa et al., 2009, 2010). These fungi have profound impacts on plant hosts in many ways: Some impact plant fitness (Rodriguez et al., 2009), some affect plant disease resistance and susceptibility (Rodriguez et al., 2009), and some decompose plant litter (Sun et al., 2011). Despite their diversity and importance, the vast majority of fungal endophytes and their ecological significance have yet to be adequately characterized.

Most studies of fungal endophytes have focused on those species that live in vascular plants, but endophytes also live in nonvascular plants including bryophytes (i.e., mosses, liverworts, and hornworts), which are a functionally important in boreal forest and tundra ecosystems where they produce much of the biomass. A great phylogenetic diversity of endophytes were found in the tissues of mosses and liverworts in boreal, temperate and tropical forests (Davis et al., 2003; Davis & Shaw, 2008; Kauserud et al., 2008; U'Ren et al., 2010). Further studies are needed to characterize the diversity, distribution, and ecological roles of endophytic fungi associated with bryophytes in different regions, including Antarctica.

The plant flora of Antarctica, which is geographically isolated from other landmasses and is the coldest continent, consists largely of bryophytes (c. 111 species of mosses, and 27 species of liverworts) (Bednarek-Ochyra et al., 2000; Ochyra et al., 2008) and includes only two native vascular plant species. As the dominant plant component in Antarctic terrestrial ecosystems, bryophytes are major primary producers and have important influences on the thermal and hydrologic cycles, and therefore likely play a significant role in regulating ecological processes. All these characteristics make Antarctica useful for studying the taxonomy, ecology, adaptation, and evolution of endophytic fungal communities associated with bryophytes. Only a few studies have reported on endophytic fungi in Antarctica, and these have been limited to a small number of bryophyte species in some scattered sites in the continental Antarctic zone (Azmi & Seppelt, 1998; McRae & Seppelt, 1999; Bradner et al., 2000; Tosi et al., 2002).

The abundance and diversity of organisms decrease from the maritime to the continental Antarctic zone (Ruisi et al., 2007). To our knowledge, no reports on the endophytic fungal diversity associated with bryophytes in the maritime Antarctic zone are available. The aim of this study was to investigate the diversity, distribution, and cold adaptation of the endophytic fungi associated with three dominant bryophytes species (the liverwort Barbilophozia hatcheri, the mosses Chorisodontium aciphyllum, and Sanionia uncinata) in the Fildes Region (62°12′-62°13′S and 58°56′-58°57′W), which represents one of the largest ice-free areas in the maritime Antarctic zone.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Reference

Study sites and sample collection

The study area is located in the Fildes Region (62°08′-62°14′S and 59°02′-58°51′W), consisting of the Fildes Peninsula, Ardley Island and adjacent islands, located in the southwestern part of King George Island, South Shetland Islands. The Fildes Region represents one of the largest ice-free areas in the maritime Antarctica and contains six permanent Antarctic stations, one of which is the Great Wall Station (China). Sampling was carried out near the Great Wall Station during China's 28th Antarctic expedition in December 2011. Fourteen liverwort and moss samples were collected from five sites in this area (Table 1).

