After the plant material has been prepared by drying and/or freezing using one of the above-mentioned techniques, a DNA extraction protocol can be implemented. Although there are a multitude of available protocols, the general methodology involves the following steps, discussed in more detail below:

• Weighing of plant tissue
• Mechanical disruption (grinding)
• (Optional) pre-treatment
• Extraction of nucleic acids from the cell
• DNA isolation and precipitation
• DNA purification

We place emphasis on the CTAB protocol due to its popularity, but also introduce other protocols that may be of interest to the reader.

• 2% w/v CTAB: a cationic detergent which, during DNA extraction, binds to the lipids in cell membranes, enhancing cell lysis, thus releasing intact nucleic acids from the nucleus and organelles
• 1.4 M NaCl: a salt which increases the ionic strength of the solution, which simultaneously induces plasmolysis, promotes separation of proteins from DNA, and aids in polysaccharide precipitation
• 100 mM Tris-HCl: a buffer (at pH ~8.0) which maintains the pH of the solution and stabilises the DNA by impeding degradation
• 20 mM EDTA (ethylenediaminetetraacetic acid): which protects the DNA by inhibiting the enzymatic activity of DNase and RNase (i.e., by chelating divalent cations, such as Mg²⁺ and Ca²⁺, which are cofactors for these enzymes)
• 0.2% ß-mercaptoethanol: which denatures polyphenols and tannins (abundant in plants), rendering it possible to separate them from the DNA in subsequent steps

CTAB buffer is added to each sample tube containing ground plant tissue and the mixture is incubated at 60–65 °C for 15–60 minutes. This can be done in an automatic shaking incubator. Alternatively, the sample tubes can be periodically shaken manually.

Alternatively, methods involving an SDS buffer can be applied (Dellaporta et al. 1983). The buffer recipe also contains NaCl, Tris-HCl, EDTA, and ß-mercaptoethanol, but differs in the application of the anionic detergent sodium dodecyl sulphate (SDS) for the disruption of cellular membranes, as well as the addition of sodium acetate (NaCH₃COO).

Assessment of the properties of each genomic DNA (gDNA) sample post-extraction – its integrity, quantity, and purity – is imperative for making decisions regarding downstream molecular work. The methods described below have some overlapping uses in terms of assessing these different properties, but we highlight which is most appropriate for each DNA quality-related aspect.

Particularly challenging types of plant tissue, as well as degraded plant material, can still yield high-quality DNA if suitably optimised protocols are followed.

For instance, seeds can be a good source of DNA if specialised protocols are used (Sudan et al. 2017). Similar to other plant tissues, seeds require different collection and storage techniques depending on their morphology. Dry seeds can usually be collected and stored for long periods without treatment before being ground and used in a DNA extraction protocol. Soft seeds in comparison may need to be flash frozen using liquid nitrogen and cryopreserved prior to DNA extraction. The watery components of fleshy or succulent plant tissues require modified approaches to speed up drying before extraction to remove polysaccharide contaminants from the DNA extract (Larridon et al. 2015; Malakasi et al. 2019).

Advances in the sensitivity of genomic sequencing and optimised DNA extraction methods make it possible to study herbarium and other dried botanical specimens (Bieker and Martin 2018; Brewer et al. 2019; Grace et al. 2021; Malakasi et al. 2019; Särkinen et al. 2012). However, using this material involves mining irreplaceable reservoirs of biological and cultural heritage (Austin et al. 2019; Freedman et al. 2018). Sampling should be restricted to the minimum size expected to yield sufficient DNA for the project and the decision on which part of the specimen to sample should be made in consultation with a collection manager or specialist (see Chapter 2 DNA from museum collections). Novel techniques have been developed for minimally destructive sampling of herbarium specimens (Shepherd 2017; Sugita et al. 2020), but these are not universally applicable. Archaeological and museum collections present similar challenges and sometimes even more sensitive decisions. Archaeological plant material can include plant micro- and macrofossils (reviewed in (Kistler et al. 2020)), such as for example grape pips (Wales et al. 2016), or even archaeological artefacts, e.g., a palm-leaf jar stopper (Pérez-Escobar et al. 2021) or paper-mulberry tapa (Peña-Ahumada et al. 2020). At the lab bench, two key obstacles should be considered: contamination and degradation of the DNA. Whereas contamination is a crucial consideration throughout the process of DNA extraction, the physical fragmentation of plant tissue requires the most consideration during experimental design and downstream HTS laboratory work since herbarium and museum samples are treated with an overall more sensitive set of protocols than standard plant tissue samples (see Chapter 2 DNA from museum collections). Any small amount of accidentally introduced nucleic acid contamination will hitchhike alongside the (most likely) degraded DNA present in the sample of interest throughout the purification procedures and has a high likelihood of being preferentially amplified. Crucially, a laboratory’s DNA extraction area should not overlap with any area where PCR amplicons are generated. Finally, a simple way to test for persistent contamination is to include extraction blanks.

Physical and chemical degradation is to be expected in herbarium and museum specimens; DNA in deceased tissue breaks down over time. The rate of physical fragmentation is related to temperature and other environmental variables, as well as the composition of the plant tissue itself. In a study of herbarium specimens, it was shown that fragment length significantly regressed against sample age going back 300 years (Weiß et al. 2016), a proxy which can be exploited as a useful starting point for making DNA quality-based lab work decisions. This is more likely to hold true within a plant clade (e.g., plant family) and with a consistent method of sample preparation. However, the relationship of increasing fragmentation with sample age is not always linear. Fixation of the plant material for accessioning in the herbarium is often the single most damaging process (Staats et al. 2011).

The CTAB extraction protocol is generally preferable for extracting fragmented DNA, as it generally gives higher yields of DNA than kit-based methods. Where fragment size distribution is predicted to be very low, a high-volume chaotropic salt used as a binding buffer in the latter stage of extraction can improve the recovery of DNA molecules (Dabney et al. 2013). Alternatively, the ratio of SPRI beads to DNA during the clean-up step can be increased to retrieve more of the shorter DNA molecules. A hallmark of chemical DNA degradation, i.e. cytosine deamination, can be addressed in downstream steps by using repair enzymes in DNA library preparation and appropriate bioinformatic treatment (Kistler et al. 2020).

A wide variety of DNA extraction protocols are available in the literature. The structural, biochemical, and genomic characteristics of plants present a particular set of challenges; isolating high purity, undamaged DNA from plant tissue is non-trivial and requires a careful and patient approach in the laboratory. Therefore, researchers must often optimise a chosen protocol for their specific experiment. Success in the primary step of a molecular workflow is crucial, unlocking the downstream steps of plant molecular identification and characterisation, and hence possibilities for addressing many exciting questions in molecular and evolutionary biology.

1. For each of the DNA-containing compartments in a plant cell, which of its characteristics deserve most consideration during DNA extraction and analysis, and why?
2. Describe the main compound classes from plant extracts that need to be removed from DNA extracts for downstream analysis. How can they be removed?
3. Describe the main difference between DNA extraction using the CTAB protocol and using a column-based extraction kit. What are the advantages and disadvantages of both?

Absorbance – A measure of the quantity of light absorbed by a sample, also referred to as optical density, measured using an absorbance spectrophotometer.

Beer-Lambert law – For a material through which light is travelling, the path length of light and concentration of the sample are both directly proportional to the absorbance of the light.

Chaotropic agent – A chemical substance which in an aqueous solution destroys the hydrogen bonds between water molecules (e.g., guanidine hydrochloride).

Cryopreservation – A preservation treatment for biological material, which involves cooling to very low temperatures (at least -80 °C, or -196 °C using e.g., liquid nitrogen).

Desiccant – A substance with a high affinity for water, such that it attracts moisture from surrounding materials, resulting in a state of dryness in its vicinity (e.g., silica gel).

DNA integrity – The level of fragmentation of extracted DNA, where minimal fragmentation of the original chromosomes equates to high DNA integrity.

Intercalating dye – A dye, whose molecular components stack between two bases of DNA, which is invaluable for DNA visualisation, yet at the same time implies a hazard for human health and demands laboratory safety considerations.

Lysate – A commonly fluid mixture of cellular contents that is the result of the disruption of cell walls and membranes via cell lysis.

Molecular marker (in a genetic context) – A sequence of DNA, which can be a single base pair, a gene, or repetitive sequence, with a known location in the genome, which tends to exhibit variation amongst individuals or taxa, such that it has useful research applications.

Organellar genome – The genetic material present in a plastid or mitochondrion, typically in the form of a small and circular genome and often in multiple copies within each organelle. These are thought to be present in eukaryotic cells as a result of endosymbiosis.

Plastome – The total genetic information contained by the plastid (e.g., chloroplast) of a plant cell.

