Chaenothecopsis xishuiensis sp. nov. to Science and Lecanora pseudargentata Newly Reported from China
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials Specimens
2.2. Morphological Study
2.3. Anatomical Study
- Moisten the selected apothecia: Apply an appropriate amount of sterile water to the well-developed apothecia with a rubber-tipped dropper and remove the ascospores after the apothecia have absorbed the water and become soft.
- Slice: Using a single-sided blade, cut the selected apothecia longitudinally so that the slices are as thin and complete as possible.
- Production: Pick up the section with the tip of a needle on a slide moistened with sterile water, cover it with a coverslip and absorb excess water with absorbent paper.
- Observation and recording: Observe the internal structure of the apothecia under a compound microscope (OLYMPUS BX53), including the colour and thickness of exciple; the colour and thickness of epithecium, hymenium, and hypothecium; the shape, size of asci, and the number of ascospores contained in them; the colour, size, and type of ascospores. Take photographs and record the relevant information.
- Colour development: Stain the prepared mount with iodine solution and observe the amyloplastic reflection of the ascospores.
2.4. Methods for the Determination of Lichen Secondary Metabolites
- Prepare the glass silicone adhesive board. Use a precoated glass silicone backing sheet (20 cm long, 10 cm wide, 0.25 mm thick). Using a pencil, carefully draw a straight line 1.5 cm from the bottom of the glass silicone board. Mark a point every 1 cm on the straight line, which will be the sample point.
- Prepare the solvent. Mix 20 mL of toluene and 3 mL of acetic acid, add to a rectangular TLC developing tank, and place in a fume cupboard.
- Prepare the samples. Take an appropriate quality of the thallus to be examined and place them separately in small centrifuge tubes. Add a suitable volume of acetone to each small centrifuge tube until the acetone covers the sample. After 10 min, the samples can be placed in order.
- Spot sampling: Use microcapillary tubes to sample separately according to the position of the sampling points on the glass silicone board. The left, right, and centre sampling points are brushed with Lethariella cladonioides to facilitate the use of split standard samples, while the remaining sampling points are sampled sequentially for testing.
- Exposure layer: After sampling, place the silicone board in a chromatography cylinder and place it 1 cm below the solvent level so that the sample origin is approximately 0.5 cm from the solvent level and make the origin line parallel to the liquid level line. When the leading edge of the solvent moves from the origin to about 1.5 cm from the top of the silicone board, remove the silicone board and dry the solvent on the board surface with a hair dryer.
- Colour rendering: Spray the silicone board with 10% sulphuric acid and observe if there are any grease spots when it is wet. Then, heat it in an oven at 94 °C for about 10–15 min until the chromatography develops well. Observe and record the colour and position of the spots under white and ultraviolet light, respectively.
- Partition: Draw tangents at one point above and one point below the chromatographic origin for colour display. The area between the top and bottom tangents is the first zone. Using the same method, draw the fourth and seventh zones of the atranorin and norstictic acid stains, respectively. Then, draw a median line between the first and fourth zones, dividing them equally into the second and third zones; draw the fifth and sixth zones between the fourth and seventh zones using the same method.
2.5. DNA Extraction, PCR Amplification, and Sequencing
- Collect apothecia and thallus in microcentrifuge tubes and add beads for thorough grinding. Add 400 μL FGA buffer and 6 μL RNaseA (10 mg/mL), vortex for 1 min, and leave for 10 min at room temperature.
- Add 130 μL LP2 buffer, mix thoroughly and vortex for 1 min.
- Centrifuge at 12,000 rpm (~13,400× g) for 5 min and transfer 300 μL of supernatant to a new tube.
- Add 1.5 times the volume of buffer LP3 and immediately vortex for 15 s.
- Add a total of 750 μL of the solution obtained in the previous step and the flocculated precipitate to an adsorbent column CB3, centrifuge at 12,000 rpm (~13,400× g) for 30 s, pour off the waste solution and place the column CB3 in a collection tube.
- Add 600 μL of rinsing solution PW to column CB3, centrifuge at 12,000 rpm (~13,400× g) for 30 s, pour off the waste solution, and place column CB3 in the collection tube. Repeat this step twice.
- Place the column CB3 back into the collection tube and centrifuge at 12,000 rpm (~13,400× g) for 2 min, discarding the waste solution. Leave the column CB3 at room temperature for a few minutes to thoroughly dry the residual rinse from the adsorbent material.
- Transfer the adsorbent column CB3 to a clean centrifuge tube, add 90 μL of elution buffer TB dropwise to the centre of the adsorbent membrane in suspension, allow to stand at room temperature for 2–5 min, centrifuge at 12,000 rpm (~13,400× g) for 2 min, and collect the solution in the centrifuge tube. Aspirate the solution from the centrifuge tube, drop it back onto the centre of the adsorbent membrane, leave at room temperature for 2–5 min, centrifuge at 12,000 rpm (~13,400× g) for 2 min, and collect the solution in a centrifuge tube. This solution contains the extracted genomic DNA.