Table 1. Data for the 14 bryophyte samples in the Fildes Region of Antarctica
Sample codeSpeciesSites (coordinates)Sampling date
374Chorisodontium aciphyllum (Hook. f. & Wils.) Broth.Ardley Island (62°13′39.29″ S, 58°56′53.51″ W)2011.12.21
375Sanionia uncinata (Hedw.) LoeskeArdley Island (62°13′39.29″ S, 58°56′53.51″ W)2011.12.21
392Sanionia uncinata (Hedw.) LoeskeArdley Island (62°13′39.29″ S, 58°56′53.51″ W)2011.12.21
417Chorisodontium aciphyllum (Hook. f. & Wils.) Broth.Ardley Island (62°13′39.29″ S,58°56′53.51″ W)2011.12.21
426Barbilophozia hatcheri (Evans) LoeskeArdley Island (62°12′44.77″ S,58°56′27.47″ W, 34 m)2011.12.21
451Chorisodontium aciphyllum (Hook. f. & Wils.) Broth.Ardley Island (62°12′44.77″S, 58°56′27.47″ W, 34 m)2011.12.21
457Barbilophozia hatcheri (Evans) LoeskeArdley Island (62°12′44.77″ S,58°56′27.47″ W, 34 m)2011.12.21
499Sanionia uncinata (Hedw.) LoeskeFiles Peninsula (62°12′52.83″S,58°57′42.00″W, 76 m)2011.12.25
500Sanionia uncinata (Hedw.) LoeskeFiles Peninsula (62°12′52.83″S,58°57′42.00″W, 76 m)2011.12.25
502Sanionia uncinata (Hedw.) LoeskeFiles Peninsula (62°12′52.83″S,58°57′42.00″W, 76 m)2011.12.25
1-1Sanionia uncinata (Hedw.) LoeskeFiles Peninsula (62°13′1.61″ S,58°57′43.51″ W, 70 m)2011.12.25
1-2Sanionia uncinata (Hedw.) LoeskeFiles Peninsula (62°12′57.05″S, 58°57′45.46″W, 13 m)2011.12.25
1-3Sanionia uncinata (Hedw.) LoeskeFiles Peninsula (62°12′57.05″S, 58°57′45.46″W, 13 m)2011.12.25
1-4Sanionia uncinata (Hedw.) LoeskeFiles Peninsula (62°12′57.05″S, 58°57′45.46″W, 13 m)2011.12.25

Isolation of endophytic fungi

The entire plants were surface-sterilized by immersion in 75% ethanol for 1 min, in 2% sodium hypochlorite for 3 min, and in 75% ethanol for 0.5 min. After the samples were surface dried with sterile paper towels, they were cut into segments of 0.1–0.3 cm. Tissue segments were then evenly placed in each 9-cm-diameter Petri dishes containing potato dextrose agar (PDA, 1.5%), tetracycline(50 mg L−1), and streptomycin sulfate (50 mg L−1). Each sample was represented by 60–100 segments, and 20–30 segments were placed on each Petri dish. Petri dishes were sealed, incubated for 2 months at 4 °C, and examined periodically. When colonies developed, they were transferred to PDA slants. Subcultures were then incubated on PDA. All of the isolates were deposited in the China Pharmaceutical Culture Collection (CPCC), Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College.

DNA extraction, amplification, and sequencing

All isolates were initially sorted into morphospecies on the basis of phenotypic characteristics including colony aspect (texture and color), cell morphology (shape), and growth rate. After this preliminary screening, representative isolates of each morphospecies were selected for molecular identification. Because the nuclear internal transcribed spacers (ITS) region and D1/D2 domain of the large-subunit rRNA gene (LSU) are the default markers for study of fungi at the species level (Seifert, 2009), we used ITS and D1/D2 gene sequences for fungal identification.

Genomic DNA was extracted with a modified CTAB method (Cubero et al., 1999). The ITS (ITS1-5.8 S-ITS2) region of the rRNA gene was amplified with the primers ITS1 and ITS4 as described by White et al. (1990). Amplification of the ITS region was performed as follows: 95 °C for 3 min, followed by 37 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 10 min. The D1/D2 domain was amplified with the primers NL1 and NL4 as described by Kurtzman & Robnett (1991). Amplification of the D1/D2 domain was performed as follows: 94 °C for 6 min, followed by 40 cycles of 94 °C for 60 s, 50 °C for 60 s, and 72 °C for 60 s; and a final extension at 72 °C for 5 min. PCR products were purified and sequenced with the same primers by Sangon Biotech Co., Ltd. (Beijing, China). The sequence data obtained in this study were deposited in GenBank under the accession numbers JX852321 to JX852420.

Molecular identification and phylogeny

Fungi were identified based on sequence similarity (i.e., assessment of the percentage of nucleotide identity with reference sequences) and the phylogenetic position. For sequence similarity determination, Megablast searches were performed in the GenBank public sequence databases to find the closest related species. Sequence data were also used to investigate phylogenetic relationships using Molecular Evolutionary Genetics Analysis (MEGA) software, version 5.0. The phylogenetic trees were constructed using the neighbor-joining (NJ) algorithm with bootstrap values calculated from 1000 replicate runs. The maximum composite likelihood model was used to estimate evolutionary distance.

