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. 2022 Nov 23;17(11):e0277778. doi: 10.1371/journal.pone.0277778

A comparison of drying methods on the quality for bryophyte molecular specimens collected in the field

Fengjiao Shen 1,#, Lin Li 1,#, Dan Wang 1, Mengzhen Wang 1, James R Shevock 2, Jiancheng Zhao 1, Shuo Shi 1,*
Editor: Rosani do Carmo de Oliveira Arruda3
PMCID: PMC9683613  PMID: 36417395

Abstract

A major challenge in extracting high-quality DNA from bryophytes is the treatment of bryophyte material in the field. The existing and commonly used treatment methods in the field have several shortcomings. Natural drying methods can lead to DNA breaks. In addition, it is highly cumbersome to carry large quantities of silica gel in the field due to its weight and high risk of contamination among samples. In this study, we explored more convenient drying methods to treat bryophyte specimens and promote more efficient DNA recovery. The quantity and quality of genomic DNA extracted from every bryophyte species using different drying methods, including hot-air drying methods (150°C, 80°C, and 40°C), natural drying method, and silica gel drying method, were measured. Spectrophotometry, electrophoresis, and PCR amplification were performed to assess the effects of different drying methods. The results of total DNA purity, total DNA concentration, PCR success, and OD 260/230 ratios suggested that the hot-air drying (40–80°C) was the best method. The morphological comparison revealed that hot-air drying at 40°C and 80°C exerted no significant adverse effects on plant morphology and taxonomic studies. Thus, this method prevents rapid DNA degradation and silica gel pollution and saves the workforce from carrying large amounts of silica gel to the field. Several inexpensive devices, such as portable hairdryers, fan heaters, and electric blankets, are available that can be easily carried to the field for drying molecular specimens.

Introduction

The drying treatment methods of bryophyte specimens greatly influence the quality of DNA while collecting specimens in the field [1, 2]. Unlike seed plant collection, traditional treatment of the bryophyte specimens does not include immediate drying in the field. Bryophyte specimens have to be dried naturally for several days; however, the natural drying treatment can cause bryophyte DNA breaks [1, 2]. It is therefore always difficult to obtain pure DNA and a high percentage of PCR from the bryophyte specimens used for molecular experiments stored in herbaria or natural history museums. The ideal treatment method used for plant molecular specimens (especially for DNA extraction from plant leaves) is the low temperature (e.g., by –80°C refrigeration, –196°C liquid nitrogen, or dry ice). However, such methods are inconvenient to be implemented in the field [3–5]. Although NaCl/CTAB (Cetyltrimethylammonium Bromide) solution can be used for fieldwork [6], it is also not convenient to carry liquids in the field, especially for large collections. Doyle and Dickson [7] found that the dried molecular specimen could be used for DNA extraction. In addition, it could be easily stored and carried, suggesting drying as one of the most appropriate methods in the field.

Although there were some case studies showed the DNA degrades during the drying process [8, 9], different drying methods and techniques have been applied to several groups of organisms, such as plants, animals, and macrofungi, to obtain molecular data in the last 30 years. Some of these methods include the alcohol method [10], silica gel method [11], diatomite and sand burying method [12], and physical and chemical desiccation [4, 13]. At present, silica gel drying is mostly used for collecting molecular specimens in the field [12, 14–18]. However, the silica gel drying method has certain limitations. First, it is inconvenient to carry silica gel in large quantities to the field, especially in remote field collection areas. Second, failure to replace silica gel following water absorption results in incomplete drying of the specimen, adversely affecting the quality of a molecular specimen [6]. Moreover, the repeated replacement of water-absorbing silica gel to ensure samples dry quickly and completely will drastically increase the fieldwork time, especially when collecting in humid environments. Third, the recycling treatment can result in cross-contamination among specimens despite specific molecular specimen bags assigned to each molecular specimen (the bag is synthesized from a breathable non-woven fabric). In conclusion, the field specimen collection step using silica gel suffers from several shortcomings during the processing of a bryophyte molecular specimen.

Considering the above problems, the hot-air drying method has emerged as a method of choice for bryophyte molecular specimens in the field. The preliminary experiment on seed plants and macrofungi showed that the high-quality DNA could be efficiently obtained from the specimen subjected to hot-air drying [19–21]. Therefore, we studied the effects of different drying methods on the quality of extracted DNA and its suitability for PCR.

Another problem encountered during bryophyte molecular specimen collection is that bryophytes are small plants and several individuals of different species can grow together as mixed populations. This makes it difficult to separate different bryophyte species properly and timely in the field. It is difficult to ensure that the molecular specimens of all species are collected in the field, if a part of the specimen is taken as a molecular specimen, as is the case with angiosperms. However, this problem could be overcome if all individuals in a bryophyte specimen were treated as one molecular sample by the hot-air drying method. Species could be separated later in the laboratory for DNA extraction and other studies. Therefore, in this study, the bryophyte specimen quality was evaluated after treated by hot-air drying method and other methods.

Materials and methods

Materials

For the study, four species were selected as different types of bryophytes based on plant size, branching pattern, and leaf texture. These included three mosses, namely Campylopus schmidii (Müll. Hal.) A. Jaeger, Polytrichum commune Hedw., Hypnum calcicola Ando, and one liverwort, Marchantia polymorpha L. All four samples were kept fresh at the beginning of the experiment. The information of voucher specimens is shown in Table 1. The voucher specimens were deposited in the herbarium of the Hebei Normal University (HBNU).

Table 1. The information of voucher specimens.

