Fig. 1.
Location of sampling sites at three mountain marshes. Red box: location of Mountain Jiri, covering five cities in the southern part of the Korean peninsula. Blue box: location of Mountain Jiri and sampling sites marked with small boxes.
a) Purple box, Wangdeungjae marsh, 35°23’21.8”N 127°47’19.0”E. b) Green box, Waegok marsh, 35°22’57.0”N 127°46’49.7”E. c) Orange box, Jeonglyeongchi marsh, 35°21’52.5”N 127°31’25.5”E.
Table I
Physicochemical measurements, sequencing results, and ecological diversity analysis of Mount Jiri marsh samples.
| x | Jeonglyeongchi | Waegok | Wangdeungjae | |
|---|---|---|---|---|
| Physico-chemical factors | Temperature (°C) | 12.75 | 16.55 | 22.93 |
| pH | 6.95 | 6.84 | 6.48 | |
| EC (µS/cm) | 32 | 36 | 96 | |
| Salinity (ppt) | 0.0 | 0.0 | 0.0 | |
| DO (mg/l) | 10.51 | 7.98 | 4.71 | |
| Turbidity (NTU) | 2.51 | 42.30 | 6.26 | |
| TN (mg/l) | 0.00 ± 0.00 | 0.00 ± 0.00 | 0.00 ± 0.00 | |
| TP (mg/l) | 1.57 ± 0.16 | 0.94 ± 0.01 | 0.00 ± 0.00 | |
| Sequencing results | Total reads | 122,953 | 113,853 | 121,392 |
| Validated reads | 98,159 | 80,099 | 22,249 | |
| Mean read length (bp) | 406.28 | 402.63 | 401.70 | |
| Maximum read length (bp) | 419 | 407 | 407 | |
| Number of OTUsa | 243 | 828 | 64 | |
| Diversity indicators | Chao1b | 243.00 | 828.00 | 64.00 |
| Shannonc | 4.84 | 6.36 | 2.97 | |
| Simpsond | 0.91 | 0.94 | 0.75 | |
| Goods Coveragee | 1.00 | 1.00 | 1.00 | |
Fig. 2.
Rarefaction curves for OTUs representing the eukaryotic microbial communities associated with the marsh samples. The OTUs were analyzed using the cluster database that was set at high identity, with the tolerance (CD-HIT) program set at a 97% sequence similarity. The Mothur platform was used to calculate the rarefaction curves and diversity indices.
a) OTUs. b) Chao1 estimator. c) Whole tree (Waegok, red curve; Jeonglyeongchi, blue curve; Wangdeungjae, orange curve). d) UPGMA tree illustrating the relationships based on weighted UniFrac distances between the eukaryotic microbial communities associated with Jeonglyeongchi, Waegok, and Wangdeungjae marshes.
Fig. 3.
Taxonomic composition of microalgal and other microbial phyla found in Jeonglyeongchi, Waegok, and Wangdeungjae marsh samples.
Table II
Relative abundance of species in the Jeonglyeongchi, Waegok, and Wangdeungjae samples.