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1. The nuclear genome of plants is hugely variable in size. To maximise retrieval of intact DNA for species with larger genomes, a higher DNA yield should be aimed for. This could affect decisions regarding input material and the number of total DNA extractions carried out per sample. The plastid genome is present in high copy numbers in plant cells, as well as being a useful unit for addressing a variety of biological questions. Therefore, it is ideal for genome skimming experiments and a valuable target in degraded material, where the (single copy) nuclear genome might be highly fragmented. The mitochondrial genome of plants is characterised by high plasticity in its genomic structure and therefore is not recommended for plant identification.
2. Problematic biomolecules in plant extracts include polyphenols, tannins, and polysaccharides. These interfere with DNA extraction buffers (such as CTAB) as well as with other buffers and enzymes used in downstream DNA analysis. They are removed from the solution by either SEVAG cleaning (in the CTAB protocol) or, basically, by column cleaning or magnetic particles (commercial kits). Polysaccharides can also be removed from the crude plant tissue prior to extraction using STE buffer. Phenolic compounds can often be removed using ß-mercaptoethanol and/or PVP. Further impurities such as secondary metabolic compounds that may interfere with enzymes in downstream protocols can often be removed using a SPRI bead clean-up protocol.
3. The CTAB protocol uses specific buffers (such as SEVAG) and DNA precipitation (involving isopropanol) to separate non-DNA and DNA biomolecules, whereas extraction kits rely on using DNA-binding columns. or magnetic particles Although the kits are much more expensive on a per-sample basis, they generally yield clean DNA with a short turnaround time (up to 6 hours). CTAB extractions are very cheap and highly scalable as they do not rely on the specifically manufactured columns or magnetic particles. However, the protocol takes at least two full days to progress from plant tissue to DNA extract. Co-precipitation of non-DNA biomolecules is often observed and therefore affects the purity of the final DNA extract. Sometimes, substantial yield losses are observed using extraction kits and this can be a key consideration when dealing with precious samples.

Museum collections of plant origin include herbaria (pressed plants), xylaria (woods), and economic botany (useful plant) specimens. They are not only places of history and display, but also of research, and contain rich repositories of molecules, including DNA. Such DNA, retrieved from historical or ancient tissue, carries unique degradation characteristics and regardless of its age is known as ancient DNA (aDNA). Research into aDNA has developed rapidly in the last decade as a result of an improved understanding of its biochemical properties, the development of specific laboratory protocols for its isolation, and better bioinformatic tools. Why are museum collections useful sources of aDNA? We identify three main reasons: 1) specimens can play a key role in taxonomic and macroevolutionary inference when it is difficult to sample living material, for example, by giving us snapshots of extinct taxa (Van de Paer et al. 2016); 2) accurate identification of specimens that were objects of debate or scientific mystery, as exemplified by misidentified type specimens of the watermelon’s progenitor (Chomicki and Renner 2015); 3) specimens can provide us with ‘time machines’ to study microevolutionary processes and diversity changes over decades- to millennia-long timeframes (Gutaker and Burbano 2017; Pont et al. 2019). In all three cases, specimens are often associated with evidence of their occurrence in space and time. For further examples see Chapter 20 Museomics, and the Glossary.

However, extracting DNA does mean the destruction of a part of the specimen. Museum curators therefore face challenges in balancing the conservation of specimens for future research with the rising demand for aDNA analysis. Increasingly, curators are also considering legal and ethical issues in sampling (Austin et al. 2019; Pálsdóttir et al. 2019). Close collaboration between the aDNA researcher and the curatorial staff of museums is therefore essential for appropriate management of these issues (Freedman et al. 2018).

With few exceptions, plant material found in museums originally grew on lands tended or owned by people for many millennia (Ellis et al. 2021). Some specimens, such as artefacts or seeds of domesticated crops have an even more direct connection to human activities. Plant specimens, along with other living things, are therefore not simply assemblages of chemical compounds such as DNA, but also embody spiritual beliefs, diverse forms of ownership, traditional knowledge, and past histories of colonialism and other forms of harm (Anderson et al. 2011; Das and Lowe 2018; Pungetti et al. 2012). The implications of this are still being worked out in dialogues between museums and affected communities, often within a decolonising framework (McAlvay et al. 2021). There are, however, immediate steps that researchers and curators can take to ensure that the use of specimens is both legal and ethical.

A first consideration is whether the plant species or artefacts (such as baskets or wooden objects) are of special significance (e.g., sacred) to the source community. Examples of sacred material include Banisteriopsis caapi, used to make ayahuasca in South America (Rivier and Lindgren 1972), or Duboisia hopwoodii (pituri), used as tobacco in Australia (Ratsch et al. 2010). An online literature search or consultation with relevant experts will give a rapid pointer, which can be followed up with source communities in the study region. Collaboration with communities and scientists in source countries is essential for acknowledging the rights to plant material (even if not legally enshrined), and can be furthered by publication of results in local languages and media. These communities also hold significant expertise on plants that will improve the quality and relevance of research (Gewin 2021).

There are international conventions that usually apply when accessing, researching, and moving plant material between institutions and countries. Researchers must also be aware of country-specific laws that may require further permits and inspections, e.g., for plants that produce controlled substances, require phytosanitary checks, or are considered invasive species. Legal elements of the Convention on Biological Diversity (CBD), Nagoya Protocol, and Convention on Trade in Endangered Species (CITES) are covered in Chapter 27 Legislation and policy as well as in other published works (e.g. McManis and Pelletier 2014, Iob and Botigué 2021). While the CBD applies to specimens received by museums from 1992, in ethical terms (and under some implementations of the Nagoya Protocol) its principles, such as benefit-sharing, also apply to pre-1992 specimens (cf. Sherman and Henry 2020).

1. Name three legal considerations and their related ethical main issues that should be taken into account for aDNA research using museum material.
2. Why is it important to process herbarium samples in a dedicated clean lab?
3. Name three benefits of getting curators involved in the early stages of research using collections.

aDNA – Ancient DNA, DNA that exhibits biochemical characteristics typical for DNA from old degraded material, i.e., damage and fragmentation, regardless of age.

Artefact – An object made by humans that is of historical or cultural importance, examples include: clothing, ornaments, utensils.

Authentication – Bioinformatic analyses that quantify damage and fragmentation of sequenced DNA to help rule out that DNA is derived from contemporary contamination.

Collection – Repository of curated biological material arranged in a systematic fashion.

Contamination – Introduction of alien tissue or DNA to a specimen or DNA isolate, examples include: microbial colonisation, human epithelium, plant-based foods, etc.

Curator – Custodian of a collection with expert knowledge about specimens, their organisation, and preservation.

Destructive sampling – Permanent removal of a fragment of a specimen of any size that will be irretrievable after biochemical characterization.

DNA damage – Typically conversion of cytosine to uracil in DNA through deamination, which accumulates with time. During sequencing, uracil is replaced with thymine, hence the common synonym, C-to-T substitutions.

Endogenous DNA – Authentic DNA from targeted individuals of a species, in contrast to exogenous DNA from associated microbes and contemporary plant and human DNA contamination.

Fragmentation – Breaks in the DNA backbone, most frequently caused by depurination, leading to shorter DNA fragments with time.

Immortalization – Molecular manipulation of DNA, for example the attachment of DNA adapters, that allows infinite re-amplification of the original DNA from a biological specimen.

Type specimen – Preserved individual plant that has defining features of that taxon that is used for the first taxonomic description of a species. This permanent feature-specimen link is recognized in a publication.

Voucher – Preserved botanical specimen kept in permanent collection and cited by research project. Vouchers will have been expertly identified and are usually annotated with collection time, place, and collector details.

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1. Legal: CITES (restriction in international trade of endangered species), Nagoya Protocol (ownership and other significance to indigenous peoples), and Drug Act (controlled substances).
2. The decay of DNA from historical plant material makes it very susceptible to contamination with exogenous modern DNA.
3. Curators can contribute (1) high-quality metadata such as collection dates and provenance, (2) knowledge of collections in-house and elsewhere, (3) knowledge of source communities and ethical and legal issues, (4) advice on choice of specimens most suitable for sampling.

The first studies conducted on DNA obtained from water samples were published in the 1990s. Cloning techniques were commonly used to investigate novel genes and functions of environmental communities at that time. Stein et al. (Stein et al. 1996) cloned DNA fragments obtained from water samples into E. coli vectors to investigate marine archaea metabolism. Relying on the development of high-throughput sequencing (HTS) technologies, it is now possible to capture and sequence almost all DNA fragments present in a water sample. A pioneering example represents the study, where Venter and colleagues sequenced sea water samples revealing diverse microbial compositions and functions (Venter et al. 2004). Further rapid development of sequencing technologies in the last decade, as well as the coinciding decrease in sequencing costs, has allowed for the incorporation of ‘environmental DNA’ (eDNA) methods (i.e., the analysis of DNA fragments isolated from environmental sample types such as water, air, and soil) into several applications, including in aquatic environmental surveys.