Species Specimen No. Collectors Collection sites Families
Campylopus schmidii A. Jaeger SFJTX003 Shen et al. Yunnan, China Dicranaceae
Polytrichum commune Hedw. 20166223 Niu Guizhou, China Polytrichaceae
Hypnum calcicola Ando 201662225 Duan Guizhou, China Hypnaceae
Marchantia polymorpha L. SFJTX004 Shen Hebei, China Marchantiaceae
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Methods

Material processing

Five drying methods, namely the hot-air drying method (150°C, 80°C, and 40°C), silica gel drying method, and natural drying method, were compared to treat bryophyte molecular specimens. The specific methods were:

  • i. In order to ensure the stability of the experimental environment and equipment, the materials were dried in the laboratory in this experiment. Before entering the laboratory, we placed the material in sealed plastic bag with small holes to keep the samples living. When the bryophyte samples arrived at the laboratory, the soil on four fresh bryophyte samples were cleaned with water. And then, the water on the bryophyte surface was sucked up by the absorbent paper.

  • ii. The material of each specimen was divided into nine parts, and each part was placed in molecular specimen bags. Three parts were used for pre-experiment, and six parts were used for the formal experiment. The fresh weight of each part of the specimen was ≥200 mg.

  • iii. In the pre-experiment, the three parts materials were placed in an electric thermostatic drying oven (DHG-9240A, Zhongyiguoke Tech. Beijing, China) at 150°C, 80°C, and 40°C, respectively, until the weight of bryophytes reached a constant value. The device can control the temperature and function like an air blast. The rate of water loss was calculated, and the time required at each temperature was recorded.

  • iv. In the formal experiment, there are six parts materials for the experiment. These three parts were placed in the oven electric thermostatic drying at 150°C, 80°C, and 40°C, respectively (the drying time were the same as pre-experiment). The fourth part material was kept in a paper bag in a cool, well-ventilated place. The fifth part material was collected in a sealed plastic bag with excess dry silica gel. The sixth part material was contained in a sealed plastic bag and placed in a –80°C refrigerator. After all of the samples, except the sixth ones, were dry, the follow-up experiment was performed.

DNA extraction and quality examination

  • i. DNA extraction

    The experimental materials of five different drying methods, with 16 repeats and 5 mg of each, were weighed. A high-throughput tissue grinding mill (SCIENTZ-48, Ningbo Xinzhi Biotechnology Co., LTD, Ningbo, China) was used to quickly grind the specimen into powder. The mCTAB method was used to extract DNA [22].

  • ii. DNA examination

    The quality of DNA was assessed using a micro-spectrophotometer (NanoDrop 2000) and agarose gel electrophoresis. Because the absorption at 230 nm can be caused by small organic compounds, we recorded the OD 260/230 ratio. The DNA concentration was used to assess the purity and amount of DNA obtained. The fragment size, degradation, and concentration of DNA were checked using 1% agarose gel electrophoresis. ImageJ (v1.4.3.67) software was used to conduct quantitative analysis on high-molecular weight genomic DNA (which is close to gel hole position in the gel image) in the total DNA agarose gel electrophoretogram results.

    STATISTICA (v10.0.228.8) was used to conduct statistical analysis of the data obtained from micro-spectrophotometer spectrophotometry (OD 260/230 ratio, total DNA concentration) and agarose gel electrophoretogram (the concentration of high-molecular weight genomic DNA). The results were analyzed using the letter-marking multiple comparison method [23] (S1 and S2 Tables).

  • iii. DNA quality examination by PCR

    To a certain extent, the quality of DNA can be reflected by the success rate of PCR amplification. The presence of an increased number of high-molecular weight genomic DNA is associated with a high amplification success rate. The PCR primers ITS-P5 “5’–3’, CCTTATCAYTTAGAGGAAGGAG” and ITS-U4 “5’–3’, RGTTTCTTTTCCTCCGCTTA” were used, which are designed for plants. The PCR amplification procedure reference to Cheng et al. [24], and the annealing temperature is 55°C. PCR products were checked using 1% agarose gel electrophoresis. The success rate of PCR was calculated to evaluate the quality of molecular samples.

Comparison of morphological characteristics

Both macroscopic and microscopic morphological characteristics are the important evidence for bryophyte species identification. It is unknown if the treatments used in this study affected the morphological identification of the specimen. Therefore, the overall plant morphology, leaf characteristics, and leaf transverse section characteristics of a single specimen after different drying methods were compared with those of the traditional naturally dried samples. If these characteristics were consistent, it indicated that different drying methods did not affect the morphological identification of the specimens.

Results

DNA quality analysis

Examination of DNA purity

The OD 260/230 ratios of four bryophytes were compared (Fig 1; S1 Table).

Fig 1. The extracted DNA purity after different drying methods (OD 260/230).

Fig 1

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Except for the fresh freezing control group, consisting of P. commune and H. calcicola, the DNA OD 260/230 ratios obtained after the hot-air drying method at 80°C were the highest. The DNA OD 260/230 ratios obtained after the silica gel drying method were the lowest. For M. polymorpha, the DNA OD 260/230 ratios obtained following the hot-air drying method at 80°C and 40°C and the natural drying method were the highest. For C. schmidii, DNA OD 260/230 ratios obtained after different drying methods showed no significant difference (p > 0.05).

Examination of DNA concentration

The DNA concentrations of four bryophytes examined by a microspectrophotometer (total DNA concentration, Fig 2) and agarose gel electrophoretogram (high-molecular weight genomic DNA) were compared (Fig 3, S1–S4 Figs).

Fig 2. The concentration of extracted total DNA after different drying methods was detected by a microspectrophotometer.

Fig 2

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Fig 3. The concentration of extracted high-molecular weight genomic DNA by agarose gel electrophoretogram after different drying methods.

Fig 3

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For P. commune, except for the fresh freezing control group, the total DNA concentration obtained after hot-air drying at 80°C was the highest. For H. calcicola, the total DNA concentration obtained after hot-air drying at 40°C, 80°C, natural drying, and silica gel drying resulted in an insignificant difference (p > 0.05). The total DNA concentrations obtained after these methods were higher than that obtained with hot-air drying at 150°C. For C. schmidii, the total DNA concentration obtained after different drying methods had no significant difference (p > 0.05). For M. polymorpha, the total DNA concentration of the five treatments had no significant difference (p > 0.05).