Effect of temperature on growth

Representative isolates of each taxon were inoculated onto the PDA plates and kept at 4, 12, 20, 27, and 35 °C in the dark. Each combination of isolate and temperature was represented by three replicate plates. Plates were inspected for 1 month, and colony diameters were measured every 5 days. The mean diameter was obtained from the three replicates. The colony diameter growth rate was calculated as the mean diameter of the isolate divided by the incubation time in days.

Data analyses

Colonization rate (CR%) was calculated as the total number of tissue segments infected by fungi divided by the total number tissue segments incubated (Petrini et al., 1982). Relative frequency (RF%) was calculated as the number of isolates of certain species divided by the total number of isolates. The endophytic fungal diversity was evaluated using the Shannon–Weiner diversity index which has two main components: evenness and the number of species (Shannon & Weiner, 1963). To evaluate the degree of community similarity of endophytic fungi between bryophyte species, Sorenson's similarity coefficient (Cs) was used and was calculated according to the formula Cs = 2j/(a + b), where j is the number of endophytic fungal species coexisting in two bryophyte species, a is the total number of endophytic fungal species in one species, and b is the total number of endophytic fungal species in the other species (Sorensen, 1948).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Reference

Colonization rate

In this study, we used a culture-dependent method to examine the endophytic fungi associated with three bryophytes species (B. hatcheri, C. aciphyllum, and S. uncinata) in the Fildes Region. From the 14 samples collected, a total of 128 isolates of fungal endophytes were obtained from 1329 tissue segments. The CR% of endophytic fungi was 12.3% for B. hatcheri, 12.1% for C. aciphyllum, and 8.7% for S. uncinata (Table 2).

Table 2. RF%, CR%, and Shannon–Weiner index (H') of endophytic fungi from three bryophyte species
TaxonNo. of isolates with the indicated taxa (RF%)
Barbilophozia hatcheri Chorisodontium aciphyllum Sanionia uncinata
Ascomycota
Annulohypoxylon sp.1 (6.7%)  
Chaetomium sp.  1 (1.2%)
Hyphodiscus sp.1 (6.7%)8 (28.6%)7 (8.2%)
Rhizoscyphus sp.1 (6.7%)9 (32.1%)1 (1.2%)
Scopulariopsis sp.  1 (1.2%)
Thelebolus sp.  1 (1.2%)
Xenopolyscytalum sp.  1 (1.2%)
Dermateaceae sp.2 (13.3%)  
Hyaloscyphaceae sp.5 (33.3%)2 (7.1%) 
Xylariaceae sp.1 (6.7%)  
Helotiales sp. 12 (13.3%)  
Helotiales sp. 21 (6.7%)  
Helotiales sp. 31 (6.7%)  
Helotiales sp. 4  1 (1.2%)
Helotiales sp. 5 1 (3.6%) 
Basidiomycota
Eocronartium sp.  1 (1.2%)
Mrakia sp.  1 (1.2%)
Rhodotorula sp. 1  1 (1.2%)
Rhodotorula sp. 2  1 (1.2%)
Sporidiobolales sp. 1 (3.6%) 
Unknown fungi
Unidentified fungus 7 (25.0%)68 (80.0%)
Total
Number of tissue segments122232975
Number of fungal isolates152885
Number of taxa9612
Colonization rate (%)12.312.18.7
Shannon index of diversity (H')1.991.500.91

Phylogenetic analyses

Fifty representative fungal isolates including all morphospecies were selected for sequence similarity analysis (Table 3) and phylogenetic reconstruction (Fig. 1), which were using the ITS rDNA sequences. The neighbor-joining tree indicated that most of culturable endophytic fungi belong to the phylum Ascomycota (41 isolates) and others belong to the phylum Basidiomycota (5 isolates) and ‘unknown fungi’ (4 isolates). The Ascomycota included four orders: Helotiales (37 isolates), Microascales (1 isolate), Sordiariales (1 isolate), and Xylariales (2 isolates). The Basidiomycota included the three orders: Cystofilobasidiales (1 isolate), Platygloeales (1 isolate), and Sporidiobolales (3 isolates). They were all supported by high bootstrap values (> 50%) in the phylogenetic tree.