Conventionally, biomonitoring of freshwater and marine environments is based on direct observation of indicator taxa to compute biotic metrics/indices. This can be time and labour intensive (Pawlowski et al. 2018). Other methods such as depletion-based electrofishing, hydroacoustics, camera traps, and gillnets are also common (Deiner et al. 2017). In recent years, eDNA methods have been added to this toolbox of available methods for biomonitoring. Species-level information on key bioindicator species has for example been obtained by using the DNA obtained from water samples (Hajibabaei et al. 2011). Other applications include population quantification (Fukaya et al. 2020), invasive species detection (Anglès d’Auriac et al. 2019), water quality monitoring (Noyer et al. 2015), and revealing food web interactions (D’Alessandro and Mariani 2021).

The main advantage of water is the ease of sample collection compared to other aquatic sample types such as sediments or biofilms, as these substrates usually require more sophisticated tools and longer sampling times (Deiner et al. 2017). A potential disadvantage is that DNA in a water column decays into undetectable levels in two weeks at most (Dejean et al. 2011; Thomsen et al. 2012), whereas in sediments and ice cores it can persist much longer (Turner et al. 2015). Thus, DNA collected from water samples typically reflects contemporary communities, whereas those collected from sediments and ice cores reflect a longer temporal scale and can be used as a source for ancient DNA (Willerslev et al. 2007) (Chapter 8 DNA from ancient sediments).

Detecting DNA in water samples obtained from aquatic environments can be challenging because it is usually present at low concentrations with an uneven spatial distribution (Ficetola et al. 2008; Goldberg et al. 2016). In this chapter, we first explain the factors affecting the detection of DNA with a specific focus on plant species for environmental applications. The literature referenced here mostly focuses on vascular plants but the general approach might be suitable for a broader group of organisms as well (Alsos et al. 2018; Apothéloz-Perret-Gentil et al. 2021; Nowak et al. 2021). We then outline the general workflow and experimental setup for collecting DNA from water and strategies to optimise its detection.

Natural processes influencing the composition and quantity of detectable DNA in a water sample can be categorised into 1) shedding of biological material from source organisms, 2) degradation, 3) transport across the water column, and 4) retention and resuspension (Harrison et al. 2019). Several biotic and abiotic environmental factors influence the rates of these processes. This creates a complex and environment-specific relationship between DNA that is detected in the water and how well this can be related to the presence and relative abundance of an aquatic organism. As almost all DNA fragments in a water sample can be detected with current sequencing technologies, establishing an optimal sampling strategy is crucial for minimising the probability of contamination and obtaining an accurate representation of biodiversity.

Recent studies that detect plant species in aquatic ecosystems via eDNA are mainly about methodological adjustments (Fujiwara et al. 2016; Gantz et al. 2018; Kuehne et al. 2020; Matsuhashi et al. 2019, 2016; Schabacker et al. 2020; Strickler et al. 2015). The technique involves three main steps: 1) collecting water samples, 2) DNA isolation and sequencing, and 3) taxonomic annotation of assembled sequences. Although substantial improvements are being made for the last step due to the developments in bioinformatics (Chapter 18 Sequence to species), sample collection, DNA isolation, and even the choice of sequencing platform (Chapter 9 Sequencing platforms and data types) can still introduce biases (Singer et al. 2019; Tsuji et al. 2019). Methodological research on these two steps is usually conducted using mesocosm and aquarium experiments focusing on spatial and temporal dynamics of DNA (Kuehne et al. 2020). In the next part of this chapter, we will describe the experimental workflow and discuss the issues related to the processes affecting DNA detection from water samples, as outlined in the previous section.

DNA isolated from water samples can be used for several downstream applications based on the specific aim of the study or survey. Currently, qPCR methods are the most commonly used method for detecting specific target taxa in water samples, while metabarcoding is used for community analyses (Chapter 11 Metabarcoding). The studies comparing the efficiency of these DNA methods with more conventional methods show varying results. For some species or taxa, DNA-based detection methods appear to outperform more conventional methods (Deiner et al. 2016; Tingley et al. 2018), though in other cases there is not necessarily an improvement in detection compared to more conventional surveys (Rose et al. 2019; Wood et al. 2019). Although DNA-based methods are constantly being improved, there are still challenges related to both false positives (when DNA is detected for a species or taxa that is known to be absent) and false negatives (when DNA is not detected for a species or taxa that is known to be present) (Beng and Corlett 2020). Therefore, at least for now, DNA-based methods for aquatic studies focusing on plants are still best coupled with conventional surveys. Based on the further development of sequencing methods and the increasing availability of reference sequence data in public databases, however, there are additional opportunities such as application of metagenomics (Chapter 12 Metagenomics), target capture (Chapter 14 Target capture), or analysis of whole plastomes (Chapter 16 Whole genome sequencing). These methods might provide the additional benefit of integrating functional information besides species detection.

1. Are water samples best collected at a single point representative of the habitat diversity as a whole? Motivate your answer.
2. Describe three biotic and three abiotic factors that can affect DNA detection rates in aquatic environments. Explain in a few sentences how these factors can result in the detection of false positives and false negatives in streaming waters.
3. List five factors that should be taken into account while designing a sampling strategy for detection of DNA from water samples.

Apoptosis – Controlled cell death which involves cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation.

Biofilm – A consortium of microorganisms where cells stick to each other and often also to a surface.

Dimictic lake – A body of freshwater whose difference in temperature between surface and bottom layers becomes negligible twice per year.

Extracellular nucleases – Enzymes that can work outside of the cell and are capable of cleaving the phosphodiester bonds between nucleotides of nucleic acids.

Mesocosm – Any outdoor experimental system that simulates the natural environment under controlled conditions.

Necrosis – Uncontrolled cell death due to the loss of osmotic control typically by swelling and bursting.

PCR inhibitors – Any factor which prevents the amplification of nucleic acids through the polymerase chain reaction.

Primer – A short single stranded nucleic acid sequence used by all living organisms in the initiation of DNA synthesis.

qPCR (Quantitative PCR) – An extension of the PCR technique which allows estimation of the initial quantity of nucleic acids in a biological sample.

Senescence – The gradual deterioration of functional characteristics with ageing (can be used both for organismal or cellular ageing).

Thermal stratification – The phenomenon in which lakes develop two discrete layers of water of different temperatures; warm on top (epilimnion) and cold below (hypolimnion).

Vector (i.e., cloning vectors) – A small piece of DNA that can be stably maintained in an organism that a foreign DNA fragment can be inserted into for cloning purposes.

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1. No. The probability of species detection depends on the presence and concentration of DNA collected in a water sample. Therefore, multiple sampling sites with replicates is highly encouraged to obtain a broader overview of the local species diversity.
2. The life history, age, and size of an organism are some of the biotic factors which can affect DNA detection rates. After release from the cell, abiotic factors such as UV, temperature, and pH can influence these rates. In streaming waters, including currents, false positives might be detected in downstream regions due to the transport of DNA. Similarly, if the DNA of the target organism degrades too quickly, it cannot be detected resulting in false negatives.
3. The scientific question, environmental conditions (physical and chemical), distance between sampling point and laboratory, and morphology and life cycle of the target organism should be considered when designing sampling strategies.

The natural presence of any plant entails the existence of a substrate where it can anchor itself and absorb nutrients for its development and survival (Wardle et al. 2004). This is most commonly the ground and specifically, soil. Nevertheless, the link between soil and plants goes beyond soil supporting plants as plants are one of the main soil-forming forces of pedogenesis through the accumulation of organic matter as well as modification of the soil biochemistry surrounding the roots (Corti et al. 2005). This process over time leads to the formation of soil layers, termed horizons, that can commonly be visibly identified (Schulz et al. 2013; Shlemon 1985; Vogt et al. 1995). Near the ground surface, the first soil horizon is an organic layer composed of growing roots and decomposing vegetative and reproductive plant material from local or regional origins, i.e., fallen debris, pollen particles, seeds (Vogt et al. 1995). Hence, this soil horizon is particularly rich in plant DNA from the environment (soil eDNA in short; Taberlet et al. 2018) and can be used as a proxy for plant identification and other biodiversity assessments (Fahner et al. 2016; Taberlet et al. 2018; Yoccoz et al. 2012).