Electrophoresis results for high-molecular weight genomic DNA showed that for P. commune, except the fresh freezing control group, the concentration of high-molecular weight genomic DNA obtained after hot-air drying at 80°C was the highest. For C. schmidii, the concentration of high-molecular weight genomic DNA obtained after hot-air drying at 40°C was the highest. For M. polymorpha, only the concentration of high-molecular weight genomic DNA obtained after 150°C is the lowest. The concentration of high-molecular weight genomic DNA of H. calcicola received after different drying methods had no significant difference (p > 0.05) (Fig 3).

PCR amplification products

The PCR amplification for samples of five different methods and the control group was conducted (S5–S8 Figs). The statistics were obtained for assessing the success rate of PCR amplification, and the results are shown in Fig 4. For the four bryophytes, there was no statistically significant difference in PCR success rate of different drying methods. However, the amplification rate of the four samples after hot-air drying at 80°C and 40°C was higher. The amplification rate of the four samples after hot-air drying at 80°C was 100%, and the amplification rate of the three samples after hot-air drying at 40°C was 100%. The success rate of PCR amplification was slightly lower after hot-air drying at 40°C; however, it was higher than that obtained from other methods.

Fig 4. Success rates of PCR amplification.

Fig 4

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Morphological comparison before and after drying

The overall plant morphology, leaf characteristics, and leaf transverse section characteristics (S9–S11 Figs) of four samples before and after different drying treatments were compared. Hot-drying at 40°C and 80°C, silica gel, and natural drying treatments resulted in insignificant differences in the morphology characteristics, especially regarding the major characteristics of identification, such as plant color, leaf morphology, the shape of the cell, and transverse section characteristics of molecular specimens. The only exception was the material dried at 150°C, which was darker in color. It can be concluded that hot drying at 40°C and 80°C, silica gel, and natural drying treatments do not affect the identification of bryophytes.

Discussion

The results showed that the effect of the hot-air drying method at 40°C and 80°C was better than that of the silica gel drying method and natural drying method. In addition, the silica gel drying method was inconvenient to perform in the field, and the natural drying method was highly affected by environmental humidity. Therefore, 40°C to 80°C hot-air drying method for specimen drying is recommended in the field to avoid rapid degradation of DNA. Moreover, this method does not damage the characteristics of the traditional morphological study. The field collection can be created in different ways, such as an electric blanket, hairdryer, or a portable fan heater. In this study, three temperatures were selected to form a temperature gradient. Among these, 150°C, 80°C, and 40°C were simulation temperatures corresponding to different distances from the vent of the portable fan heater.

In addition to drying methods, plants should begin the drying process as soon as possible after the field collection event. The best practice is to begin the drying process on the day of collection. The following points should be noted during the specific implementation of the operation: 1) Specimens stored in open packets can offer more airflow, especially if the packets are placed upright to aid drying. 2) If the sample is wet, for example, collected from water, it should be carefully processed during hot drying to keep a lower temperature. The samples should be squeezed first to release most of the excess water. 3) Because high temperature and humidity can damage the molecular material, large quantities of materials should not be placed in the same sample bag or packet.

Interestingly, the results of our study resemble those obtained for angiosperms [20] and mushrooms [21]. In these two studies, temperatures used for molecular specimen drying were 40°C and 70°C, respectively. And cut into pieces should be needed to reduce the drying time and improve the molecular specimen quality. At present, lichen specimens, similar to bryophyte specimens, are dried primarily by the natural drying method. It is challenging to extract DNA from lichen specimens, which dried naturally, after being collected for two years [25]. Moreover, there exist several other limitations in treating animal molecular specimens [26]. The results of the drying method in this study and the above research provide a reference for the treatment of other organism DNA specimens, e.g., algae, lichen, and animals.

Conclusions

It is demonstrated in this study that the hot-air drying (40–80°C) offered the best results for drying bryophyte molecular specimens as soon possible after a collecting event. This method causes little damage to the DNA in bryophyte samples and is also convenient to operate. It is recommended that this method be used in the future for drying bryophyte specimens in the field.

Supporting information

S1 Table. Comparison of OD 260/230 values of the four bryophytes’ DNA after different drying treatments.

150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying; F, fresh sample; a,b,c,d The superscript of same letters indicate that there is no statistically significant difference (P>0.05), the superscript of different letters indicate that there is a statistically significant difference (P<0.05).

(DOCX)

Click here for additional data file. (13.7KB, docx)
S2 Table. Comparisons of extract DNA concentrations of the four bryophytes after different drying treatments.

150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying; F, fresh sample; DNA-N means total DNA concentration form Nanodrop 2000 micro-spectrophotometer and DNA-G means long fragment DNA from agarose gel electrophoretogram. a,b,c,d The superscripts of the same letters indicate that there is no statistically significant difference (P>0.05). The superscripts of the different letters indicate that there is a statistically significant difference (P<0.05) −1.

(DOCX)

Click here for additional data file. (14.7KB, docx)
S1 Fig. Agarose gel electrophoresis of Genomic DNA of C. schmidii obtained from different drying methods.

Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

(TIF)

Click here for additional data file. (2.1MB, tif)
S2 Fig. Agarose gel electrophoresis of Genomic DNA of P. commune obtained from different drying methods.

Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

(TIF)

Click here for additional data file. (2.3MB, tif)
S3 Fig. Agarose gel electrophoresis of Genomic DNA of H. calcicola obtained from different drying methods.

Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

(TIF)

Click here for additional data file. (2.2MB, tif)
S4 Fig. Agarose gel electrophoresis of Genomic DNA of M. polymorpha obtained from different drying methods.

Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

(TIF)

Click here for additional data file. (1.8MB, tif)
S5 Fig. Agarose gel electrophoresis of PCR products of C. schmidii obtained from different drying methods.

Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

(TIF)

Click here for additional data file. (2.2MB, tif)
S6 Fig. Agarose gel electrophoresis of PCR products of P. commune obtained from different drying methods.

Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

(TIF)

Click here for additional data file. (4.5MB, tif)
S7 Fig. Agarose gel electrophoresis of PCR products of H.calcicola obtained from different drying methods.

Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

(TIF)

Click here for additional data file. (1.5MB, tif)
S8 Fig. Agarose gel electrophoresis of PCR products of M. polymorpha obtained from different drying methods.

Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

(TIF)

Click here for additional data file. (3.3MB, tif)
S9 Fig. The morphological characters of overall plants.

Note, C, C. schmidii; P, P. commune; H, H. calcicola; M, M. polymorpha; 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying. Bar scales C/P/H = 1 mm; M = 1 cm.

(TIFF)

Click here for additional data file. (19.7MB, tiff)
S10 Fig. The morphological characters of leaves.

Note, C, C. schmidii; P, P. commune; H, H. calcicola; M, M. polymorpha; 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying. Bar scales C/P = 0.5 mm; H = 0.1 mm; M = 1 mm.

(TIFF)

Click here for additional data file. (14MB, tiff)
S11 Fig. The morphological characters of transverse sections.

Note, C, C. schmidii; P, P. commune; H, H. calcicola; M, M. polymorpha; 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying. Bar scales = 50 μm.

(TIFF)

Click here for additional data file. (14.6MB, tiff)
S1 Raw images

(PDF)

Click here for additional data file. (20MB, pdf)

Acknowledgments

Thanks to Dr. Jing-Yuan Niu and Dr. Xu-Hong Duan for collecting the specimens; Dr. Hai-Yan Liu, for assistance with statistical analysis; Dr. Shi-Liang Zhou, for his helpful advice and revision of the draft. We thank TopEdit (www.topeditsci.com) for linguistic assistance during the preparation of this manuscript.

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

The authors report the following sources of funding: National Natural Science Foundation of China [31370237 and 31370184], Youth Foundation of Education Department of Hebei Province, No. QN2017342, http://jyt.hebei.gov.cn/, awarded to SS, Natural Science Foundation of Hebei Province under Grant, No. C2019205175, https://kjt.hebei.gov.cn/jjb/, awarded to LL, Innovation Fund Project for Graduate Student of Hebei Province, No. CXZZBS2019088, http://jyt.hebei.gov.cn/, awarded to FS, and Innovation Funding Program for Graduate Students of Hebei Normal University, No. CXZZSS2021066, http://www.hebtu.edu.cn/. awarded to FS. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Rosani do Carmo de Oliveira Arruda

29 Aug 2022

PONE-D-22-14636A comparison of drying methods on the quality for bryophyte molecular specimens collected in the fieldPLOS ONE

Dear Dr. Shuo Shi

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Additional Editor Comments:

REVIEWER 1.

Title: 1. The title of manuscript is little inappropriate and incomplete.

Introduction

The text should be more inclined towards the problems associated with the sampling and DNA isolation from bryophytes species. What are the methods traditionally used for the collection of bryophytes sample? Why different treatment is required and what are their drawbacks?

Materials and Methods

1. Line 76-77: What is the location or collection site of the studied bryophytes specimen?

2. Line 79-80: All four samples were kept fresh at the beginning of the experiment…??? Means stored in -80oC

3. Line 86: The collected bryophytes samples from field contains mud, dirt and microscopic organisms which may cause undesirable PCR amplification. What are the steps taken to clean the collected samples?

4. Line 94-100: Some parts should be reconsidered and rewritten. Experiment design is not clear from the text. From the text it appears that samples are dried in oven at 150°C, 80°C, and 40°C, respectively and then kept in a paper bag in a cool, well-ventilated place, second in a sealed plastic bag with excess silica gel, and third in a sealed plastic bag in a –80°C refrigerator and then again dried at 150°C, 80°C, and 40°C. That is bit confusing.

5. Line 121: Please specify the sequence and Tm value of PCR primers ITS-P5 and ITS-U4 used in present study.

6. Line 124-127: Please specify the method used for evaluation of effect of different drying method on the morphological characteristics of bryophytes.

7. What concentration of gel is used for the gel electrophoresis (DNA isolation and PCR products)??

Results:

1. Line 131: The ratio of the spectrophotometric absorbance of sample at 260/280 and 260/230 are generally used to determine the purity of nucleic acid contaminated by phenol or proteins and organic acids respectively. The present study includes DNA purity result based on only the 260/230 ratio.

2. S1 Table: Title should be completed, must be well understandable without the text. What is the difference between DNA purity and DNA concentration (Table 1& 2)? Please provide details of abbreviations (C, P, H, M, N, S, F) in the footnote of table.

3. Figure (1-9): Legends should be completed and well understandable without text.

4. Line 133-138 (Table 1): Spectrophotometer, nanodrops and gel electrophoretogram are used to determine the concentration of DNA. The extracted DNA having a ratio of absorbance A260/280- 1.8 and A260/230- 2.0-2.2 are considered as pure. The A260/230 values of three samples are less than 2.0. The contamination of organic acids might be present in sample. Please provide the justification of statement in your manuscript

5. Instead of long fragment DNA author can use “high-molecular weight genomic DNA”.

6. Provide details (name, collection site, specimen no. family, etc.,)in tabular form about the bryophytes specimens used in present study.

Discussion

Please cite the literature associated with problems arises during collection storage and DNA isolation from bryophyte specimens and how this study provides solutions to those problems. Please give conclusion and future prospects of your study.

REVIEWER 2.

The manuscript brings a simple but important technical advance in the area of molecular studies of bryophytes. Considering the findings presented here, it is possible to carry out the field collection more easily and, at the same time, guarantee the obtaining of quality DNA samples for molecular investigations.

Some minor revisions are necessary:

1- Page 5, line 102: write only "DNA extraction"

2- Page 8, lines 147 to 152: for H. calcicola, the results for hot-air drying at 40°C was not commented. The results obtained for M. polymorpha was not commented. Need to reference that the paragraph is the analysis of Fig. 2.