Table 3. Molecular identification of endophytic fungi isolated from three bryophyte species
Host speciesRepresentative isolate codeGene typeGenBank accession numberClosest related species/GenBank accession numberMaximum identity (%)Identification
B. hatcheri I12F-02255ITS JX852325 Uncultured Hyaloscyphaceae (FJ475648)476/513 (92.8%)Hyaloscyphaceae sp.
D1/D2 JX852375 Uncultured soil fungus (EU692229)544/569 (95.6%)
I12F-02256ITS JX852326 Uncultured Helotiales (FM997939)494/502 (98.4%)Helotiales sp. 1
D1/D2 JX852376 Uncultured fungus (GU174403)575/587 (98.0%)
I12F-02258ITS JX852328 Rhizoscyphus ericae (AY394907)496/502 (98.8%)Rhizoscyphus sp.
D1/D2 JX852378 Uncultured soil fungus (EU692281)568/572 (99.3%)
I12F-02260ITS JX852330 Annulohypoxylon sp. (JQ327866)517/523 (98.9%)Annulohypoxylon sp.
D1/D2 JX852380 Xylariaceae sp. (AB746916)548/562 (97.5%)
I12F-02261ITS JX852331 Xylariaceae sp. (AY315402)510/528 (96.6%)Xylariaceae sp.
D1/D2 JX852381 Xylariaceae sp. (JF773597)547/559 (97.9%)
I12F-02269ITS JX852339 Hyphodiscus sp. (AB546944)506/521 (97.1%)Hyphodiscus sp.
D1/D2 JX852389 Hyphodiscus hymeniophilus (GU727550)566/574 (98.6%)
I12F-02296ITS JX852366 Uncultured soil fungus (GU083257)520/529 (98.3%)Helotiales sp. 2
D1/D2 JX852416 Uncultured Helotiales (JF449495)556/564 (98.6%)
I12F-02297ITS JX852367 Mollisia minutella (FR837920)472/518 (91.1%)Dermateaceae sp.
D1/D2 JX852417 Uncultured fungus (EU292358)543/558 (97.3%)
I12F-02299ITS JX852369 Uncultured fungus (HQ260283)512/525 (97.5%)Helotiales sp. 3
D1/D2 JX852419 Uncultured Helotiales (EU046067)562/567 (99.1%)
C. aciphyllum I12F-02251ITS JX852321 Hyphodiscus sp. (AB546944)499/512 (97.5%)Hyphodiscus sp.
D1/D2 JX852371 Hyphodiscus hymeniophilus (GU727550)573/578 (99.1%)
I12F-02263ITS JX852333 Rhizoscyphus ericae (AM887700)511/518 (98.6%)Rhizoscyphus sp.
D1/D2 JX852383 Uncultured soil fungus (EU691216)573/575 (99.7%)
I12F-02268ITS JX852336 Uncultured Hyaloscyphaceae (FJ475648)476/513 (92.8%)Hyaloscyphaceae sp.
D1/D2 JX852386 Uncultured soil fungus (EU692229)553/583 (94.8%)
I12F-02280ITS JX852350 Uncultured fungus (JN889728)321/366 (87.7%)Sporidiobolales sp.
D1/D2 JX852400 Basidiomycota sp. (AB558448)538/612 (87.9%)
I12F-02283ITS JX852353 Uncultured fungus (FJ237219)806/857 (94.0%)Unidentified fungus
D1/D2 JX852403 Uncultured fungus (FJ456973)339/415 (81.7%)
I12F-02289ITS JX852359 Fungal endophyte (HQ335303)509/511 (99.6%)Helotiales sp. 5
D1/D2 JX852409 Uncultured fungus (EU292389)560/571 (98.1%)
S. uncinata I12F-02253ITS JX852323 Uncultured fungus (FJ237219)804/857 (93.8%)Unidentified fungus
D1/D2 JX852373 Uncultured fungus (FJ456973)339/415 (81.7%)
I12F-02254ITS JX852324 Hyphodiscus sp. (AB546944)504/517 (97.5%)Hyphodiscus sp.
D1/D2 JX852374 Hyphodiscus hymeniophilus (GU727550)577/584 (98.8%)
I12F-02259ITS JX852329 Mrakia psychrophilia (EU224267)597/598 (99.8%)Mrakia sp.
D1/D2 JX852379 Mrakia sp. (EU680778)612/617 (99.2%)
I12F-02262ITS JX852332 Uncultured fungus (AB520435)506/545 (92.8%)Eocronartium sp.
D1/D2 JX852382 Eocronartium muscicola (AY512844)581/594 (97.8%)
I12F-02273ITS JX852343 Fungal endophyte (HQ335299)497/499 (99.6%)Xenopolyscytalum sp.
D1/D2 JX852393 Xenopolyscytalum sp. (HE603984)558/565 (98.8%)
I12F-02277ITS JX852347 Rhodotorula sp. (JF805370)407/492 (82.7%)Rhodotorula sp. 1
D1/D2 JX852397 Basidiomycota sp. (JQ768846)577/594 (97.1%)
I12F-02278ITS JX852348 Rhodotorula sp. (DQ870625)543/609 (89.2%)Rhodotorula sp. 2
D1/D2 JX852398 Rhodotorula sp. (EU075184)560/603 (92.9%)
I12F-02284ITS JX852354 Uncultured Helotiales (HQ212246)523/528 (99.1%)Helotiales sp. 4
D1/D2 JX852404 Uncultured fungus (EU292389)565/577 (97.9%)
I12F-02285ITS JX852355 Rhizoscyphus ericae (AM887700)512/519 (98.7%)Rhizoscyphus sp.
D1/D2 JX852405 Uncultured soil fungus (EU691216)565/578 (97.8%)
I12F-02287ITS JX852357 Thelebolus microsporus (GQ483644)518/519 (99.8%)Thelebolus sp.
D1/D2 JX852407 Thelebolus ellipsoideus (FJ176895)567/570 (99.5%)
I12F-02288ITS JX852358 Chaetomium murorum (HM365268)504/532 (94.7%)Chaetomium sp.
D1/D2 JX852408 Chaetomium globosum (AB449688)553/564 (98.0%)
I12F-02300ITS JX852370 Scopulariopsis hibernica (FJ946484)583/592 (98.5%)Scopulariopsis sp.
D1/D2 JX852420 Scopulariopsis sp. (HQ676488)527/538 (98.0%)
image