Since the first isolation of DNA from soil bacteria, soil eDNA has gained attention for the assessment of terrestrial environments for several reasons: soil is virtually everywhere, it is easy to collect and transport, harbors signals from above and below biota including both active and dormant cells, and is a non-invasive sample collection technique (Torsvik et al. 1990; Yoccoz 2012); for more on soil eDNA applications see Chapter 24 Environment and biodiversity assessments). Soil eDNA assessments targeting modern plant diversity commonly employ samples that are collected near the surface (organic horizont). However, some studies may refer to sediments which can lead to confusing eDNA samples coexisting in underground environments (Kristensen and Rabenhorst 2015). Although both soil and sediments are products of mineral weathering (Wood 1987), in soils the deposition of these products happens in situ and remains on the surface, while in sediments these products are transported and redeposited elsewhere in layers over time, e.g., the ground or the bottom of a lake or stream (Burdige 2020). Moreover, sediments in general have very different organic content, particle size and mineralogy, and lesser organismal activity than soil, although the transition from soil to sediment can be gradual and depends on the eco-physiological characteristics of the regional environment (e.g., tropical vs. boreal forest; Shackley 1975; Smol et al. 2001). Yet, during flooding events sediments can be transported very rapidly from one place to another while sedimenting in new layers mixed with soil (Baldwin and Mitchell 2000). In these contexts, soil and sedimentary eDNA samples may have a mix of different spatio-temporal signals when it comes to the reconstruction of terrestrial or aquatic environments (Deiner et al. 2017; Thomsen and Willerslev 2015). Ancient sedimentary DNA (sedaDNA) is commonly sampled from bottom sediment layers in either aquatic or terrestrial environments (Parducci et al. 2018), and its temporal signal is usually correlated with sampling depth (Willerslev and Cooper 2005). For more on sedaDNA and its applications see Chapter 8 aDNA from sediments. Sedimentary DNA (sedDNA) usually refers to modern sediments that were either recently deposited or signal contemporary environments. Plant biodiversity assessments of modern environments often employ surface lake sediments (Andersen et al. 2012; Pedersen et al. 2015; Willerslev et al. 2014) as it captures current biodiversity from the entire watershed catchment area (Alsos et al. 2018). This chapter focuses on modern DNA isolated from soil eDNA.

Further, studies may also refer to bulk soil DNA when using soil samples to identify unknown communities, especially in forensic contexts (Boggs et al. 2019; Gothwal et al. 2007; Meiklejohn et al. 2018). Bulk DNA is commonly used in contexts where known taxa are mixed, molecularly identified (usually by metabarcoding), and then studied. There is no consensus on the precise use of these different terms, and the terminology often reflects disciplinary backgrounds and study approaches (Kristensen and Rabenhorst 2015). Yet, it is worth noting that all terms mentioned so far are not mutually exclusive nor encapsulate a particular environment. For example, soil may also be used in aquatic contexts when pedogenic processes lead to horizon differentiation, e.g., estuarine substrata (Wardle et al. 2004). Thus, careful interpretation of the context in which the term is employed is recommended to ensure correct interpretation of data and studies.

Molecular (plant) identification using soil or sediment eDNA relies on isolating DNA traces from roots, debris, seeds, and pollen (Levy-Booth et al. 2007), which signal diverse spatial and temporal origins, i.e., local or regional, ancient or contemporary. When these plant parts settle into the ground, DNA can be present either in intact cells (intracellular DNA or iDNA) or free in the environment following cell lysis or rupture (extracellular DNA or exDNA; Nagler et al. 2018). The largest fraction of eDNA in underground environments is exDNA that originates from bacteria and fungal soil communities (Levy-Booth et al. 2007; Nagler et al. 2018; Pietramellara et al. 2009; Poté et al. 2009).

The state of DNA in the soil is subject to intrinsic and extrinsic DNA properties related to the origins of the DNA as well as factors influencing its decay (Barnes et al. 2014; Lacoursière-Roussel and Deiner 2021; Sirois and Buckley 2019). For more on leaf DNA decay together with organic horizon formation, see the infographic. Soil eDNA is therefore a combination of iDNA and exDNA, that can degrade rapidly or persist over time. Intrinsic DNA properties that can affect its persistence in the ground include characteristics such as DNA GC content, purity, and weight (Nielsen et al. 2000; Pietramellara et al. 2009; Sirois and Buckley 2019; Taberlet et al. 2018; Vuillemin et al. 2017). Intrinsic DNA properties are those of the organism that affect the magnitude of DNA deposition such as life history traits like biomass, feeding, social, nesting, burrowing, hibernation, etc. Extrinsic DNA properties are more related to abiotic and biotic processes operating in the ground, e.g., soil mineralogy, organic components, pH, electrostatic properties, moisture, the presence/absence of UV radiation, bioturbation, enzymatic activity by microbial communities, and decomposition (Cozzolino et al. 2007; Gardner and Gunsch 2017; Gulden et al. 2005; Levy-Booth et al. 2007; Prosser and Hedgpeth 2018; Saeki et al. 2011). Examples of biotic processes operating in natural environments can be found in the infographic.

iDNA persists due to protection from the cell wall and membranes against abiotic processes. Cells are more likely to remain intact in the ground if there is decreased enzymatic activity as a result of rapid soil desiccation, low temperatures, or extreme pH values (Pietramellara et al. 2009; Taberlet et al. 2018). exDNA is more likely to persist when it binds to surface-reactive particles and hydrophobic soil components such as clay, sand, silt, and humic acids (Levy-Booth et al. 2007; Pietramellara et al. 2009). DNA may also indirectly persist via bacterial integration of DNA fragments (Levy-Booth et al. 2007). Bacterial enzymatic activity plays a central role in DNA degradation in soil (Blum et al. 1997). DNase is secreted copiously to access the phosphorus and nitrogen from the DNA and acts more rapidly on DNA at higher temperatures (Levy-Booth et al. 2007). Since both the temperature and underground biota activity levels are higher in tropical climates, there are generally increased degradation rates in tropical vs. boreal soils. Soil types may also affect degradation rates (Sirois and Buckley 2019) using a controlled microcosm reported that synthetic DNA degraded slower in forest than in agricultural soils where tillage and other disruptive processes can affect persistence. Predicting the origins and persistence of eDNA remains a thorny issue, mainly because of the complex nature of the properties involved (Barnes and Turner 2015; Deiner et al. 2017).

Plant eDNA bound to soil particles can originate from multiple taxa and multiple vegetative parts, each one with particular mechanisms to bind, persist and degrade in soil substrates. Plant DNA persistence within soil allows us to harvest its botanical memory for identifying vegetation through time. Indeed, comparisons of plant identifications through both visual vegetation surveys and soil eDNA assessments have shed light on the temporal signals stored in top soils. In boreal areas, plant identification through soil eDNA signal mostly registered contemporary vegetation (Ariza et al. 2022; Edwards et al. 2018; Yoccoz et al. 2012), however, taxa surveyed up to 30 years ago was also reported, suggesting that soil eDNA harbors more of a contemporary memory (Ariza et al. 2022). The extent of this memory effect across soil types and environments is poorly understood while its implications are relevant for society (e.g., biodiversity assessments and monitoring, forensics, biosafety). For more on applications of soil eDNA see Chapter 24 Environment and biodiversity assessments.

The flora and study area are key in any study to ensure sound conclusions. Below you will find considerations that can help you to answer common questions when designing field and wet lab experiments.

1. The laboratory technician hands you an extraction protocol that has been used previously to extract DNA from soil and sediments. How do you know if this protocol will extract both iDNA and exDNA? Motivate your answer.
2. You are designing your soil eDNA study for a plant taxon that is distributed heterogeneously across plots. Describe the soil sampling strategy that will take into account the target taxon distribution.
3. You want to reconstruct vegetation types based on soil eDNA targeting the trnL P6 loop. This marker will not allow you to identify all taxa to species level. Will this affect your ability to determine the vegetation types? Motivate why or why not?

Bioturbation – Biological processes involved in the dissemination of genetic media through terrestrial media.

DNA degradation – Refers to the physical changes of the DNA molecule.

DNA decay – Refers to the reduction in detectable quantity of eDNA.

DNA persistence – Refers to the amount of DNA that remains detectable across time.

DNA polymorphism – Presence of two or more variants of a particular DNA sequence.

Horizon – A layer parallel to the soil surface whose physical, chemical and biological characteristics differ from the layers above and beneath.

Power analysis – Probability of detecting an effect, given that the effect is really there. Can also be seen as rejecting the null hypothesis when it is in fact false.

Pedogenesis – The process of soil formation as regulated by the effects of place, environment, and history.

Rarefaction curves (in ecology) – A technique to assess species richness given the number of samples collected.

