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Species delimitation and phylogenetic analysis of endophyte community

Article title: Geographical and temporal changes of foliar fungal endophytes associated with the invasive plant Ageratina adenophora

Journal name: Microbial Ecology

Authors: Liang Mei1,2, Ming Zhu3, De-Zhu Zhang1,2, Yong-Zhou Wang1,2, Jing Guo1,2, and Han-Bo Zhang1,2

Author affiliations:

1Laboratory of Conservation and Utilization for Bio-resources and Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming 650091, P. R. China

2School of Life Science, Yunnan University, Kunming, Yunnan Province 650091, P. R. China

3School of Medicine, Yunnan University, Kunming, Yunnan Province 650091, P. R. China

Author to whom correspondence should be sent: Han-Bo Zhang, email: . Tel: 86-0871-5034282. Mailing address: School of Life Science, Yunnan University, Kunming, China, 650091

To identify the endophytes isolated from A. adenophora, we firstly observed the endophytes for their morphology of colony and microscopic characteristics including hypha and spore. However, because many endophytes do not sporulate in culture and lack the taxonomic characters needed for identification, their species delimitation were mainly based on gross colony features which results in 76 taxonomic units (data not shown), fewer greatly than that based on molecular sequence data (see discussion below). Therefore, only genotypes identified from the nuclear ribosomal internal transcribed spacer region (ITS) have been provided. These genotypes do not mean the traditional species boundary but just were used, for the convenience, to represent the biodiversity dynamics along the invasion of A. adenophora in this paper.

Genomic DNA of each endophyte was isolated using the CTAB method [9]. The rRNA genes of the internal transcribed spacer (ITS) were amplified with ITS4 and ITS5 primers [8]. Subsequently, 2 μl of the PCR fragment from each isolate was digested with 1.0 U of RsaI, BsuRI, and HinfI (Bio Basic, Markham, Canada), respectively, in a 15 μl reaction system [4], resolved by electrophoresis on 3% agar gels with ethidium bromide and photographed by using GIS system (Tanon, Shanghai, China). The patterns of separated DNA fragments were analyzed manually, and an operational taxonomic unit (OTU) was defined as a group of isolates that have indistinguishable pattern of restriction-enzyme digested products. Representative PCR product from each OTU was purified and sequenced by BGI-SEQUENCING (Shenzhen, China). The obtained sequences were further edited by deleting primer sequences using Editseq and Seqman software in the DNASTAR package (DNASTAR, Madison, WI). ITS consensus sequences were trimmed to homologous starting and ending points, such that length variations reflected only insertion or deletion events.

For searching the closest match to ITS sequence, we did the nucleotide BLAST analysis with unmodified options from the GenBank of NCBI (Bethesda, Maryland, USA). Since some GenBank records are misidentified and misnamed [2, 5], only sequences from type species were downloaded as recommended previously [5]. If such sequences are unavailable, only the published closest match with the species name (or generic name) was downloaded. In cases one sequence could retrieve one more published matches with different species name (or generic name), all matches with the same identity score were downloaded. If no such matches appeared in the first 100 target sequences via BLASTing, the closest match was downloaded and these sequences were categorized as the ones from unknown fungi. All downloading sequences and our representative sequences were used to construct phylogenetic trees using the PHYLIP package [3] with the Jukes–Cantor evolutionary model and the neighborjoining method. Bootstrap analysis was carried out for 1000 replicates. The nucleotide sequences reported in this study have been deposited in GenBank under accession numbers KC507193-KC507297.

At division level, endophyte community mainly included Sordariomycetes and Dothideomycetes, similar to those reported in most territorial plant species [1-2, 6]. Few genera, including Collectotrichum, Nemania, Phomopsis and Xylaria are those numerical members (Fig. S1, Table S1). Colletotrichum contained 132 isolates and occurred in 21 sites. They were further separated into 5 groups. Based on the morphology and ITS sequence, position of group one (65 strains, 8 OTUs) is determined to be C. gloeosporioides because all of the isolates that we accept in this clade shared the string 5’–GGGCGGGT–3’, a string appears to be specific to members of the C. gloeosporioides complex [7]. Group 2 was close to C. boninense (31 strains, 5 OTUs), group 3, to C. acutatum (strains, 3 OTUs), and group 4, to C. thailandicum ( strains, 1 OTU). Although the remaining 12 strains (Group 5, 4 OTUs) were phylogenetically close to C. gloeosporioides (AJ301979), they were excluded from the analysis of geographically genetic differentiation of C. gloeosporioides because all of these strains have no sequence 5’–GGGCGGGT–3’ which is specific to species C. gloeosporioide as Weir et al [7] demonstrated previously.

Fig. S1 Phylogenetic relationship of representative ITS gene sequences of foliar endophytes isolated from A. adenophora. Site and sampled strain number are indicated in the following parentheses of the representative strain of each OTU. Protozoan Brachylecithum lobatum (AY222144) is used as an outgroup. Bootstrap values (1,000 replications) of equal to or >50% are indicated at branch nodes. GenBank accession no. of reference sequences are shown in parentheses. The scale bar represents 5% estimated sequence divergence. Shadowed part included those from Colletotrichum.