Figure 1. Phylogenetic tree of the endophytic fungi isolated from three bryophytes and other fungal species based on the ITS (ITS1-5.8S-ITS2) region sequences of rDNA. The tree was constructed with the neighbor-joining method. Bootstrap support values are indicated for major nodes having values ≥ 50%.

Download figure to PowerPoint

The endophytic fungi were identified to lower taxonomic levels by means of the phylogenetic analysis and similarity comparison. The 50 isolates were identified to 21 taxa: 11 to genus (i.e., Annulohypoxylon sp., Chaetomium sp., Eocronartium sp., Hyphodiscus sp., Mrakia sp., Rhizoscyphus sp., Rhodotorula sp.1, Rhodotorula sp.2, Scopulariopsis sp., Thelebolus sp., and Xenopolyscytalum sp.), 3 to family (i.e., Dermateaceae sp., Hyloscyphaceae sp., and Xylariaceae sp.), 6 to order (i.e., Helotiales sp. 1, Helotiales sp. 2, Helotiales sp. 3, Helotiales sp. 4, Helotiales sp. 5, and Sporidiobolales sp.), and 1 to ‘unknown fungi’ (unidentified fungus) (Table 3). For example, isolate I12F-02258 showed the highest similarity (99.8%) with Rhizoscyphus ericae (AY394907) and clustered with R. ericae in one group with high bootstrap values (99%) in the phylogenetic tree (Fig. 1); isolate I12F-02258 was therefore identified as Rhizoscyphus sp.