3- Page 8, lines 153 to 155: consider reviewing the comparison between the results for the fresh freezing control group and hot-air drying at 80°C for M. polymorpha; in table S2 it is clear that, for this species, the only treatment that showed a significant difference (p > 0.05) was drying with hot air at 150°C.

4- Page 8, lines 161 to 163: consider reviewing the comparison of the success rate of PCR amplification after hot-air drying at 80°C and the other treatments; in Fig. 4 (and supplementary figures as well) it is not possible to detect a difference between this treatment and others in some cases.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Title:

1. The title of manuscript is little inappropriate and incomplete.

Introduction

The text should be more inclined towards the problems associated with the sampling and DNA isolation from bryophytes species. What are the methods traditionally used for the collection of bryophytes sample? Why different treatment is required and what are their drawbacks?

Materials and Methods

1. Line 76-77: What is the location or collection site of the studied bryophytes specimen?

2. Line 79-80: All four samples were kept fresh at the beginning of the experiment…??? Means stored in -80oC

3. Line 86: The collected bryophytes samples from field contains mud, dirt and microscopic organisms which may cause undesirable PCR amplification. What are the steps taken to clean the collected samples?

4. Line 94-100: Some parts should be reconsidered and rewritten. Experiment design is not clear from the text. From the text it appears that samples are dried in oven at 150°C, 80°C, and 40°C, respectively and then kept in a paper bag in a cool, well-ventilated place, second in a sealed plastic bag with excess silica gel, and third in a sealed plastic bag in a –80°C refrigerator and then again dried at 150°C, 80°C, and 40°C. That is bit confusing.

5. Line 121: Please specify the sequence and Tm value of PCR primers ITS-P5 and ITS-U4 used in present study.

6. Line 124-127: Please specify the method used for evaluation of effect of different drying method on the morphological characteristics of bryophytes.

7. What concentration of gel is used for the gel electrophoresis (DNA isolation and PCR products)??

Results:

1. Line 131: The ratio of the spectrophotometric absorbance of sample at 260/280 and 260/230 are generally used to determine the purity of nucleic acid contaminated by phenol or proteins and organic acids respectively. The present study includes DNA purity result based on only the 260/230 ratio.

2. S1 Table: Title should be completed, must be well understandable without the text. What is the difference between DNA purity and DNA concentration (Table 1& 2)? Please provide details of abbreviations (C, P, H, M, N, S, F) in the footnote of table.

3. Figure (1-9): Legends should be completed and well understandable without text.

4. Line 133-138 (Table 1): Spectrophotometer, nanodrops and gel electrophoretogram are used to determine the concentration of DNA. The extracted DNA having a ratio of absorbance A260/280- 1.8 and A260/230- 2.0-2.2 are considered as pure. The A260/230 values of three samples are less than 2.0. The contamination of organic acids might be present in sample. Please provide the justification of statement in your manuscript

5. Instead of long fragment DNA author can use “high-molecular weight genomic DNA”.

6. Provide details (name, collection site, specimen no. family, etc.,)in tabular form about the bryophytes specimens used in present study.

Discussion

Please cite the literature associated with problems arises during collection storage and DNA isolation from bryophyte specimens and how this study provides solutions to those problems. Please give conclusion and future prospects of your study.

Reviewer #2: The manuscript brings a simple but important technical advance in the area of molecular studies of bryophytes. Considering the findings presented here, it is possible to carry out the field collection more easily and, at the same time, guarantee the obtaining of quality DNA samples for molecular investigations.

Some minor revisions are necessary:

1- Page 5, line 102: write only "DNA extraction"

2- Page 8, lines 147 to 152: for H. calcicola, the results for hot-air drying at 40°C was not commented. The results obtained for M. polymorpha was not commented. Need to reference that the paragraph is the analysis of Fig. 2.

3- Page 8, lines 153 to 155: consider reviewing the comparison between the results for the fresh freezing control group and hot-air drying at 80°C for M. polymorpha; in table S2 it is clear that, for this species, the only treatment that showed a significant difference (p > 0.05) was drying with hot air at 150°C.

4- Page 8, lines 161 to 163: consider reviewing the comparison of the success rate of PCR amplification after hot-air drying at 80°C and the other treatments; in Fig. 4 (and supplementary figures as well) it is not possible to detect a difference between this treatment and others in some cases.

**********

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2022 Nov 23;17(11):e0277778. doi: 10.1371/journal.pone.0277778.r002

Author response to Decision Letter 0

14 Oct 2022

Dear Editor,

Re: Manuscript ID: PONE-D-22-14636 and Title: A comparison of drying methods on the quality for bryophyte molecular specimens collected in the field.

Thank you for your letter and the reviewers’ comments concerning our manuscript. Those comments are valuable and very helpful. We have read through the comments carefully and have made corrections. Based on the instructions provided in your letter, we will upload the files named “Response to Reviewers”, “Revised Manuscript with Track Changes”, and “Manuscript”. In the “Revised Manuscript with Track Changes”, we have modified the original text in revision mode. The responses to the reviewer’s comments are marked in red and presented in the “Response to Reviewers”. We have revised the article format according to the Journal Requirements.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The ORCID iD of the corresponding author, Shuo Shi, is https://orcid.org/0000-0001-8428-7261. The original uncropped and unadjusted gel results images were reported in the original gel files, original gel S1-8.pdf (according to the S1-8 Figs in the file Supporting Information).

REVIEWER 1.

Title: 1. The title of manuscript is little inappropriate and incomplete.

Introduction

The text should be more inclined towards the problems associated with the sampling and DNA isolation from bryophytes species. What are the methods traditionally used for the collection of bryophytes sample? Why different treatment is required and what are their drawbacks?

Re:

The questions raised by Reviewer1 are critical. The traditional method used for bryophyte collection in the field is natural drying treatment. However, the silica gel drying method is used primarily for collecting bryophyte molecular specimens in the field. Compared to the molecular speciments dried with silica gel in the field, it is more difficult to isolate pure DNA and PCR amplification from the herbaria specimens which dried by natural drying treatment. In the long-term experimental practice, we found that treatment methods in the field had a significant impact on the molecular specimens. Therefore, this study aimed to explore the effects of different drying methods on bryophyte specimens in the field. We have supplemented it in the preface according to your suggestions.