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1. By checking if there is a step that can lyse the cells to extract iDNA. This step can be grinding, sonication, thermal shocks, or chemical treatments such as with chloroform.
2. To take into account heterogeneity the strategy is to take many subsamples and mix them.
3. Soil eDNA using trnL P6 loop will not give you accurate species lists in most floras, but rather lists of genera with occasional low-level or higher-level identifications. Most vegetation types are characterized by a few key species only, so having limited taxonomic resolution of your identifications is unlikely to affect the overall vegetation type calling. However in some floras or vegetation types this approach will be insufficient, e.g., for those characterized by specific taxa in locally speciose genera.

Molecular methods can contribute to the analysis of pollen both by identifying which species are present (qualitative) as well as by giving a measure of the abundance of different pollen species (quantification). While DNA metabarcoding methods are currently most often used (Table 1), DNA barcoding techniques have also been applied to target specific species from a mixture, while metagenomics now allows for pollen quantification. For a review of these different sequencing methods, see Chapter 10 DNA barcoding, Chapter 11 Amplicon metabarcoding, and Chapter 12 Metagenomics.

1. What are the main advantages of molecular pollen identification over traditional (microscopic) methods? Justify your answer.
2. Pollen is dispersed by various vectors. There are two main types of pollination strategies in land plants, please name them and also explain the importance of the difference between the two in terms of DNA yield.
3. Which four factors make the quantification of pollen grains using metabarcoding problematic?

Anemophilous – Wind-pollinated.

Bead beating – The application of beads to break open the outer cell wall of pollen grains.

Hirst-type pollen trap – Volumetric air sampler that is one of the standard devices for monitoring airborne pollen and spores.

cpDNA – Chloroplast DNA.

Entomophilous – Insect-pollinated.

Exine – Outer wall of pollen grains. Composed mainly of sporopollenin that is extremely resistant to degradation. The exine of pollen grains has to be broken to release the DNA from the organic material within the grains.

Palynology – The science that studies both living and fossil spores, pollen grains, and other microscopic structures (e.g., chironomids, dinocysts, acritarchs, chitinozoans, scolecodonts).

Pollen grains – The male gametophyte of seed plants; source and carrier for the male gametes (spermatozoids or sperm cells).

Pollenkitt – The outermost hydrophobic lipid layer mostly present on entomophilous pollen grains.

Sporopollenin – A chemically inert biological polymer that is a component of the outer wall (see Exine) of a pollen grain.

Super-resolution microscopy – Technique in optical microscopy that allows visualisation of images with resolutions up to 140 nm, much higher than those imposed by the diffraction limit. This technique also allows visualisation of internal structures.

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1. A higher taxonomic resolution can be achieved using molecular methods such as metabarcoding. Furthermore, pollen analysis requires highly trained experts that have to spend considerable time to analyse a single sample and therefore molecular techniques are faster, especially with a large number of samples.
2. Entomophilous (insect collected) and anemophilous (wind dispersed) pollen. The presence of pollenkitt on entomophilous pollen grains influences the amount of DNA that can be obtained per pollen grain.
3. Copy number, DNA preservation, DNA isolation technique, and amplification bias.

DNA-based methods for the molecular identification of plant products can help us to address food and medicine authenticity issues at each stage in the supply chain (Di Bernardo et al. 2007). Documentation and requirements for DNA-based detection methods for food authentication are defined in collaborative activities by the European Committee for Standardization (CEN) and the International Organization for Standardization (ISO). Both rapid and accurate identification of plant products are crucial for the the herbal drug industry (Mishra et al. 2016), where DNA-based authentication is recognised as a sensitive approach to identify edible and medicinal plant species, cultivars, and to detect their substitutes and adulterants in crude or processed products independent from life stage, tissue type, and physiological conditions of their constituents (Howard et al. 2020; Lo and Shaw 2018a, 2019; Mishra et al. 2016; Pawar et al. 2017; Raclariu et al. 2018; Techen et al. 2014). Molecular methods were integrated in the Pharmacopoeia of China in 2020 (Pharmacopoeia Committee of P. R. China 2020) and validated by the British Pharmacopoeia Commission in 2018 (British Pharmacopoeia Commission 2018) . In addition to species authentication, genetic identification of medicinal plants can assist in the field of pharmacophylogenomics and bioprospecting to discover new plant pharmaceutical resources (Hao et al. 2015) (Chapter 22 Healthcare).

The majority of standardised DNA-based authentication methods for the inspection and regulation of food and plant-medicines use well-established PCR-based techniques for DNA amplification as these are sensitive, specific, and simple (Hirst et al. 2019). PCR-based authentication methods are standardised in the CEN international legislation mainly for the detection of genetically modified foods (GMOs) including soybean, hazelnut, almond, rapeseed, etc. (Grohmann and Seiler 2019).

DNA barcoding methods are also established for the identification of unique medicinal and edible plant species (Ichim 2019). High resolution melting (HRM) in combination with DNA barcoding (Bar-HRM) can also be used to identify barcode differences in complex botanical matrices and assess the quality of crude materials in the herbal supply chain (Mezzasalma et al. 2017) (See Chapter 13 Barcoding - High resolution melting).

High-throughput sequencing (HTS) methods such as amplicon metabarcoding are also powerful tools for the authentication of herbal end products, post-marketing control, pharmacovigilance, and the assessment of species composition in botanical medicines, such as in traditional Chinese medicines (TCMs) (Arulandhu et al. 2017; De Boer et al. 2015; Juul et al. 2015; Lo and Shaw 2018b; Omelchenko et al. 2019; Raclariu et al. 2018; Seethapathy et al. 2019). An example of the discriminatory power of DNA metabarcoding is revealed in a study in which 15 highly processed TCM ingredients could be identified as species and genera listed on CITES appendices I and II (Coghlan et al. 2012).

In addition to PCR-based techniques, the detection of single nucleotide polymorphisms (SNPs) is frequently used for the molecular identification and authentication of various food commodities using small DNA fragments (Di Bernardo et al. 2007; Lo and Shaw 2019). Several assay types are commercially available for SNP chips and similar technologies (Hirst et al. 2019). Metagenomics is also promising for the qualitative and quantitative analysis of processed food and medicine matrices (Raime et al. 2020).

DNA-based methods for molecular plant identification depend on well-curated nucleotide sequence repositories. In addition to GenBank (Benson et al. 2018) and the Barcode of Life database (BOLD) (Ratnasingham and Hebert 2007), the Medicinal Materials DNA Barcode Database (MMDBD) has been proposed as a sequence reference platform to identify medicinal plant, animal, and fungi species (Wong et al. 2018).

Successful DNA extraction is the foundation for any further downstream analysis (Corrado 2016; Elsanhoty et al. 2011; Pinto et al. 2007; Turkec et al. 2015). Since food and medicine products can differ in molecular characteristics and structural form, the choice for a DNA isolation strategy must be sample specific. DNA extraction from most food products are based on DNA isolation techniques originally designed in the 1980s (Dellaporta et al. 1983; Lockley and Bardsley 2000), though these protocols are now typically adapted to include polymerases resistant to the inhibitors commonly found in a wide range of food and medicinal products (Omelchenko et al. 2019). Frequently used DNA extraction procedures are phenol-chloroform, detergent, and protease-based extraction methods and solid-phase extraction methods (see Chapter 1 DNA from plant tissue).

It is in the interest of both biodiversity conservation and public safety that DNA-based techniques are further developed to screen food and medicine sourced from the global market (Han et al. 2016; Ichim and de Boer 2020; Seethapathy et al. 2019). Standards for taxon-specific PCR techniques are described for some food plants, like soybean, hazelnut, almond, and rapeseed, but these are established mostly for allergen and GMP testing (Grohmann and Seiler 2019). HTS techniques are the only control tests that ensure the potential identification of all species in complex medicine and food products without prior knowledge on expected adulterants. These have become increasingly popular and more affordable methods for commercial laboratories. Today, laboratories offer analysis for meat, plants (including spices and herbs), fish, and crustaceans (Hirst et al. 2019). However, market tests have come with a higher detection limit and cannot guarantee a 100% confirmation of the presence or absence of an adulterant species (Hirst et al. 2019). Thus, the gap between the expectations and reality for DNA testing of food and medicine is the availability of commercial tests, the limit for detection and quantification, the specificity and the comparison with accurate reference materials and databases. In all these applications, however, the extraction of sufficient quantities of (reasonably) high-quality DNA are necessary. We can expect however that as the interest in using DNA-based methods for the authentication of food and medicine grows, that further development in methods related to extracting DNA from these often-challenging sources will also progress.

1. The quality of DNA from food and medicinal sources is a critical factor for DNA-based analyses. Which factors can influence the quality of nucleic acids extracted from foods and plant-based medicines?
2. What is the first step when choosing a DNA isolation technique for your samples?
3. What methods can be used for measuring DNA quality after isolation?

Bioprospecting - The exploration of biodiversity for new resources of social and commercial value.