| Taxonomy | Relative abundance (%) | ||||||
|---|---|---|---|---|---|---|---|
| Phylum | Class | Order | Family | Species | Jeonglye ongchi | Waegok | Wangdeungjae |
| Annelida | – | Haplotaxida | Enchytraeidae | Mesenchytraeus pelicensis | 0 | 0.05 | 0 |
| Annelida | – | Haplotaxida | Naididae | Dero sp. | 0 | 0.17 | 9.98 |
| Apicomplexa | – | – | – | Apicomplexan Acarus | 0 | 0.01 | 0 |
| Apicomplexa | – | – | Sphaerocystidae | Paraschneideria metamorphosa | 0 | 0.19 | 0 |
| Apicomplexa | Coccidia | Eucoccidiorida | Cryptosporidiidae | Cryptosporidiidae environmental | 0 | 3.09 | 0.93 |
| Apicomplexa | Coccidia | Eucoccidiorida | Eimeriidae | Eimeriidae environmental | 5.43 | 1.11 | 0 |
| Apicomplexa | Coccidia | Eucoccidiorida | Eimeriidae | Eimeria sp. | 0 | 0 | 26.17 |
| Arthropoda | – | Cyclopoida | Cyclopidae | Paracyclops chiltoni | 0 | 0.16 | 0 |
| Arthropoda | Arachnida | – | Anystidae | Anystis sp. | 0 | 0.03 | 0 |
| Arthropoda | Arachnida | – | Hygrobatidae | Hygrobates norvegicus | 5.73 | 0 | 0 |
| Arthropoda | Insecta | Diptera | Chironomidae | Micropsectra sp. | 0.31 | 0 | 0 |
| Arthropoda | Insecta | Diptera | Chironomidae | Monodiamesa sp. | 0 | 0.05 | 0 |
| Arthropoda | Insecta | Diptera | Culicidae | Aedes albopictus | 0 | 34.77 | 0 |
| Ascomycota | – | – | – | Uncultured ascomycete | 0 | 0.04 | 0 |
| Ascomycota | Saccharomycetes | Saccharomycetales | Debaryomycetaceae | [Candida] schatavii | 0.07 | 0 | 0 |
| Ascomycota | Sordariomycetes | – | – | Leptosporella sp. | 0 | 0.11 | 0 |
| Ascomycota | Sordariomycetes | Chaetosphaeriales | Chaetosphaeriaceae | Thozetella pandanicola | 0 | 1.24 | 0 |
| Ascomycota | Sordariomycetes | Diaporthales | Diaporthaceae | Diaporthe amygdali | 0 | 0.02 | 0 |
| Ascomycota | Sordariomycetes | Hypocreales | Nectriaceae | Fusarium oxysporum | 0 | 0.05 | 0 |
| Ascomycota | Sordariomycetes | Xylariales | – | Discosia querci | 0 | 0.01 | 0 |
| Bacillariophyta | Bacillariophyceae | – | – | Achnanthidium daonense | 0 | 0.04 | 0 |
| Bacillariophyta | Bacillariophyceae | – | – | Achnanthidium digitatum | 0 | 0.11 | 0 |
| Bacillariophyta | Bacillariophyceae | – | – | Achnanthidium minutissimum | 0 | 0.25 | 0 |
| Bacillariophyta | Bacillariophyceae | – | – | Achnanthidium straubianum | 0 | 0.12 | 0 |
| Bacillariophyta | Bacillariophyceae | – | Bacillariaceae | Nitzschia acidoclinata | 0 | 0.04 | 0 |
| Bacillariophyta | Bacillariophyceae | – | Bacillariaceae | Nitzschia dissipata | 0 | 0.18 | 0 |
| Bacillariophyta | Bacillariophyceae | – | Cymbellaceae | Cymbella aspera | 0 | 1.45 | 0 |
| Bacillariophyta | Bacillariophyceae | – | Cymbellaceae | Cymbopleura naviculiformis | 0 | 0.82 | 0 |
| Bacillariophyta | Bacillariophyceae | – | Cymbellaceae | Placoneis elginensis | 0 | 0.09 | 0 |
| Bacillariophyta | Bacillariophyceae | – | Gomphonemataceae | Gomphonema affine | 0.57 | 0.36 | 0 |
| Bacillariophyta | Bacillariophyceae | – | Gomphonemataceae | Gomphonema cf. | 0 | 0.18 | 0 |
| Bacillariophyta | Bacillariophyceae | Eunotiales | Eunotiaceae | Eunotia sp. | 0.24 | 0.14 | 0.81 |