Fungal diversity

The fungal taxa isolated from B. hatcheri, C. aciphyllum, and S. uncinata were 9, 6, and 12, respectively (Table 2). Only two taxa coexisted in all three bryophyte species, and these were Hyphodiscus sp. and Rhizoscyphus sp. Six taxa were found only in the liverwort B. hatcheri. Two taxa were found only in the moss C. aciphyllum, and nine taxa were found only in the moss S. uncinata (Table 2).

The dominant fungal endophyte was Hyaloscyphaceae sp. in B. hatcheri,Rhizoscyphus sp. in C. aciphyllum, and one unidentified fungus in S. uncinata, and their relative frequencies were 33.3%, 32.1%, and 80.0%, respectively (Table 2). Hyphodiscus sp. was also a frequent taxon with relative frequencies of 6.7% in B. hatcheri, 28.6% in C. aciphyllum, and 8.2% in S. uninata. In contrast, several fungi were infrequent. For example, Chaetomium sp., Eocronartium sp., Scopulariopsis sp., Thelebolus sp., and Xenopolyscytalum sp. were isolated only one time (Table 2).

The Shannon–Weiner diversity index was used to evaluate and compare the diversity of the endophytic fungus community between bryophyte species. The indices were highest for B. hatcheri, lowest for S. uncinata, and intermediate for C. aciphyllum (Table 2).

The Sorenson's similarity coefficients for the endophytic fungi from the three bryophyte species were 0.19, 0.33, and 0.40, respectively. Similarity was highest between B. hatcheri and C. aciphyllum, intermediate between C. aciphyllum and S. uncinata, and lowest between B. hatcheri and S. uncinata. The results suggest that the distribution of the isolated fungal taxa on three bryophytes was obviously different.

Effect of temperature on growth rate in vitro

Based on their responses to temperature, the endophytic fungi could be divided into three groups according to Gounot (1986) and Robinson (2001). Nine fungal taxa were psychrophiles with an optimum temperature for growth of ≤ 15 °C and a maximum temperature for growth of ≤ 20 °C: Eocronartium sp., Mrakia sp., Rhodotorula sp.1, Rhodotorula sp.2, Xylariaceae sp., Helotiales sp. 1, Helotiales sp. 2, Sporidiobolales sp., and one unidentified fungus; eleven fungal taxa were psychrotrophs with an optimum temperature for growth of 15–20 °C and a maximum temperature for growth of > 20 °C: Annulohypoxylon sp., Chaetomium sp., Dermateaceae sp., Hyphodiscus sp., Rhizoscyphus sp., Thelebolus sp., Xenopolyscytalum sp., Hyaloscyphaceae sp., Helotiales sp. 3, Helotiales sp. 4, and Helotiales sp. 5; Scopulariopsis sp. was mesophile with an optimum temperature above 20 °C (Table 4).

Table 4. In vitro growth rate (mm day−1) of endophytic fungi isolated from three bryophytes as affected by temperatures
TaxonIsolates testedTemperature (°C)
412202735
  1. Values are means of three replicate plates containing PDA.