Materials and Methods

1. Line 76-77: What is the location or collection site of the studied bryophytes specimen?

Re:

According to your suggestion, the name, collection site, specimen no., and family of the studied bryophytes specimen have been added to the manuscript (Table 1).

2. Line 79-80: All four samples were kept fresh at the beginning of the experiment…??? Means stored in -80℃

Re:

We are so sorry for misleading your understanding because our expression is not accurate enough. In order to ensure the stability of the experimental environment and equipment, the materials were dried in the laboratory in this experiment. Before entering the laboratory, the specimens were placed in sealed plastic bags with small holes to keep the samples living. In lines 79-80, the fresh material means the living bryophyte collected from the field. We have added the sentences to “Material processing-i” of the manuscript.

3. Line 86: The collected bryophytes samples from field contains mud, dirt and microscopic organisms which may cause undesirable PCR amplification. What are the steps taken to clean the collected samples?

Re:

We are sorry for the unclear description of the collected samples cleaning method. Bryophyte cleaning steps are as follows: 1) The bryophyte was moved from the substrate, and the soil and gravel were shaken out; 2) Bryophyte specimens were rinsed in the pure water, and the sand attached in the plant were removed by tweezers. Repeat this step 2-3 times; 3) Use absorbent paper to blot the water. In the manuscript, we have added some sentences to “Material processing-i”.

This method could flush out most microscopic organisms, but not all of them. In the subsequent PCR, we used a plant-specific DNA barcode by PCR primers ITS-P5 and ITS-U4 [24]. The microorganisms could hardly be amplified by this pair of primers. To make the article more understandable, we have supplemented the primers’ peculiarities as follows:

The PCR primers ITS-P5 “5’–3’, CCTTATCAYTTAGAGGAAGGAG” and ITS-U4 “5’–3’, RGTTTCTTTTCCTCCGCTTA” were used, which are designed for plants. The PCR amplification procedure reference to Cheng et al [24], and the annealing temperature is 55°C.

4. Line 94-100: Some parts should be reconsidered and rewritten. Experiment design is not clear from the text. From the text it appears that samples are dried in oven at 150°C, 80°C, and 40°C, respectively and then kept in a paper bag in a cool, well-ventilated place, second in a sealed plastic bag with excess silica gel, and third in a sealed plastic bag in a –80°C refrigerator and then again dried at 150°C, 80°C, and 40°C. That is bit confusing.

Re:

We are so sorry for misleading your understanding. According to your suggestion, we have rewritten “Line 94-100” as follows:

iv. In the formal experiment, there are six parts materials for the experiment. These three parts were placed in the oven electric thermostatic drying at 150°C, 80°C, and 40°C, respectively (the drying time were the same as pre-experiment). The fourth part material was kept in a paper bag in a cool, well-ventilated place. The fifth part material was collected in a sealed plastic bag with excess dry silica gel. The sixth part material was contained in a sealed plastic bag and placed in a –80°C refrigerator. After all of the samples, except the sixth ones, were dry, the follow-up experiment was performed.

5. Line 121: Please specify the sequence and Tm value of PCR primers ITS-P5 and ITS-U4 used in present study.

Re: The Tm value of primers is the theoretical value calculated according to the sequence of primers. However, in an experiment, it is often necessary to explore the anneal temperature through the Tm value of primers. We already had the proper anneal temperature of this pair of primes for bryophytes from the preliminary experiments. According to your suggestion, we supplemented the sequence and the anneal temperature in the manuscript. As follow: “The PCR amplification procedure reference to Cheng et al. [24], and the annealing temperature is 55°C.”

6. Line 124-127: Please specify the method used for evaluation of effect of different drying method on the morphological characteristics of bryophytes.

Re:

Thanks for your suggestion. Key characters used to identify taxa (families, genera, species) and the characteristics commonly used for bryophyte morphological description were compared among the results of different treatments. The character states of traditional naturally dried specimens, were observed and compared with the ones after hot-air and silica gel dried treatments. We have rewritten it as follows:

Both macroscopic and microscopic morphological characteristics are the important evidence for bryophyte species identification. It is unknown if the treatments used in this study affected the morphological identification of the specimen. Therefore, the overall plant morphology, leaf characteristics, and leaf transverse section characteristics of a single specimen after different drying methods were compared with those of the traditional naturally dried samples. If these characteristics were consistent, it indicated that different drying methods did not affect the morphological identification of the specimens.

7. What concentration of gel is used for the gel electrophoresis (DNA isolation and PCR products)??

Re:

The concentration of agarose gel of electrophoresis is 1%. We have added it to the manuscript.

Results:

1. Line 131: The ratio of the spectrophotometric absorbance of sample at 260/280 and 260/230 are generally used to determine the purity of nucleic acid contaminated by phenol or proteins and organic acids respectively. The present study includes DNA purity result based on only the 260/230 ratio.

Re: As the reviewer said, the ratio of the spectrophotometric absorbance of the sample at 260/280 and 260/230 are generally used to determine the purity of nucleic acid. For the 260/280 ratio, a value of ~1.8 is generally accepted as “pure” for DNA and a ratio of ~2.0 is generally accepted as “pure” for RNA. A 260/280 ratio lower than 1.8 indicates significant protein contamination. Protein contamination is usually due to inadequate DNA extraction or unstable operation. It cannot be used to assess the quality of experimental materials. Pure nucleic acid solutions typically have 260/230 values in the range of 2.0–2.2. If the ratio is lower, it may indicate the presence of contaminants such as EDTA, carbohy-drates, and phenolic compounds, all of which absorb at 230 nm. In the drying process, these compounds may be degraded and complexed with DNA, resulting in a low value of 260/230. Thus, the present study manuscript includes DNA purity results based on only the 260/230 ratio.