| Bacillariophyta | Bacillariophyceae | Naviculales | – | Humidophila australis | 0 | 0.03 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | – | Uncultured Halamphora | 0 | 0.03 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Amphipleuraceae | Halamphora sp. | 0 | 0.11 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Naviculaceae | Pinnunavis sp. | 0 | 0.18 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Naviculaceae | Navicula sp. | 0 | 0.04 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Neidiaceae | Neidium hitchcockii | 0 | 0.01 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Neidiaceae | Neidium sp. | 0 | 0.11 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Pinnulariaceae | Pinnularia cf. | 0 | 0.11 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Pinnulariaceae | Pinnularia microstauron | 0 | 0.51 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Pinnulariaceae | Pinnularia subgibba | 0.34 | 0 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Pinnulariaceae | Pinnularia viridiformis | 0 | 0.04 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Sellaphoraceae | Sellaphora cf. | 0 | 0.01 | 0 |
| Bacillariophyta | Bacillariophyceae | Naviculales | Sellaphoraceae | Sellaphora pupula | 0 | 0.04 | 0 |
| Bacillariophyta | Bacillariophyceae | Surirellales | – | Surirella brebissonii | 0 | 0.75 | 0 |
| Bacillariophyta | Bacillariophyceae | Surirellales | – | Surirella cf. | 0 | 0.08 | 0 |
| Bacillariophyta | Bacillariophyceae | Surirellales | – | Surirella sp. | 0 | 0.09 | 0 |
| Bacillariophyta | Bacillariophyceae | Thalassiophysales | Catenulaceae | Amphora copulata | 0 | 0.21 | 0 |
| Bacillariophyta | Coscinodiscophyceae | – | Aulacoseiraceae | Aulacoseira alpigena | 0 | 0.12 | 1.83 |
| Bacillariophyta | Coscinodiscophyceae | – | Aulacoseiraceae | Aulacoseira sp. | 0 | 0 | 7.77 |
| Bacillariophyta | Coscinodiscophyceae | Chaetocerotales | Chaetocerotaceae | Uncultured Chaetoceros | 0 | 0.02 | 0 |
| Bacillariophyta | Fragilariophyceae | Fragilariales | Fragilariaceae | Fragilaria vaucheriae | 0 | 0.29 | 0 |
| Bacillariophyta | Fragilariophyceae | Tabellariales | Tabellariaceae | Tabellaria flocculosa | 0.23 | 0.44 | 0 |
| Basidiomycota | Agaricomycetes | Agaricales | – | Inocybe spuria | 0 | 0.01 | 0 |
| Basidiomycota | Agaricomycetes | Polyporales | – | Fibroporia gossypium | 0.11 | 0.08 | 0 |
| Basidiomycota | Tremellomycetes | – | – | Holtermanniella nyarrowii | 0.04 | 0 | 0 |
| Basidiomycota | Tremellomycetes | Cystofilobasidiales | Cystofilobasidiaceae | Cystofilobasidium macerans | 0.31 | 0.01 | 0 |
| Basidiomycota | Tremellomycetes | Filobasidiales | – | Solicoccozyma terricola | 0.21 | 0 | 0 |
| Basidiomycota | Tremellomycetes | Filobasidiales | Filobasidiaceae | Filobasidium magnum | 0.15 | 0 | 0 |
| Basidiomycota | Tremellomycetes | Tremellales | – | Cryptococcus carnescens | 0.02 | 0 | 0 |
| Basidiomycota | Tremellomycetes | Tremellales | – | Papiliotrema flavescens | 0.03 | 0 0 | |
| Blastocladiomycota | – | – | – | Uncultured Blastocladiomycota | 0 | 0.11 | 0 |
| Chlorophyta | – | – | – | Chlorophyta sp. | 0 | 0.01 | 0 |
| Chlorophyta | Chlorophyceae | – | Microsporaceae | Microspora sp. | 0 | 0 | 1.24 |
