eDNA-based monitoring of parasitic plant (Sapria himalayana)

Sapria himalayana Griffith., is a root parasitic plant that is exceptionally beautiful and odd-looking and found in Southeast Asia. Now these plants are at risk of extinction as they face a large number of different threats. Appropriate measures and conservation plans are needed and one crucial key for successful conservation is species monitoring. The flower is the only part of S. himalayana that is visible during a short period of time of the year. Thus, conducting a visual survey in the field at the other times of the year would be difficult. DNA from living organisms could be found accumulating in environment and so-called environmental DNA (eDNA). Here, an eDNA-based method was developed to specifically monitor S. himalayana in nature. Detecting the specifically generated amplicons allowed us to monitor the presence of S. himalayana at any time of the year. This developed method would increase the conservation success of the S. himalayana.

plants would be only possible after their flowers emerge. Currently applied DNA-based methods work reasonably well to enumerate species for natural resource management and conservation. Advanced molecular techniques facilitate the estimating and monitoring of biodiversity, especially the increasing application of environmental DNA or eDNA. This has proven to be a sensitive, effective and convenient method with increased speed [9][10][11] . DNA from living organisms including animals, plants and fungi could be found to accumulate in environment and so-called environmental DNA (eDNA) 12,13 . Using eDNA for species monitoring is performed by detecting DNA fragments that organisms of interest release into the environments. DNA found in environments could originate from various sources. The use of eDNA for species monitoring and detection is becoming more popular, with an increasing number of studies dedicated to both testing and applying these methods. However, these are mainly applied to aquatic organisms, including various fish, amphibians, and mammals 11,[14][15][16] . To date, no study has used eDNA for parasitic plants.
In the present study, the eDNA-based method for monitoring root parasitic plants species using hermit's spittoon (S. himalayana) as a model species has been developed and tested. Species-specific primer pairs that amplify DNA fragments only in S. himalayana were used to detect species in combination with quantitative real-time PCR (qPCR). An eDNA detection method from soil for S. himalayana was established first. Next, distribution surveys were conducted based on eDNA for this species in the Doi Suthep-Pui National Park, Chiang Mai, Thailand when both the flowers of S. himalayana were visible and no visible trace remained above-ground. The results were compared to visual observation during the time when its flowers could be spotted in order to prove that the adapted protocols, could be used to detect S. himalayana eDNA in both aboveground and belowground stages of life.

Results
S. himalayana DNA was successfully amplified in soil samples taken from the sites where we spotted the S. himalayana buds and the host Tetrastigma species (SSH1-SSH3). The target species was detected in all three sampling sites. No amplified DNA was found in samples from where S. himalayana was not observed (NSH and SBF), in all three replicates, indicating that there is consistency in detection of the target species. Similarly, there was no amplification of the target species in all replicates observed from negative samples ( Table 1).
Except for the negative control sites (NSH and SBF), the qPCR results detected the S. himalayana within all soil samples ( Table 1). The sequences of the amplicons were a 100% match with both the reference sequences from tissue samples of the S. himalayana and from GenBank. The number of cycles in qPCR analysis which was required for detection was also consistently between 15-16 cycles for all eDNA triplicates of the SSH1 soil sample, this was slightly different from the value obtained from the tissue-derived DNA (13-14 cycles) ( Table 1). With optimal PCR efficiency, this indicates a minimal difference of around 1 order of magnitude in DNA concentration. Additionally, the S. himalayana DNA was successfully amplified in all three replicates on individual soil samples SSH2 and SSH3 with 23-24 cycles (difference of ~2-3 orders of magnitude in DNA concentration) (Fig. 2). S. himalayana eDNA was also amplified in two out of three qPCRs from soil samples collected at a distance of within 5 m from the buds/flowers (Table 1) with 32 and 34 qPCR cycles, as expected for eDNA extracted from diluted environmental samples. No positive PCRs were obtained from DNA extracts from NSH and SBF, where there is no record of hermit's spittoon and its host.  www.nature.com/scientificreports www.nature.com/scientificreports/ The DNA dilution series (1/10 1 -1/10 5 ) was used in qPCRs and found that the number of qPCR cycles increased as the DNA concentration went down (Table 1 and Fig. 2). Comparing the required number of cycles in qPCR analysis for detection at the SSH1site with the DNA from tissue would indicate a minimal difference of  Table S2). www.nature.com/scientificreports www.nature.com/scientificreports/ around 1 in the order of magnitude in DNA concentration which is unsurprising as we collected the soil samples directly from the S. himalayana buds/flowers. Whereas, the required number of qPCR cycles at the site around 5 m away from the buds/flowers indicates a minimal difference of around 4-5 orders of magnitude in DNA concentration. One of three PCRs was found to be negative in the soil sample collected from around 5 m away from the buds/flowers. This could be a result of diluted environmental samples rather than a false negative. While, in water sampling, increasing the volume of sample may reduce the rate of false negatives (e.g. 11 ), there is no such report in soil sampling.
It can be seen that the required number of qPCR cycles for SSH2 detection (23 cycles) and SSH3 (24 cycles) were similar with a DNA concentration of around 1/10 3 . Although, when the soil samples were collected in October 2016, the flowers had already emerged and could be seen on the ground and thus, differences in the flower stages between sites were observed. At the SSH2 and SSH3 sites, most of them were still buds (globose with white and pink bracts), while all S. himalayana spotted at the SSH1 site were either in bloom or the flowers dehisced and became dark in colour. The differences in flower stages may lead to the difference of qPCR cycles required for detection in SSH1, SSH2 and SSH3 sites. Thus, further investigation would be interesting and should be carried out to better understand the matter.
The molecular approach, based on eDNA extraction from soil, is already commonly used to characterise soil microorganisms and its application is now being used to characterise other soil organisms 17 . eDNA detection is conducted in a variety of environments such as agricultural fields, deserts, forests, the Arctic and Antarctic 18,19 . The availability of an eDNA-based method will provide new options for monitoring and surveying root parasitic plants. Applying the method for detection of root parasitic weeds will be also useful. This can refer to detecting parasitic weeds such as Striga and Orobanche spp., which are difficult to control as their life cycles are mainly underground. This leads to difficulty diagnosing infection already done before the parasites emerge 20 . The method can be also used to detect other rare and economically parasitic plants such as Rafflesia and Balanophora.