2.6. Alignment and Phylogenetic Tree Construction
3. Results
3.1. Phylogenetic Analysis
3.2. Taxonomy
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Species | GenBank Accession Numbers |
---|---|
Chaenothecopsis consociata (Nádv.) A.F.W. Schmidt | AY795851 |
Chaenothecopsis debilis (Sm.) Tibell | AY795853 |
Chaenothecopsis epithallina Tibell | AY795856 |
Chaenothecopsis fennica (Laurila) Tibell | AY795858 |
Chaenothecopsis golubkovae Tibell & Titov | AY795859 |
Chaenothecopsis haematopus Tibell | AY795861 |
Chaenothecopsis kilimanjaroensis Temu & Tibell | MN575660 |
Chaenothecopsis kilimanjaroensis Temu & Tibell | MN575662 |
Chaenothecopsis montana Rikkinen | JX119105 |
Chaenothecopsis pusilla (Ach.) A.F.W. Schmidt | AY795866 |
Chaenothecopsis pusiola (Ach.) Vain. | JX119106 |
Chaenothecopsis sitchensis Rikkinen | JX119102 |
Chaenothecopsis subparoica (Nyl.) Tibell | AY795869 |
Chaenothecopsis vainioana (Nádv.) Tibell | JX119107 |
Chaenothecopsis viridialba (Kremp.) A.F.W. Schmidt | JX000103 |
Chaenothecopsis viridireagens (Nádv.) A.F.W. Schmidt | JX119108 |
Mycocalicium albonigrum (Nyl.) Fink | AF223969 |
Mycocalicium americanum (R. Sant.) Tibell | AY795879 |
Mycocalicium hyaloparvicellulum Daranag. & K.D. Hyde | NR_168156 |
Mycocalicium subtile (Pers.) Szatala | AF225440 |
Phaeocalicium curtisii (Tuck.) Tibell | KT695401 |
Phaeocalicium populneum (Brond. ex Duby) A.F.W. Schmidt | AY795874 |
Phaeocalicium praecedens (Nyl.) A.F.W. Schmidt | KC590481 |
Sphinctrina anglica Nyl. | OQ718101 |
Sphinctrina leucopoda Nyl. | AY795875 |
Sphinctrina tubaeformis A. Massal. | OQ717614 |
Sphinctrina turbinata (Pers.) De Not. | OQ718102 |
Chaenothecopsis xishuiensis Z.F. Jia | MW017534 |
Talaromyces acaricola Visagie, N. Yilmaz & K. Jacobs | MW269298 |
Lecanora argentata (Ach.) Malme | MN387067 |
Lecanora cenisia Ach. | EU558541 |
Lecanora chlarotera Nyl. | MN654577 |
Lecanora conizaeoides Nyl. ex Cromb. | AF189717 |
Lecanora farinacea Fée | JN943726 |
Lecanora formosa (Bagl. & Carestia) Knoph & Leuckert | KT453771 |
Lecanora hybocarpa (Tuck.) Brodo | EF105412 |
Lecanora intricata (Ach.) Ach. | AF070022 |
Lecanora intumescens (Rebent.) Rabenh. | AY541254 |
Lecanora somervellii Paulson | MH512979 |
Lecanora praepostera Nyl. | MN586973 |
Lecanora pseudargentata Lumbsch | OQ832601 |
Lecanora pseudargentata Lumbsch | MH714514 |
Lecanora rugosella Zahlbr. | MK573926 |
Lecanora solaris L.S. Yakovchenko & E.A. Davydov | MH512982 |
Lecanora polytropa (Ehrh.) Rabenh. | MZ243614 |
Protoparmelia ochrococca (Nyl.) P.M. Jørg., Rambold & Hertel | KP822293 |
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Li, M.; Ju, Y.; Jia, Z. Chaenothecopsis xishuiensis sp. nov. to Science and Lecanora pseudargentata Newly Reported from China. Diversity 2023, 15, 893. https://doi.org/10.3390/d15080893
Li M, Ju Y, Jia Z. Chaenothecopsis xishuiensis sp. nov. to Science and Lecanora pseudargentata Newly Reported from China. Diversity. 2023; 15(8):893. https://doi.org/10.3390/d15080893
Chicago/Turabian StyleLi, Min, Yanhu Ju, and Zefeng Jia. 2023. "Chaenothecopsis xishuiensis sp. nov. to Science and Lecanora pseudargentata Newly Reported from China" Diversity 15, no. 8: 893. https://doi.org/10.3390/d15080893
APA StyleLi, M., Ju, Y., & Jia, Z. (2023). Chaenothecopsis xishuiensis sp. nov. to Science and Lecanora pseudargentata Newly Reported from China. Diversity, 15(8), 893. https://doi.org/10.3390/d15080893