| Chlorophyta | Chlorophyceae | Chlamydomonadales | Chlamydomonadaceae | Chlamydomonas sp. | 0 | 0.05 | 1.24 |
| Chlorophyta | Chlorophyceae | Chlamydomonadales | Chlorococcaceae | Chlorococcum sp. | 0 | 0.04 | 0 |
| Chlorophyta | Chlorophyceae | Sphaeropleales | – | Dictyococcus sp. | 0 | 0.08 | 0 |
| Chlorophyta | Chlorophyceae | Sphaeropleales | – | Bracteacoccus deserticola | 0 | 0 | 0.09 |
| Chlorophyta | Chlorophyceae | Sphaeropleales | Neochloridaceae | Neochloris sp. | 0.22 | 0 | 0 |
| Chlorophyta | Chlorophyceae | Sphaeropleales | Scenedesmaceae | Scenedesmus sp. | 0 | 0 | 1.71 |
| Chlorophyta | Chlorophyceae | Sphaeropleales | Scenedesmaceae | Asterarcys quadricellulare | 0 | 0.02 | 0 |
| Chlorophyta | Trebouxiophyceae | – | Coccomyxaceae | Coccomyxa simplex | 0.15 | 0.01 | 0 |
| Chlorophyta | Trebouxiophyceae | Chlorellales | Chlorellaceae | Chlorella vulgaris | 0 | 0.01 | 0.44 |
| Chlorophyta | Ulvophyceae | Ulotrichales | – | Tupiella speciosa | 0.12 | 0.03 | 0 |
| Chlorophyta | prasinophytes | – | – | Monomastix opisthostigma | 0 | 0.05 | 0 |
| Chordata | Amphibia | Caudata | Salamandridae | Cynops pyrrhogaster | 0.05 | 0 | 0 |
| Chordata | Mammalia | Cetacea | Delphinidae | Lagenorhynchus obliquidens | 0 | 0 | 2.45 |
| Chytridiomycota | – | – | – | Uncultured Chytridiomycota | 1.31 | 1.09 | 0.44 |
| Chytridiomycota | – | – | – | Uncultured rhizosphere | 0 | 0.01 | 0 |
| Chytridiomycota | Chytridiomycetes | – | – | Catenomyces sp. | 0 | 0.08 | 0 |
| Chytridiomycota | Chytridiomycetes | – | – | Rhizophlyctis rosea | 0 | 0 | 1.58 |
| Chytridiomycota | Chytridiomycetes | Chytridiales | – | Chytridiales sp. | 0 | 0.41 | 0 |
| Chytridiomycota | Chytridiomycetes | Chytridiales | – | Uncultured Chytridiales | 0 | 0.46 | 0 |
| Chytridiomycota | Chytridiomycetes | Chytridiales | – | Uncultured Chytriomyces | 0 | 0.23 | 0 |
| Chytridiomycota | Chytridiomycetes | Chytridiales | – | Chytriomyces sp. | 0.12 | 0.09 | 10.94 |
| Chytridiomycota | Chytridiomycetes | Chytridiales | – | Obelidium mucronatum | 2.62 | 0.58 | 0 |
| Chytridiomycota | Chytridiomycetes | Chytridiales | – | Rhizoclosmatium globosum | 9.07 | 0 | 0 |
| Chytridiomycota | Chytridiomycetes | Chytridiales | Chytridiaceae | Chytridiaceae sp. | 0 | 0.11 | 0 |
| Chytridiomycota | Chytridiomycetes | Cladochytriales | – | Nowakowskiella elegans | 0 | 0.02 | 0 |
| Chytridiomycota | Chytridiomycetes | Cladochytriales | – | Nowakowskiella hemisphaerospora | 0.43 | 0.17 | 0 |
| Chytridiomycota | Chytridiomycetes | Cladochytriales | – | Nowakowskiella multispora | 0 | 1.61 | 0 |
| Chytridiomycota | Chytridiomycetes | Cladochytriales | – | Nowakowskiella sp. | 0 | 0.16 | 0 |
| Chytridiomycota | Chytridiomycetes | Cladochytriales | Cladochytriaceae | Cladochytrium replicatum | 0 | 0.02 | 0 |
| Chytridiomycota | Chytridiomycetes | Cladochytriales | Cladochytriaceae | Cladochytrium tenue | 0 | 0.01 | 0 |
| Chytridiomycota | Chytridiomycetes | Rhizophydiales | – | Rhizophydiales sp. | 0 | 0.08 | 0 |
| Chytridiomycota | Chytridiomycetes | Rhizophydiales | – | Uebelmesseromyces sp. | 0 | 0.94 | 0 |
| Chytridiomycota | Chytridiomycetes | Rhizophydiales | Kappamycetaceae | Kappamyces laurelensis | 0.13 | 0.01 | 0 |