Floral and soil materials and DNA extraction. S. himalayana buds were collected at Doi Suthep-Pui
National Park, Chiang Mai, Thailand. Soil samples were collected from three different sites and also at Doi Suthep-Pui National Park, Chiang Mai, Thailand. Soil samples came from the following key areas: where we spotted the S. himalayana buds and the host Tetrastigma species (called SSH), from where we did not see both buds and host (called NSH), and from where we found another parasitic plant, Balanophora fungosa J. R. Forst. & G. Forst. (called SBF). Three triplets of soil samples were taken per site in October 2016 (when flowers were emerging) and again in April 2017 (when there were no flowers). In November 2017, the studied sites were visited for visual detection only and no further soil samples were collected.
All three soil samples per site were pooled, mixed and air-dried at 30-40 °C for 24-48 h. Dried soil was ground into fine powder which was subsequently used for the DNA extraction 21 . DNA was extracted from 500 mg of soil per sample using the NucleoSpin ® Soil Kit (Macherey Nagel ™ ) according to the manufacurer's protocols. Each pooled sample was extracted in triplicates and then ready to be used in next analysis.

Species-specific primers designing.
To design primers specific to S. himalayana, we sequenced the partial ITS region from three individuals of each S. himalayana and B. fungosa, which is the closest related parasitic plant to our target species in the studied area. S. himalayana and B. fungosa buds/flowers were collected at Doi Suthep-Pui National Park, Chiang Mai, Thailand. The total DNA was extracted from tissue samples using the Nucleospin Plant II kit (Macherey-Nagel, Germany) according to the manufacturer's protocol. From searching through the public databases, there was no available sequence of the parasitic plants species collected from the studied area. We therefore amplified and sequenced the partial ITS regions with the primers B330F 5′ TGACGGGTGACGGAGAATTAGG 3′ and B1764R 5′ CAATAATCCTTCCGCAGGTTCACC 3′, both of which were modified from previous sequences of parasitic plants retrieved from GenBank (Table S1). For qPCR analysis, species specific primers Sapria_ITSF 5′ TGTCGGATTTTCCGTCTCATCC 3′ and Sapria_ITSR 5′ GTCACACGATTAATCGCTCGTACA3′ were designed using the PrimerBlast software (http://www.ncbi. nlm.nih.gov/tools/primer-blast/) to target a short (191 bp) fragment of the ITS region of S. himalayana using sequences from the consensus sequence generated by this work (Table S1) and also GenBank and checked against all other S. himalayana sequences in GenBank at the time.
The specificity of the primers was tested by comparing the sequences to other Sapria species including B. fungosa species which had been found in the studied area. The designed primers contain no less than 5 mismatches with non-target species. qPCR analysis. The qPCR was conducted in a 20 μL reaction volume containing 10 μL of ABI TaqMan Universal Master Mix II, 1 μL of each primer, and 2 μL of DNA extract. The qPCR conditions were as follows: 10 min at 95 °C and 50 cycles of 30 s at 95 °C, and 45 s at 51 °C, and 30 s at 72 °C. All samples were run as triplicates. Amplifications were conducted using the Rotor-Gene Q System (Qiagen, Germany), and Cq values were automatically set using the system software. In all qPCR, the R 2 values for the standard curve were ≥0.97 and efficiency 90-97%.
Three replicates in each qPCR set contained only reagents but no DNA were used as a negative control whilst one tube that contained all reagents and S. himalayana extracted DNA template was used as a positive control. The absence/presence calls was determined from data of the post-PCR read. Only the target amplified above the target's threshold obtained from the default analysis settings in the Rotor-Gene Q software version 2.3.1 (Qiagen, Germany), in the target species' eDNA was called present 22 .