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Table S1 Percentage of endophyte community at genus level

Genus / OTUs / MJP / ES / YJ / PR / SL / XM / DT / CYI / CYII / GM / FQ / XY / NH / LF / XD / MN / LG / XS / JD / TL / BG / Total / Percentage
Colletotrichum / AX10, AX21, AX34, AX36, AX38, AX81, AX96, AX115, AX118, AX129, AX152, AX159, AX165, AX182, AX188, CT5, FT2, HT10, JK41, JT2, SS7 / 4 / 4 / 17 / 2 / 11 / 12 / 6 / 4 / 8 / 6 / 3 / 14 / 5 / 3 / 1 / 1 / 1 / 5 / 11 / 12 / 2 / 132 / 28.51%
Nemania / JK15, AX40, SS29, AX48, AX54, AX45,CT6 / 1 / 7 / 1 / 2 / 1 / 4 / 4 / 2 / 3 / 5 / 6 / 2 / 5 / 11 / 7 / 1 / 4 / 2 / 1 / 69 / 14.90%
Phomopsis / AX91, JK9, AX134, ET1, AX177, AX97, AX2 / 7 / 11 / 2 / 1 / 4 / 2 / 2 / 5 / 2 / 2 / 3 / 2 / 14 / 3 / 1 / 61 / 13.17%
Xylaria / AX55, AX46, AX43, AX41, AX13, HB3, AX64, AX114 / 2 / 1 / 2 / 1 / 2 / 1 / 2 / 3 / 2 / 1 / 4 / 1 / 1 / 23 / 4.97%
Nigrospora / GT1, FT4, AX181, AX164, SS10 / 2 / 2 / 1 / 11 / 3 / 19 / 4.10%
Trametes / SS2,AX131 / 1 / 2 / 4 / 10 / 17 / 3.67%
Cercophora / AX150, AX12 / 3 / 2 / 5 / 10 / 2.16%
Corprinopsis / JK84 / 9 / 1 / 10 / 2.16%
Schizophyllum / AX15, AX143, AX158 / 1 / 5 / 1 / 1 / 1 / 9 / 1.94%
Rachicladosporium / HX6, AX4 / 1 / 1 / 1 / 1 / 2 / 2 / 8 / 1.73%
Guignardia / AX162, MT2, AX5 / 1 / 1 / 2 / 1 / 1 / 1 / 1 / 8 / 1.73%
Phialophora / AX163, AX146, AX1 / 2 / 1 / 1 / 1 / 1 / 1 / 7 / 1.51%
Phanerochaete / AX95, AX156, JK3 / 1 / 1 / 1 / 1 / 1 / 2 / 7 / 1.51%
Botryosphaeria / DT1 / 3 / 2 / 5 / 1.08%
Alternaria / JK91, AX35 / 1 / 4 / 5 / 1.08%
Chaetomium / AX121, SS5 / 1 / 1 / 1 / 3 / 0.65%
Arthrinium / AX148, AX147, / 3 / 3 / 0.65%
Hypoxylon / AX111 / 2 / 1 / 3 / 0.65%
Nodulisporum / AX76, JK24, SS33 / 1 / 1 / 1 / 3 / 0.65%
Didymella / AX82, SS14 / 1 / 1 / 1 / 3 / 0.65%
Neofusicoccum / GT4 / 2 / 2 / 0.43%
Psathyrella / AX85, AX136 / 1 / 1 / 2 / 0.43%
Tinctoporellus / AX99 / 1 / 1 / 2 / 0.43%
Fusarium / AX202 / 1 / 1 / 0.22%
Clonostachys / AX50 / 1 / 1 / 0.22%
Acremonium / AX119 / 1 / 1 / 0.22%
Sordaria / JK39 / 1 / 1 / 0.22%
Zopfiella / AX157 / 1 / 1 / 0.22%
Coniochaeta / SS12 / 1 / 1 / 0.22%
Cladosporium / JK36 / 1 / 1 / 0.22%
Stagonospora / AX113 / 1 / 1 / 0.22%
Leptosphaeria / AX166 / 1 / 1 / 0.22%
Paraconiothyrium / JK40 / 1 / 1 / 0.22%
Trichocladium / AX104 / 1 / 1 / 0.22%
Preussia / SS13 / 1 / 1 / 0.22%
Peniophora / AX138 / 1 / 1 / 0.22%
Lentinus / AX170 / 1 / 1 / 0.22%
Polyporus / AX151 / 1 / 1 / 0.22%
unknow fungi / AX8, AX7,AX107, AX11, AX108, AX112, AX102, AX122, AX128, AX142, JK27, AX168, AX144 / 2 / 2 / 15 / 2 / 4 / 3 / 1 / 4 / 2 / 1 / 1 / 37 / 7.99%
Total / 463

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