| Chytridiomycota | Chytridiomycetes | Rhizophydiales | Rhizophydiaceae | Rhizophydium planktonicum | 0 | 0 | 0.51 |
| Chytridiomycota | Chytridiomycetes | Rhizophydiales | Rhizophydiaceae | Rhizophydium sphaerotheca | 0 | 2.14 | 0 |
| Chytridiomycota | Chytridiomycetes | Spizellomycetales | – | Fimicolochytrium alabamae | 0.18 | 0.15 | 0 |
| Chytridiomycota | Monoblepharidomycetes | Monoblepharidales | – | Hyaloraphidium curvatum | 0.09 | 0.02 | 0 |
| Chytridiomycota | Monoblepharidomycetes | Monoblepharidales | Harpochytriaceae | Harpochytrium sp. | 0 | 0.06 | 0 |
| Chytridiomycota | Monoblepharidomycetes | Monoblepharidales | Oedogoniomycetaceae | Oedogoniomyces sp. | 0 | 0.01 | 0 |
| Eustigmatophyceae | – | – | – | Uncultured eustigmatophyte | 0 | 0.04 | 0 |
| Gastrotricha | – | Chaetonotida | Chaetonotidae | Chaetonotus cf. | 0 | 24.43 | 0 |
| Mollusca | Bivalvia | Veneroida | Sphaeriidae | Pisidium walkeri | 0 | 0.03 | 0 |
| Nematoda | Chromadorea | Monhysterida | Monhysteridae | Eumonhystera cf. | 0.46 | 0 | 22.27 |
| Platyhelminthes | – | Catenulida | Catenulidae | Catenula turgida | 0.07 | 0 | 0 |
| Platyhelminthes | – | Catenulida | Stenostomidae | Stenostomum sp. | 0 | 0.06 | 6.62 |
| Platyhelminthes | – | Rhabdocoela | Typhloplanidae | Phaenocora sp. | 0 | 0.02 | 0 |
| Platyhelminthes | – | Tricladida | Planariidae | Phagocata sibirica | 68.64 | 0 | 0 |
| Streptophyta | – | Brassicales | Brassicaceae | Brassica napus | 0 | 0 | 0.34 |
| Streptophyta | – | Caryophyllales | Polygonaceae | Persicaria virginiana | 1.96 | 0.01 | 0 |
| Streptophyta | – | Ericales | Styracaceae | Styrax americana | 0 | 0.04 | 0 |
| Streptophyta | – | Malpighiales | Salicaceae | Populus trichocarpa | 0 | 2.84 | 0 |
| Streptophyta | – | Piperales | – | Aristolochiaceae environmental | 0.16 | 0 | 0 |
| Streptophyta | Liliopsida | Poales | Poaceae | Stipa narynica | 0 | 15.14 | 0 |
| Streptophyta | Zygnemophyceae | Desmidiales | – | Uncultured Closterium | 0.43 | 0.06 | 0 |
| Streptophyta | Zygnemophyceae | Desmidiales | Closteriaceae | Closterium moniliferum | 0 | 0.21 | 0 |
| Streptophyta | Zygnemophyceae | Desmidiales | Closteriaceae | Closterium venus | 0 | 0 | 0.62 |
| Streptophyta | Zygnemophyceae | Desmidiales | Desmidiaceae | Euastrum affine | 0 | 0 | 2.02 |
| Xanthophyceae | – | – | – | Xanthophyceae sp. | 0 | 0.05 | 0 |
Fig. 4.
Molecular phylogenetic analysis by the maximum likelihood (ML) tree. Numbers at the nodes indicate bootstrap probabilities (> 50 v%) of the ML analyses (1,000 replicates).
a) Phylogenetic relationship between all species identified using a BLASTN search within the NCBI database. Seventeen phyla corresponded to the species names listed in the phylogenetic tree.
b) Phylogenetic distances between the identified microalgal species (pink branch, Bacillariophyta; green branch, Chlorophyta).
Fig. 5.
Composition of microalgal species grown in each culture medium and identified using the Illumina MiSeq analysis (M). Four culture media were used: Blue Green-11 (BG11) medium, Optimum Haematococus Medium (OHM), Bold Basal medium (BB), and Diatom Medium (DM).