Annulohypoxylon sp.I12F-022600.971.271.34--
Chaetomium sp.I12F-022882.372.553.640.53-
Eocronartium sp.I12F-022621.761.531.22--
Hyphodiscus sp.I12F-022510.741.131.10--
I12F-022651.061.191.22--
I12F-022720.931.021.07--
I12F-022750.890.921.02--
Mrakia sp.I12F-022591.390.84---
Rhizoscyphus sp.I12F-022631.001.041.430.15-
I12F-022810.760.941.040.30-
I12F-022581.111.381.470.41-
Rhodotorula sp. 1I12F-022770.821.020.97--
Rhodotorula sp. 2I12F-022780.380.770.71--
Scopulariopsis sp.I12F-023000.500.661.171.36-
Thelebolus sp.I12F-022872.652.803.97--
Xenopolyscytalum sp.I12F-022730.930.901.27--
Dermateaceae sp.I12F-022970.280.300.87--
Hyaloscyphaceae sp.I12F-022570.761.051.370.56-
Xylariaceae sp.I12F-022611.431.441.34--
Helotiales sp. 1I12F-022560.750.940.85--
Helotiales sp. 2I12F-022960.450.520.32--
Helotiales sp. 3I12F-022990.190.360.48--
Helotiales sp. 4I12F-022840.961.522.39--
Helotiales sp. 5I12F-022891.111.202.13--
Sporidiobolales sp.I12F-022800.830.530.14--
Unidentified fungusI12F-022530.330.410.35--

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Reference

Colonization rate

The CR% of the three bryophyte species by endophytic fungi were low (8.7–12.3%) in the present study. Davis & Shaw (2008) have revealed the CR% of endophytic fungi associated with bryophytes in tropical and temperate ecosystems: The CR% was 85.2% for 26 species in New Zealand, 96.6% for 18 species in North Carolina, 68.8% for 14 species in Germany, 60.9% for 22 species in Pacific Northwest. The above results show that the CR% of bryophytes by endophytic fungi is lower in the Antarctic ecosystem than in tropical and temperate ecosystems. These low CR% in this study suggest that a combination of abiotic conditions in Antarctica (i.e., dry, cold, oligotrophic, and UV radiation) may limit the survival of the endophytic fungi in bryophytes. A similar trend was also found in vascular plant species. The CR% of the endophytic fungi in some vascular plant species in temperate environments ranged from 36.7% to 100% (Huang et al., 2008; Gazis & Chaverri, 2010; Sun et al., 2012), while the CR% of endophytic fungi was 34.8% for Colobanthus quitensis (Rosa et al., 2010) and 9.4% for Deschampsia antarctica (Rosa et al., 2009) in Antarctica.

Endophytic fungal composition

In the present study, 128 fungal isolates were identified, and these belonged to 20 fungal taxa in the Ascomycota, Basidiomycota, and 1 unknown fungus. The results indicate that the maritime Antarctic bryophytes are an interesting niche that harbors diverse fungal endophytes. Similar results were reported in previous studies of fungal endophytes in Antarctica. Azmi & Seppelt (1998) isolated fungi from the mosses Bryum pseudotriquetrum, Ceratodon purpureus, and Grimmia antarctica in the Windmill Islands region (66°17′S and 110° 32′E) and identified 21 fungal taxa. McRae & Seppelt (1999) studied the fungal diversity of the mosses G. antarctici and B.  pseudotriquetrum in the Windmill Islands (66°17′S and 110°33′E) and identified 10 fungal taxa. Bradner et al. (2000) identified the microfungi isolated from the moss Bryum argenteum at Marble Point (77°24′S and 163°48′E) and found a new fungal isolate Embellisia species. Tosi et al. (2002) investigated the microfungi associated with eight moss species (B. pseudotriquetrum, Syntrichia princeps, Schistidium antarctici, Bargenteum, Sarconeurum glaciale, C. purpureus, Hennediella heimii, and Campylopus pyriformis) in Victoria Land (74–76°S and 162–165°E) and identified 28 fungal taxa belonged to 18 genera. The above results concerning fungal endophytes of bryophytes do not support the hypothesis that the diversity of organisms decreases from the maritime to the continental Antarctic zone (Ruisi et al., 2007).