2. S1 Table: Title should be completed, must be well understandable without the text. What is the difference between DNA purity and DNA concentration (Table 1& 2)? Please provide details of abbreviations (C, P, H, M, N, S, F) in the footnote of table.

Re:

Thanks for your advice. Purity is to detect the contamination of other small molecules in the nucleic acid solution. Concentration is the total amount of nucleic acid in the detection solution. In order to make it easier for readers to understand, we have revised it according to your suggestions:

We have rewritten the title of S1 Table and S2 Table, and add the abbreviations (C, P, H, M, N, S, F) in the table’s footnote. The title of S1 Table was changed to “Comparison of OD 260/230 values of the four bryophytes’ DNA after different drying treatments”. The title of S2 Table was changed to “Comparisons of extract DNA concentrations of the four bryophytes after different drying treatments”.

3. Figure (1-9): Legends should be completed and well understandable without text.

Re:

According to your suggestion, we have rewritten the legends of the figures.

4. Line 133-138 (Table 1): Spectrophotometer, nanodrops and gel electrophoretogram are used to determine the concentration of DNA. The extracted DNA having a ratio of absorbance A260/280- 1.8 and A260/230- 2.0-2.2 are considered as pure. The A260/230 values of three samples are less than 2.0. The contamination of organic acids might be present in sample. Please provide the justification of statement in your manuscript

Re:

To ensure the consistency of the experimental methods, we used mCTAB (Li et al. 2013) method to extract DNA from the four samples. Due to the significant differences in the systematic position of the four samples, the plant morphology and chemical composition were also different. For example, Marchantia polymorpha belongs to Marchantiophyta, and the plant morphology is thallus. In contrast, the other three species belong to Bryophyta, and their morphology are cormus. There are different optimal DNA extraction protocols for different samples. But we tried to use one of them to ensure experiment processing consistency. In the practical application of a large number of species in the future, it is also necessary to use one method to extract DNA for most species. Perhaps the method we used was not optimal for extracting the three mosses’ DNA. This is the reason of A260/230 values of the three samples are less than 2.0. However, the purpose of this study is not to compare different methods of DNA extraction, but to compare different effects of treatment methods for the moss specimens. For this study, the consistent extraction operation scheme can ensure the comparison of the results obtained.

5. Instead of long fragment DNA author can use “high-molecular weight genomic DNA”.

Re:

Excellent advice! We have use “high-molecular weight genomic DNA” instead of “long fragment DNA”.

6. Provide details (name, collection site, specimen no. family, etc.,)in tabular form about the bryophytes specimens used in present study.

Re: According to your suggestion, the name, collection site, specimen no., and family of the studied bryophytes specimen have been added to the manuscript. (Table 1) And as the revision for your first question of methods and materials.

Discussion

Please cite the literature associated with problems arises during collection storage and DNA isolation from bryophyte specimens and how this study provides solutions to those problems. Please give conclusion and future prospects of your study.

Re:

This study aimed to explore the effects of different drying methods on bryophyte specimens, especially in the field. The long-time preserving methods and DNA extraction methods comparations are not the questions of this study, although we compared the DNA extraction methods before deciding which one to use.

We have added the conclusion following your suggestion, which is as follows:

It is demonstrated in this study that the hot-air drying (40–80°C) was the best method for drying bryophyte molecular specimens in the field. This method causes little damage to the DNA in bryophyte samples and is also convenient to operate. It is recommended that this method be used in the future for drying bryophyte specimens in the field.

Regarding prospects, we mentioned in the last paragraph of the discussion that the method could provide a reference scheme for animals, bacteria, lichens, algae, etc.

Thank you for your comments concerning our manuscript. Those comments are valuable and very helpful.

REVIEWER 2.

The manuscript brings a simple but important technical advance in the area of molecular studies of bryophytes. Considering the findings presented here, it is possible to carry out the field collection more easily and, at the same time, guarantee the obtaining of quality DNA samples for molecular investigations.

Re:

Thanks for your suggestions. We have answered your questions one by one, as follows

Some minor revisions are necessary:

1- Page 5, line 102: write only “DNA extraction”

Re: Thanks for your advice. We have changed it to “DNA extraction”.

2- Page 8, lines 147 to 152: for H. calcicola, the results for hot-air drying at 40°C was not commented. The results obtained for M. polymorpha was not commented. Need to reference that the paragraph is the analysis of Fig. 2.

Re: According to your suggestion, the results for hot-air drying at 40°C for H. calcicola and the results of M. polymorpha have been supplemented to the manuscript as follows:

For H. calcicola, the total DNA concentration obtained after hot-air drying at 40°C, 80°C, natural drying, and silica gel drying resulted in an insignificant difference (p > 0.05).

For M. polymorpha, the total DNA concentration of the five treatments had no significant difference (p > 0.05).

3- Page 8, lines 153 to 155: consider reviewing the comparison between the results for the fresh freezing control group and hot-air drying at 80°C for M. polymorpha; in table S2 it is clear that, for this species, the only treatment that showed a significant difference (p > 0.05) was drying with hotair at 150°C.

Re: According to your suggestion, we have changed the results for M. polymorpha to “For M. polymorpha, only the concentration of high-molecular weight genomic DNA obtained after 150°C is the lowest.”

4- Page 8, lines 161 to 163: consider reviewing the comparison of the success rate of PCR amplification after hot-air drying at 80°C and the other treatments; in Fig. 4 (and supplementary figures as well) it is not possible to detect a difference between this treatment and others in some cases.

Re：

Thank you for your advice. In Figure 4, there are two reasons for the little difference in amplification efficiency of the concentrated drying methods:

1) As mentioned in the discussion, the natural drying method is greatly affected by the environmental humidity, and the silica gel drying is greatly affected by the replacement frequency of operation. At the experiment’s time, the laboratory’s ambient humidity was only about 20-40%. So the specimens of the natural drying method could be dried more quickly than the spencimens in the field with high humidity. When using silica gel for drying, the frequency of material replacement in this experiment is higher than in the field of mass operation. So the natural drying rate and silica gel drying rate in this experiment are higher than in the field collection.