Pharmacophylogenomics - Plant pharmacophylogenomics is a field established by combining the fields of ethnopharmacology, plant systematics, phytochemistry, pharmacology, and bioinformatics. It is the application of phylogenomics to the study of pharmaceuticals.

Pharmacopoeia - From the obsolete typography pharmacopœia, literally, “drug-making”. In its modern technical sense, it is a book containing directions for the identification of compound medicines, and is published by the authority of a government or a medical or pharmaceutical society.

Pharmaphylogenetics - Field of research focusing on the phylogenetic correlation between phylogeny, chemical constituents, and pharmaceutical effects of medicinal plants.

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1. Sample source and processing, collection and storage, homogenisation, and the presence of contaminants.
2. One should firstly consider whether it is a complex mixture or a pure product, and the degree of processing (form and degree of homogeneity).
3. Absorbance methods, agarose gel electrophoresis, and fluorescent DNA-intercalating dyes.

1. Name one sampling limitation of working with faecal samples as compared to other types of samples (e.g., gut contents) and give suggestions on how to overcome this limitation.
2. How does prior information of studied species ecology aid in sampling design?
3. Contamination can occur during sample collection, sample preprocessing, and DNA extraction. Describe the main type of contamination during each phase and how it can be prevented.

Coprolites – Fossilised faeces.

Near-infrared spectroscopy (NIRS) – A non-destructive and fast technique utilising the near-infrared region of the electromagnetic spectrum.

RNAlater – Non-toxic aqueous reagent for storage purposes, preserving RNA and DNA.

Stable isotopes – Non-radioactive elements.

Zoonotic disease – Infectious disease caused by pathogens jumping from non-human hosts to humans.

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1. Possible challenges and solutions: It is difficult to obtain fresh faecal samples → one can use pointing dogs; Problem of using relatively long DNA barcoding fragments → use primers that can amplify shorter regions; Overlapping habitats of closely related species → use additional molecular markers to identify species (though this increases the cost and time necessary); Faecal samples only provide a snapshot of the entire diet → take multiple samples from the same individual and/or sample a larger number of individuals over a longer period and larger geographical area.
2. It helps to narrow down the study areas for field collection. This reduces the manpower, resources, and time needed, increasing the chance of finding samples. When applying for permits, you can point out how to keep the disturbance of animals in the field to a minimum with this knowledge, which will increase the chances of obtaining permission.
3. During sample collection → wear gloves, ensure that samples are not collected from wet soil, practice good collection hygiene; During sample preprocessing → remove outer layers that were in close contact with the environment, work in flow-hood and a PCR-free lab; During DNA extraction → include extraction controls, avoid using extraction kits with plant-based or other types of contaminants.

Sedimentary ancient DNA studies aim to reconstruct the biology and ecology of past environments using the DNA present in the sediment record. Compared to modern soil and sedimentary DNA (see Chapter 4 DNA from soil), these analyses can be more challenging due to the prolonged exposure of the DNA to degradation processes. This has major implications for the scope of the study and the appropriate study design, which will be discussed in this chapter.

1. Name and explain two main advantages of using sedaDNA as a proxy for past plant presence compared to pollen. Motivate your answer.
2. Imagine you have a long lake sediment core that is thought to be between 50 000 and 10 000 years old. What dating methods could be used to date this core and why?
3. What are the main sources of bias when working with sedaDNA (name at least 3) and how can you limit the resulting false positives?

Alkylation – Addition or substitution of an alkyl group (C_nH_2n+1) to an organic molecule.

Accelerator Mass-Spectrometry (AMS) dating – A dating method that determines the age of an organic material (i.e., macroscopic remains of plants or animals) by measuring their radiocarbon concentration.

Cell lysis – The process whereby the membrane(s) of a cell breaks down, thereby releasing the cell contents.

exDNA – Extracellular DNA; all DNA located outside cell membranes.

Geochemical fingerprinting – A method using chemical signals to infer the origin, the formation and/or the environment of a geological sample.

Half-life – The time necessary for half of a radioactive atom’s nucleus to decay by emission of matter and energy to form a new daughter product. The half-life is specific to a radioactive element, and can be used for dating purposes.

iDNA – Intracellular DNA; all DNA present within cell membranes.

Lake catchment – Area of land from which water and surface runoff drains into a lake.

Luminescence dating – A group of methods to determine how long ago mineral grains were last exposed to sunlight or sufficient heating by measuring the luminescence emitted by the mineral grain upon stimulation.

Metabarcoding – Method for the simultaneous identification of many taxa within the same complex DNA extract. This is achieved by high throughput sequencing (HTS) of amplicons from taxonomic marker genes (barcodes).

Next Generation Sequencing (NGS) – Massively parallel sequencing technology allowing high throughput of DNA.

Nucleases – Diverse group of enzymes able to hydrolyze the phosphodiester bonds of DNA and RNA thereby cleaving them into smaller fragments.

Optically stimulated luminescence (OSL) dating – Dating method that determines the age of a sample by measuring the luminescence it emits in response to visible or infrared light.

Palaeoecology – The study of the relationship between past organisms and their ancient environments.

Permafrost – Soil, sediment, or rock that is continuously exposed to temperatures of < 0 °C for at least two consecutive years.

Radioactive isotope – An atom with excess nuclear energy and prone to undergo radioactive decay.

Reference library – A database of known DNA sequences with their taxonomic identifications, used in bioinformatics as a reference to identify the DNA sequences obtained in a sedaDNA study.

sedaDNA – Sedimentary ancient DNA; this is the aged and degraded DNA from dead organisms now incorporated in the sediment record, either as iDNA in dead tissues, or as exDNA free in the sediment matrix or adsorbed to sediment particles.

Shotgun sequencing – A method for the random sequencing of all of the DNA within a DNA extract.

Taphonomic processes – The processes involved in the transfer, deposition and preservation or organismal remains, including DNA.

Target capture – A technique that allows the capture of the DNA of interest by hybridization to target-specific probes (baits).

Tephrochronology – A geochronological technique that uses layers of tephra (volcanic ash from a single volcanic eruption) to create a chronological framework for the sedimentary record.

Thermoluminescence (TL) dating – Dating method that determines the age of a sample by measuring the luminescence it emits in response to heat.

Total DNA – The intracellular and extracellular DNA combined.

Tree-ring dating – Also called dendrochronology; a method of dating tree rings to the exact year they were formed.

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1. Possible advantages of sedaDNA compared to pollen as a proxy for past plant presence are: the possibility of detecting past plant presence even in the absence of visible remains; less labour-intensive as taxonomic identification is automated; in principle, no prior taxonomic knowledge is needed for the data generation with sedaDNA (although it is highly called for in the interpretation of the data); and it is possible to obtain a higher taxonomic resolution depending on the choice of marker.
2. For mineral-rich sediments, luminescence dating can be used as this method can be applied to sediments from a few decades old to over a million years old, and is based on the phenomenon that mineral crystals absorb electrons from ionising radiation of surrounding sediments over time. For sediment rich in organic materials, AMS radiocarbon dating of identified macroscopic remains (with calibration) is a good option. Radiocarbon dating is based on the concentration of C¹⁴ in organismic remains. The half-life of C¹⁴ (5730 years) makes it an appropriate method for samples under 50,000 years old. To increase confidence in the dating results, multiple dating techniques could be used for creating an age model for the core.
3. Biases when working with sedaDNA can come from: taphonomic processes including differential DNA degradation and preservation, choice of metabarcoding primers, completeness of reference library, and contamination during sampling, DNA extraction and other lab processes. False positives can be limited by inclusion of multiple replicates and controls and prevention of contamination at every step of the experimental design, preparation of an appropriate reference database, and checking if the identifications fit with what is known for the age and location of the sample by a taxonomic expert.

The revolution in genome-wide screening has vastly reduced the price for sequencing, with enormous implications in the biomedical field, industry, biodiversity monitoring, as well as in plant identification. The first plant genome (Arabidopsis thaliana L.) was sequenced using Sanger sequencing. This took 10 years to complete with an associated cost of approximately $100,000,000 (Arabidopsis Genome Initiative 2000). With current high-throughput sequencing (HTS) methods, this same genome now takes 1 week to sequence and assemble, and costs $1000 (Michael et al. 2018). Plant genomes and DNA sequences are however under-represented in the literature in comparison to other organisms such as microorganisms and animals, and most reported plant genomes belong to angiosperms with relatively small genomes. This is due to a number of confounding factors that make the sequencing of plant genomes particularly difficult including the extraction of sufficient quantities of high-quality DNA that is not irreparably damaged (Inglis et al. 2018) (see Chapter 1 DNA from plant tissue), the size and complexity of the genome (i.e., gene islands, high GC content, transposable elements), heterozygosity, and polyploidy (Chen et al. 2018) (see Chapter 16 Whole genome sequencing). Advances in high molecular weight DNA extraction, high throughput sequencing technologies, and bioinformatics approaches to deal with heterozygosity and assembly in recent years have alleviated these challenges tremendously, with enormous strides in the generation of high quality genomic datasets to test research hypotheses. In this chapter, we review the different sequencing technologies most commonly utilised today, beginning with Sanger sequencing, the current method of choice for small-scale projects. We then discuss HTS approaches, including next-generation sequencing and third-generation PCR-free sequencing methods (van Dijk et al. 2018). The underlying principles of these technologies are discussed and linked to their advantages and disadvantages in considering which method is best suited for different sequencing projects.