Table III
Illumina MiSeq (M) and culture-based analyses of microalgae from Jeonglyeongchi, Waegok, and Wangdeungjae marsh samples.
| Species | Accession number | Jeonglyeongchi | Waegok | Wangdeungjae | |||
|---|---|---|---|---|---|---|---|
| M | CB | M | CB | M | CB | ||
| Achnanthidium daonense | KJ658413 | – | – | + | – | – | – |
| Achnanthidium digitatum | KX946582 | – | – | + | – | – | – |
| Achnanthidium minutissimum | MH358459 | – | – | + | – | – | – |
| Achnanthidium straubianum | KY863467 | – | – | + | – | – | – |
| Nitzschia acidoclinata | KT072971 | – | – | + | – | – | – |
| Nitzschia dissipata | AJ867018 | – | – | + | + | – | – |
| Cymbella aspera | KJO11615 | – | – | + | – | – | – |
| Cymbopleura naviculiformis | AM501997 | – | – | + | – | – | – |
| Placoneis elginensis | AM501953 | – | – | + | – | – | – |
| Gomphonema affine | MN197879 | + | – | + | – | – | – |
| Gomphonema cf. | AM502005 | – | – | + | – | – | – |
| Eunotia sp. | KJ961696 | + | – | + | – | + | – |
| Humidophila australis | KM116120 | – | – | + | – | – | – |
| Uncultured Halamphora | MK656307 | – | – | + | – | – | – |
| Halamphora sp. | MG027261 | – | – | + | – | – | – |
| Pinnunavis sp. | KJ961669 | – | – | + | – | – | – |
| Navicula sp. | MK177604 | – | – | + | – | – | – |
| Neidium hitchcockii | KU674393 | – | – | + | – | – | – |
| Neidium sp. | KU674445 | – | – | + | – | – | – |
| Pinnularia cf. | JN418569 | – | – | + | – | – | – |
| Pinnularia microstauron | AM501981 | – | – | + | – | – | – |
| Pinnularia subgibba | KT072984 | + | – | – | – | – | – |
| Pinnularia viridiformis | AM501985 | – | – | + | – | – | – |
| Sellaphora cf. | EF151967 | – | – | + | – | – | – |
| Sellaphora pupula | AJ544653 | – | – | + | – | – | – |
| Surirella brebissonii | KX120739 | – | – | + | – | – | – |
| Surirella cf. | KX120782 | – | – | + | – | – | – |
| Surirella sp. | KX120781 | – | – | + | – | – | – |
| Amphora copulata | MG027291 | – | – | + | – | – | – |
| Aulacoseira alpigena | AY569578 | – | – | + | – | + | – |
| Aulacoseira sp. | AY569587 | – | – | – | – | + | – |
| Uncultured Chaetoceros | MH023058 | – | – | + | – | – | – |
| Fragilaria vaucheriae | AM497736 | – | – | + | – | – | – |
| Tabellaria foecu losa | MH356258 | + | – | + | – | – | – |
| Chlorophyta sp. | MK929233 | – | – | + | – | – | – |
| Microspora sp. | AF387160 | – | – | – | – | + | – |
| Chlamydomonas sp. | MH683856 | – | – | + | + | + | + |
| Chlorococcum sp. | MK954470 | – | – | + | + | – | – |
| Dictyococcus sp. | HM852440 | – | – | + | – | – | – |
| Bracteacoccus deserticola | JQ259938 | – | – | – | – | + | – |
| Neochloris sp. | AB917132 | + | + | – | – | – | – |
| Scenedesmus sp. | MHO 10849 | – | – | – | – | + | + |
| Asterarcys quadricellulare | MN179327 | – | – | + | – | – | – |
| Coccomyxa simplex | MH196858 | + | – | + | – | – | – |
| Chlorella vulgaris | MK652782 | – | – | + | + | + | – |
| Tupiella speciosa | MF000567 | + | – | + | – | – | – |
| Monomastix opisthostigma | FN562445 | – | – | + | – | – | – |