2) In this study, we performed PCR amplification with plant-specific primers of DNA barcode, ITS. Compared with other methods that are difficult to amplify, the advantages of this method are simple operation and high amplification efficiency.

In conclusion, although there was no significant difference in the PCR success rate of different drying methods, the DNA with a low PCR rate had been significantly damaged. Although different drying methods seem to have little difference in effect, they can reflect the good quality of DNA from the 40-80°C hot-air drying method. We have revised the article as follows based on your suggestions:

The PCR amplification for samples of five different methods and the control group was conducted (S5–S8 Figs). The statistics were obtained for assessing the success rate of PCR amplification, and the results are shown in Fig 4. For the four bryophytes, there was no statistically significant difference in PCR success rate of different drying methods. However, the amplification rate of the four samples after hot-air drying at 80°C and 40°C was higher. The amplification rate of the four samples after hot-air drying at 80°C was 100%, and the amplification rate of the three samples after hot-air drying at 40°C was 100%. The success rate of PCR amplification was slightly lower after hot-air drying at 40°C; however, it was higher than that obtained from other methods.

Your comments are valuable and very helpful to us. Thank you!

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Decision Letter 1

Rosani do Carmo de Oliveira Arruda

3 Nov 2022

A comparison of drying methods on the quality for bryophyte molecular specimens collected in the field

PONE-D-22-14636R1

Dear Dr. Shi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

The manuscript contains information relevant to the study of DNA in Bryophytes and will certainly have a positive impact on research on this topic. All suggestions made by the reviewers of the manuscript were accepted and carried out by the authors.All queries were kindly explained and resolved. The methodological issues raised were resolved and also answered by the authors. Thus, I consider that the answers were sufficient, and that the article is ready to be accepted for publication.

Reviewers' comments:

The manuscript contains information relevant to the study of DNA in Bryophytes and will certainly have a positive impact on research on this topic. All suggestions made by the reviewers of the manuscript were accepted and carried out by the authors.All queries were kindly explained and resolved. The methodological issues raised were resolved and also answered by the authors. Thus, I consider that the answers were sufficient, and that the article is ready to be accepted for publication.

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Acceptance letter

Rosani do Carmo de Oliveira Arruda

15 Nov 2022

PONE-D-22-14636R1

A comparison of drying methods on the quality for bryophyte molecular specimens collected in the field

Dear Dr. Shi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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PLOS ONE Editorial Office Staff

on behalf of

Dr. Rosani do Carmo de Oliveira Arruda

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Comparison of OD 260/230 values of the four bryophytes’ DNA after different drying treatments.

    150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying; F, fresh sample; a,b,c,d The superscript of same letters indicate that there is no statistically significant difference (P>0.05), the superscript of different letters indicate that there is a statistically significant difference (P<0.05).

    (DOCX)

    Click here for additional data file. (13.7KB, docx)
    S2 Table. Comparisons of extract DNA concentrations of the four bryophytes after different drying treatments.

    150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying; F, fresh sample; DNA-N means total DNA concentration form Nanodrop 2000 micro-spectrophotometer and DNA-G means long fragment DNA from agarose gel electrophoretogram. a,b,c,d The superscripts of the same letters indicate that there is no statistically significant difference (P>0.05). The superscripts of the different letters indicate that there is a statistically significant difference (P<0.05) −1.

    (DOCX)

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    S1 Fig. Agarose gel electrophoresis of Genomic DNA of C. schmidii obtained from different drying methods.

    Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

    (TIF)

    Click here for additional data file. (2.1MB, tif)
    S2 Fig. Agarose gel electrophoresis of Genomic DNA of P. commune obtained from different drying methods.

    Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

    (TIF)

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    S3 Fig. Agarose gel electrophoresis of Genomic DNA of H. calcicola obtained from different drying methods.

    Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

    (TIF)

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    S4 Fig. Agarose gel electrophoresis of Genomic DNA of M. polymorpha obtained from different drying methods.

    Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

    (TIF)

    Click here for additional data file. (1.8MB, tif)
    S5 Fig. Agarose gel electrophoresis of PCR products of C. schmidii obtained from different drying methods.

    Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

    (TIF)

    Click here for additional data file. (2.2MB, tif)
    S6 Fig. Agarose gel electrophoresis of PCR products of P. commune obtained from different drying methods.

    Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

    (TIF)

    Click here for additional data file. (4.5MB, tif)
    S7 Fig. Agarose gel electrophoresis of PCR products of H.calcicola obtained from different drying methods.

    Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

    (TIF)

    Click here for additional data file. (1.5MB, tif)
    S8 Fig. Agarose gel electrophoresis of PCR products of M. polymorpha obtained from different drying methods.

    Note, 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying.

    (TIF)

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    S9 Fig. The morphological characters of overall plants.

    Note, C, C. schmidii; P, P. commune; H, H. calcicola; M, M. polymorpha; 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying. Bar scales C/P/H = 1 mm; M = 1 cm.

    (TIFF)

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    S10 Fig. The morphological characters of leaves.

    Note, C, C. schmidii; P, P. commune; H, H. calcicola; M, M. polymorpha; 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying. Bar scales C/P = 0.5 mm; H = 0.1 mm; M = 1 mm.

    (TIFF)

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    S11 Fig. The morphological characters of transverse sections.

    Note, C, C. schmidii; P, P. commune; H, H. calcicola; M, M. polymorpha; 150, 150°C hot-air drying; 80, 80°C hot-air drying; 40, 40°C hot-air drying; N, natural drying; S, silica gel drying. Bar scales = 50 μm.

    (TIFF)

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    S1 Raw images

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    Attachment

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    Attachment

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    Attachment

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    Data Availability Statement

    All relevant data are within the paper and its Supporting information files.

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