The sequencing platform that is ultimately chosen by a scientist depends on a number of factors. This can include (but is not limited to) the scientific question being considered, the quality of target DNA (see Chapter 1 DNA from plant tissue), costs, as well as in-house expertise and/or availability of existing platforms. In all cases, however, the quality and sequencing depth of target DNA should be considered. For DNA that is primarily expected to exist in shorter sequences (i.e., samples that are expected to be degraded from herbarium or ancient sources), then technologies requiring long reads are often not necessary, and Illumina sequencing or Ion Torrent technologies may be sufficient. If however one wishes to avoid any PCR bias or acquire long reads, then using PacBio or Nanopore is advisable. Finally, it may even be useful to use two different types of sequencing to overcome each technology’s respective limitations. For example, in whole genome sequencing, hybrid methods combining Illumina with PacBio are commonly used to ensure long reads and high accuracy.

In the last decades, developments in sequencing platforms have primarily focused on increasing the throughput and accuracy of sequencing output, increasing the length of reads, and reducing costs. We can expect the field to continue developing further in this direction, with a focus in particular on the miniaturisation of these platforms for more on-site work, as well as better automation and integration of analytical software and data analysis pipelines. In particular, miniaturisation and automatization of data analysis can be expected to have major impacts in regulatory fields related to both food safety and trade, where the ability for non-specialists to rapidly test on-site for the presence/absence of species will be extremely useful (see Chapter 22 Healthcare and Chapter 23 Food safety). Further development of HTS technologies to be used at the single-cell level and in functional studies can be also expected.

1. What method(s) are most commonly used for whole genome projects of plants and why? Which sequencing method(s) are most currently most commonly used for library preparations and why?
2. In a scenario where you may want to include amplicons and primers of different length when creating a library for sequencing, how would you adapt your library setup? How would it affect your sequencing costs?
3. Why do Nanopore and PacBio-based technologies provide longer reads than Illumina or Ion torrent-based technologies? Why are these longer reads especially useful for projects in plant identification?

Allopolyploid hybrids – A polyploid species with multiple sets of chromosomes that originate from different species. If the hybrid is derived from two diploid species, the resulting tetraploid is fertile. These allopolyploid hybrids may be at least partially reproductively isolated from the parent species from which they are derived, and allopolyploid speciation is the best known route to hybrid speciation in plants.

Bridge amplification – A method used in Illumina sequencing to create DNA clusters with 1000s of double-stranded copies of the target DNA in flow cells. After amplification and generation of these clusters is complete, the reverse strand is washed away and sequencing by synthesis takes place.

Capillary gel electrophoresis (CGE) – An analytical method for the separation of charged molecules. DNA is separated according to size with this technique, with only nanogram quantities necessary for the input. Single-base pair resolution can be achieved on fragments up to several hundred base pairs in length.

Circular consensus sequencing (CCS) – Developed by PacBio and also known as HiFi reads, involves the circulation of a target DNA strand by ligating the ends of the strand (called a SMRTbell). This SMRTbell can be read multiple times by a DNA polymerase, dramatically reducing the error rate in the generated sequence.

Electrolytic solution – An electrically conductive solution. This conductivity is often due to the presence of ions in solution (for example dissociated Na⁺ and Cl- ions), though non-ionic solutions can also be conductive.

Epigenetic modifications – Alterations in gene expression and cellular function without changes to the original DNA sequence. Three mechanisms for epigenetic modifications so far identified include DNA methylation, histone modification, and non-coding RNA (ncRNA)-associated gene silencing.

Insulating solid – A solid material that an electric current cannot pass through.

Ion-sensitive field-effect transistor (ISEFT) – A field effect transistor that can measure ion concentrations in solution. Changes in the H⁺ concentration result in a pH change in solution that results in changes in the current that is detected. This technology is used in Ion Torrent sequencing platforms to identify when a base pair is added to a growing DNA double strand and is the basis for identifying the target DNA sequence.

Phred quality scores – Scores to measure the confidence of the nucleobase identifications generated from DNA sequencing methods. They are widely accepted for assessing the quality of reads.

Rolling circular amplification (RCA) – Where a linear single-stranded DNA molecule is firstly circularised and then copied multiple times as a single sequence (Johne et al. 2009). With the nanopore platform, RCA allows the same DNA sequence to be read multiple times to give a higher read accuracy.

Single molecular real time sequencing (SMRT) – A term coined by PacBio to describe their sequencing technologies. In contrast to second generation sequencing methods, SMRT technologies possess single-molecule sensitivity and provide the sequence readout in real time, dramatically increasing the sensitivity and turnaround times for DNA sequencing.

Zero mode waveguide (ZMW) – Nanosized wells that can be etched into different materials, with attoliter (10^-21 L) volumes. ZMW technology differentiates a fluorescent molecule that is floating in solution from a fluorescently-labelled nucleotide that is located at the bottom of the well. This technology is used by PacBio for the single-molecule detection of fluorescently-labelled nucleotides that are added to immobilised DNA at the bottom of these wells so that nucleotide incorporation can be detected in real time.

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1. For whole genome projects, PacBio and Nanopore technologies are the most commonly used technologies. This is because both are long read technologies, which reduces the bioinformatic challenges related to assembling 1000s of short contigs together for assembling a whole genome. For library preparations, Illumina platforms are still the most commonly used due to their relatively competitive costs, high accuracy, and available support from a range of analysis tools and pipelines.
2. It is important to create equimolar pools so that the total number of DNA molecules is normalised across a library, so that one result does not dominate the others. However, even after normalisation of concentrations, it may still be the case that amplicons of very different length will not be amplified with the same efficiency. Additionally, using primers of roughly the same length so that their annealing temperatures are approximately the same is also important in order to avoid PCR bias within the same library. Thus, in a scenario with amplicons and/or primers of very different length, it is often best to put those amplicons in separate libraries. However, when amplicons and primers are of reasonably similar size, pooling the library samples can be an effective method to reduce sequencing costs.
3. Nanopore and PacBio based technologies provide longer reads than Illumina or Ion torrent based technologies since both have platforms available that do not require for a sample to be fragmented. These long reads can be especially useful for projects in plant identification when working with sequences that are particularly repetitive or have very large genomes. Additionally, PacBio technologies can also be used when longer amplicons are required for the phasing of haplotypes for instance or when tracing polyploid ancestry.

The method of identifying living organisms to species level using DNA sequences has been coined DNA barcoding (Hebert et al. 2003). It makes use of short (< 1000 bp), agreed-upon regions of the genome (a ‘barcode’) that evolve quickly enough to differ among closely related species (Kress et al. 2005). A generated barcode sequence from a sample allows for identification by matching the sequence against a reference library of sequences. Reference sequence libraries comprise sequences generated from vouchered and expert-identified materials in natural history collections, which are available through public sequence repositories or tailored databases. In other words, DNA barcodes function as molecular identifiers for individual species, in the same way as machine-readable black-and-white barcodes are used in the retail industry to identify products (Veldman et al. 2014).

DNA-based typing for species identification focused first on microbial organisms (Olive and Bean 1999). DNA barcoding as a concept distinct from DNA-based typing or phylogenetic analysis of taxon accessions was popularised by Hebert et al. (2003), who proposed to use the mitochondrial gene CO1 as the standard barcode for all animals. Despite initial scepticism (Rubinoff et al. 2006; Will and Rubinoff 2004), DNA barcoding was readily embraced by the scientific community. Assessments have since shown that CO1 can be used to distinguish over 90% of species in many animal groups: among these spiders (Barrett and Hebert 2005), birds (Hebert et al. 2004b), amphibians (Smith et al. 2008), and butterflies (Burns et al. 2008).

In recent years, the barcoding movement has grown substantially, and worldwide efforts coordinated by the Consortium for the Barcode of Life (CBOL) are now being focused on barcoding all organisms (Hobern and Hebert 2019; Hobern 2020). The amount of sequencing data derived from DNA barcoding is exponentially increasing, and it is now considered a mainstream taxonomic tool. Although DNA barcoding does not replace the need for traditional taxonomy, it does highlight the need for robust species descriptions to enable accurate identification of species from “orphan” barcodes (sequences from unnamed species). Integrative taxonomy, which is achieved by combining evidence from morphology, ecology, phylogenetics and DNA barcoding, is critically important to speed up species discovery in the light of biodiversity loss (Padial et al. 2010; Schlick-Steiner et al. 2010).

