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가설 설정
- Both species were reciprocally monophyletic for both ITS and LSU, with low intraspecific and high interspecific variation, validating the morphological identification. These sequence data and the phylogenetic tree served as the framework to which we determined whether GenBank sequences are misidentified.
제안 방법
- [19]. After verification via gel electrophorese on a 1% agarose gel and the PCR product purified using the Expin PCR Purification Kit (GeneAll Biotechnology, Korea), DNA sequencing was performed with an ABI3700 automated DNA sequencer (Applied Biosystems, USA) at Macrogen (Seoul, Korea).
- This problem of misidentification is made worse since both species are sympatric and have a global distribution [9, 17, 22]. In this study, we have rigorously reexamined Bjerkandera specimens from Korea and verified the distinguishing morphological characters separating these two species (Fig. 1, Table 2). We also found that DNA data are useful to distinguish between B.
- NJ analyses were performed using p-distances, substitutions including transitions and transversions, pairwise deletion of missing data, and 1,000 bootstrap replicates. ML was performed using the combined rapid bootstrap and search for the best-scoring ML tree analysis, the GTRGAMMA model of sequence evolution, and 1,000 bootstrap replicates. Both rooted and unrooted analyses were performed on the datasets to enhance our ability to identify distantly related species that were mislabeled as Bjerkandera.
- Other previous studies also raise the problem of identification using environmental DNA data and BLAST for identification [28]. To clarify the issue, we performed a phylogenetic analysis of our Bjerkandera ITS data, adding data from T. cucumeris. We found that 1,024 sequences of T.
- We chose Bjerkandera because (i) there are only two species, (ii) the two species are highly similar and easily misidentified by non-specialists despite distinguishing morphological characters, and (iii) the results have implications to genomic and biotechnological research. To complete these goals, we first identified true B. adusta and B. fumosa samples through rigorous morphological observation, followed by DNA sequencing to build a framework for comparison. Two molecular markers, the internal transcribed spacer (ITS) and the 28S nuclear ribosomal large subunit (LSU), were sequenced since they are the two most common genes used in fungal systematics [5, 23, 24].
대상 데이터
- All specimens used in this study were collected throughout the Korean Peninsula between 1989 and 2013, dried, and deposited in the Seoul National University Fungal Collection (SFC) (Table 1). Specimens labeled as Bjerkandera were rigorously reexamined based on distinguishing morphological characters to determine their true species identification.
이론/모형
- Based on a previous phylogenetic study, Phanerochaete chrysosporium was selected as the outgroup for rooted phylogenetic analyses [14]. Intra- and interspecific pairwise distances were calculated in MEGA5 using the p-distance model, substitutions including transitions and transversions, and pairwise deletion of gaps.
성능/효과
- Based on intraspecific p-distances of B. adusta and B.fumosa from step two (ITS: <6%; LSU: <3%), to be conservative, we downloaded all sequences that had a p-distance of <8% (92% similarity) for ITS and <5% (95% similarity) for LSU.
- Based on our search criteria (see Materials and Methods section), a total of 121 unique ITS and 15 unique LSU sequences were identified to be highly similar to B. adusta and B. fumosa and included in the final phylogenetic analyses. For ITS, 90 sequences were shown to be B.
- fumosa which identities were verified using morphology. Both species were reciprocally monophyletic for both ITS and LSU, with low intraspecific and high interspecific variation, validating the morphological identification. These sequence data and the phylogenetic tree served as the framework to which we determined whether GenBank sequences are misidentified.
- Our analysis had three steps. First, phylogenetic trees for ITS and LSU were built using only specimens of B. adusta and B. fumosa which identities were verified using morphology. Both species were reciprocally monophyletic for both ITS and LSU, with low intraspecific and high interspecific variation, validating the morphological identification.
- For ITS, the intraspecific variation of Korean B. adusta and B. fumosa was 0.0–0.55% and 0.0%, respectively, whereas the interspecific variation was 5.15– 5.89%.
- For LSU, the intraspecific variation of Korean B. adusta and B. fumosa was 0.0–0.16% for both species, and the interspecific variation was 1.44–1.78%.
- fumosa, one sequence was an unidentified species (FJ820598). For LSU, two sequences were misidentified and shown to be B. adusta: Antrodia malicola (AY333836) and an unidentified fungal species (JQ249221). The remaining 13 sequences were not closely related to Bjerkandera.
- We found that the number of misidentified sequences of Bjerkandera in GenBank is substantial. More ITS sequences (95 sequences) were present in GenBank compared with LSU (29 sequences), and as such, the problem of misidentification was more evident for ITS sequences. Our discussion of misidentification herein focuses on ITS.
- 1). The final identification recognized 18 B. adusta and 6 B. fumosa. One specimen of B.
- The intraspecific variation of ITS for B. adusta and B. fumosa was 0.0–5.48% and 0.0–1.86%, respectively, and the interspecific variation was 3.53–7.85%.
- The intraspecific variation of LSU for B. adusta and B. fumosa was 0.0–2.45% and 0.0–0.55%, respectively, whereas the interspecific variation was 1.14–2.38%.
- The results revealed that B. fumosa was more commonly misidentified as B. adusta (n = 4) as opposed to the opposite case (n = 1) (Fig. 2B). This is likely due to B.
- S6). These results indicate that T. cucumeris and B. adusta are distinguishable with molecular data, and the problem was due to misidentified sequences.
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