Of the 21 fungal taxa in the present study, only two were previously reported as endophytes associated with mosses in Antarctica: Thelebolus and Chaetomium species were isolated from the mosses in the continental Antarctic zone (Azmi & Seppelt, 1998; McRae & Seppelt, 1999; Tosi et al., 2002). Endophytic Hyphodiscus and Xylariaceae have been documented in bryophytes in other ecosystems (Davis et al., 2003; Davis & Shaw, 2008; Kauserud et al., 2008). Additionally, two fungal taxa were reported to be associated with mosses in the previous studies, but they are not known to be endophytic; for example, Eocronartium muscicola is a parasitic bryophilous basidiomycete and has been found on at least 21 moss species (Boehm & McLaughlin, 1988, 1989), and R. ericae is an ericoid mycorrhizal fungus living in the tissues of the liverwort Cephaloziella varians in the maritime and sub-Antarctic (Upson et al., 2007; Newsham, 2010). Some fungal species were isolated from nonbryophyte samples in Antarctica, for example Scopulariopsis species (Bialasiewicz & Czarnecki, 1999), and the yeast Mrakia and Rhodotorula species (Connell et al., 2008; Loque et al., 2010). Species of Annulohypoxylon, Dermateaceae, Helotiales, and Hyaloscyphaceae were also reported to be endophytes of vascular plants in other ecosystems (Zhang et al., 2009; Gazis & Chaverri, 2010; Kernaghan & Patriquin, 2011). In the present study, Xenopolyscytalum species and one unidentified fungus (represented by isolates I12F-02253, -02276, -02282, and -02283) were first reported as fungal endophytes of plants. This unidentified fungus showed very low levels of sequence similarity with fungal species in the NCBI, suggesting that this taxon may be indigenous to Antarctica, and more taxonomic studies are necessary to determine its correct identification.

Values of the Shannon–Weiner diversity index, which was used to evaluate and compare the fungal diversity between the three bryophyte species, ranged from 0.91 to 1.99 in this study. Comparable Shannon–Weiner diversity indices (1.44 ± 0.3) were reported for endophytic fungi in the native Antarctic vascular plant species C. quitensis (Rosa et al., 2010). In Antarctica, several abiotic stresses, such as cold and UV radiation, may lead to such low diverse endophytic fungal community that only a few stress-adapted fungal groups could survive in this environment.

The Sorenson's similarity coefficients for the endophytic fungi from the three plant species ranged from 0.18 to 0.40 in this study. These low Sorenson's similarity coefficients indicate that the fungi have different distributions and perhaps host specificity. This contrast to the findings by Davis & Shaw (2008) who reported that endophytic fungi of liverworts are restricted by geography but not by host.

Effect of temperature on growth in vitro

The results of growth temperature tests suggest that most of the endophytic fungi from bryophytes in the Fildes Region possess a remarkable ability to grow when temperatures are low. Tosi et al. (2002) found that most fungi isolated from mosses in Victoria Land could grow ≤ 5 °C and had their optimum between 10 and 24 °C, indicating that most endophytic fungi isolated from mosses in continental Antarctica are also psychrophilic and psychrotrophic. These psychrophilic and psychrotrophic endophytic fungi in bryophytes may rely on possible physiological (e.g., the accumulation of trehalose, the alteration of membrane lipid composition, antifreeze proteins, and cold-active enzymes) and morphological adaptations (e.g., the predominance of sterile fungi) to grow at low temperature. These physiological and morphological mechanisms have been reported in Antarctic fungi from other environmental samples (Robinson, 2001; Ruisi et al., 2007). Additionally, life history of inhabit plant's tissue may help fungal endophytes coping with cold stress in the environments.

The effect of the endophytic fungi on Antarctic bryophytes remains unknown. Although endophytes may improve the tolerance of their hosts to abiotic stress (Khan et al., 2012), further study is needed to determine whether the fungal endophytes could increase the tolerance of their bryophyte hosts to stress conditions in the Antarctica. It will be more interesting to study the physiology, evolution, and ecological role of these fungal endophytes in the future.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Reference

This research was supported by National Infrastructure of Microbial Resources (No. NIMR-2011-3), the National Natural Science Foundation of China (NSFC) (nos: 30970038 and 31170041), National S&T Major Special Project on Major New Drug Innovation (nos: 2012ZX09301002-001 and 2012ZX09301002-003), and Special Fund for Health-scientific Research in the Public Interest (No.201002021). We thank the Chinese Arctic and Antarctic Administration (CAA) and Shunan Cao in China's 28th Antarctic expedition team for sample collection.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Reference
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