Species delimitation is a central tenet of taxonomy (see Chapter 17 Species delimitation). Traditionally, species were identified, described and classified based mainly on their morphological characters. This is more difficult when it comes to cryptic, hybridising or highly convergent species (Struck et al. 2018). Combining characters, such as molecular data and behaviour, can provide further confidence when attempting to distinguish between species (Schlick-Steiner et al. 2010). However, species delimitation remains fundamentally difficult due to the fact that it is unclear how a species should be defined (de Queiroz 2007). The assumption that species are fixed entities underpins every international agreement on biodiversity conservation, all national environmental legislation and the efforts of many individuals and organisations to safeguard plants and animals (Garnett and Christidis 2017). However, one of the major unresolved questions in science is ‘What is a species?’, even though this is one of the most important concepts in biology (Kennedy and Norman 2005). Species concepts differ, and a single definition that fits all organisms has not been found (de Queiroz 2007).

Most species concepts agree on species being evolving metapopulations (de Queiroz 2007), and this implies that genetic variation exists both within and between species. Advanced approaches using many accessions as well as many loci, such as species delimitation based on multispecies coalescent theory, can enhance species identification resolution. However, more data also adds new challenges, and inferred structure due to population-level processes and that due to species boundaries are hard to distinguish (Sukumaran and Knowles 2017). Initial studies on DNA barcoding suggested a significant barcoding ‘gap’ between intra- and interspecific variation (Barrett and Hebert 2005; Hebert et al. 2004a, 2003), but these studies have been criticised for undersampling both intraspecific and interspecific divergence (Meyer and Paulay 2005). A DNA barcoding reference database for identification that would include all species references should also contain multiple accessions of populations to ensure that intraspecific and interspecific variation can be distinguished In absence of this ideal situation, many studies use more or less arbitrary cut-off percentages for sequence divergence (Blaxter et al. 2005; Ghorbani et al. 2017a; Veldman et al. 2017). Species assignments in DNA barcoding are hypotheses similar to species assignments based on morphology.

To identify an unknown DNA barcode using a reference library, one can use several approaches to look at the interrelatedness of the samples (see Chapter 18 Sequence to species). Many databases including GenBank and BOLD (Ratnasingham and Hebert 2007) make use of the similarity based BLAST (Altschul et al. 1990). Similarity based on genetic distance can be used as above and for phylogenetic tree reconstruction (Hebert et al. 2003). Disadvantages of using distance based information are 1) these do not yield diagnostic characters for species distinction (DeSalle et al. 2005); 2) similarity scores do not always give the nearest neighbour as the closest relative (Koski and Golding 2001); and 3) a lack of an objective set of criteria to delineate taxa when using distances (Goldstein et al. 2000). Other common approaches are based on characters instead of distances, and these rely on phylogenetic methods using maximum likelihood (Felsenstein 1973), parsimony (Nixon 1999), Bayesian statistics (Huelsenbeck and Ronquist 2001), or multispecies coalescent methods (Yang and Rannala 2017). These phylogenetic methods are implemented in RAxML (Stamatakis 2006; Swofford 2002), PAUP* (Swofford 2002), MrBayes (Huelsenbeck and Ronquist 2001) and BPP (Yang 2015), respectively. Character-based tree building overcomes many of the shortcomings of distance-based results, but tree-based methods have limitations if single gene trees are used to infer phylogenetic relationships. Another limitation of tree building for species identification is that evolution at the species level is not hierarchical. Applying hierarchical methods and terms, such as trees, classification and monophyly for delimitation of species, is not reflective of the evolutionary history of individuals and populations within a species (DeSalle et al. 2005). Veldman et al. (2014) summarised several suggestions to minimise the effect of these drawbacks, such as a diagnostic system including other lines of evidence (DeSalle et al. 2005), a probabilistic modelling approach (Knowles and Carstens 2007), the use of dominant and codominant multi-locus markers (Hausdorf and Hennig 2010) and new heuristic methods without fixed species assignments (O’Meara 2010).

The mitochondrial genome in plants evolves far too slowly to allow it to distinguish between species (Cho et al. 2004). Phylogenetic studies of plants focused early on plastid markers as well as the ribosomal DNA (Palmer et al. 1988; Ritland and Clegg 1987). In the search for alternatives to the popular animal marker COI, various genes and non-coding regions in the plastid genome were proposed (CBOL Plant Working Group 2009; Fazekas et al. 2008, 2009; Hollingsworth 2011; Kress and Erickson 2007; Kress et al. 2005). In its most basic definition, a barcode must differ between species so that species can be identified. However, a barcode should not differ much within species, and not be too different between species within the same genus or family because this would make it more difficult to assign a unknown to a group with confidence.

The plastid marker rbcL was for example good to infer relationships between angiosperm families (Soltis et al. 1999) but varies too little for species discrimination in many plant genera (China Plant BOL Group et al. 2011). In addition to being sufficiently rapidly evolving, a barcode must also be flanked by conserved regions of the genome that can function as universal amplification primer binding sites. A single primer pair that would amplify any of over 350,000 species of plants would be ideal (Kress et al. 2005). The plastid coding region matK for example has variation between species, but it can be difficult to amplify universally (de Boer et al. 2014; Kool et al. 2012) (de Boer et al. 2014; Piredda et al. 2011; Sass et al. 2007). Insufficient primer universality makes this marker difficult to use in large-scale studies across families, although using target-group specific-primers for amplification is often successful (Mahadani and Ghosh 2013; Palhares et al. 2015; Purushothaman et al. 2014; Wallace et al. 2012). Other considerations can also affect barcode marker choice.

The nuclear ribosomal marker ITS, and specifically nrITS2, is used commonly in barcoding and metabarcoding studies (China Plant BOL Group et al. 2011; Ivanova et al. 2016; Raclariu et al. 2017, 2018). nrITS2 has been long advocated as a secondary marker to plastid barcodes (China Plant BOL Group et al. 2011). However, nrDNA has limitations for phylogenetic inference that also apply to barcoding (Álvarez and Wendel 2003), including alignment difficulties and limited utility in phylogenetic inference between closely related and/or recently diverged taxa (Manzanilla et al. 2018). It is also a challenge to determine the orthology and the paralogy of nrDNA sequences in the case of hybridization events or incomplete lineage sorting (Bailey 2003; Fehrer et al. 2007; Soltis and Kuzoff 1995). nrITS is also present in multiple copies, and these copies can belong to different parental lineages in hybrids, and PCR amplification success of these copies is unrelated to whether these copies are functionally transcribed or not, which in turn has an influence on the substitution rate of these sequences (Kool et al. 2012). Bailey et al. (2003) emphasised that especially for allopolyploids nrDNA might not be the optimal choice to assess species trees, which applies equally well to species assignment in DNA barcoding studies.

The strict requirements for both universality and high variability for potential universal barcodes has led some to label DNA barcoding a “search for the Holy Grail” (Rubinoff et al. 2006). Since there is still no single plant barcoding locus combining variability and universality, the current consensus is that a combination of two or more markers should be used for standard barcoding applications (CBOL Plant Working Group 2009; China Plant BOL Group et al. 2011; Hollingsworth 2011). Thus, where the animal community is entirely focused on using standardized and defined markers for species discrimination, the plant community has a looser vision for DNA-based identification of plants with tailored solutions based on their study objective. Plant DNA-based identification incorporates plastid, nuclear ribosomal and nuclear sequence data ranging from barcodes through plastomes, genome skimming and target capture to whole genomes (Bohmann et al. 2020; Coissac et al. 2016; Hollingsworth et al. 2016; Manzanilla et al. 2018).

The core plant DNA barcoding markers are rbcL and matK (CBOL Plant Working Group 2009). nrITS (or nrITS2 only) is the third most commonly used barcode (China Plant BOL Group et al. 2011; Hollingsworth 2011; Kress et al. 2005). The trnL-F spacer, psbA-trnH, and rpoC1 (Ghorbani et al. 2017b; Kool et al. 2012; Kress et al. 2005) are also reported in literature, and the trnL P6 loop is the standard barcode for plant metabarcoding studies (Taberlet et al. 2012, 2007).

When choosing appropriate markers for a plant DNA barcoding study it is important to consider the following questions:

What is the necessary taxonomic level of identification? For composition studies of a flora or vegetation, genus-level identifications are often sufficient. Species-level identification can however be important for other questions. Identifying all angiosperms in Greenland is more straightforward than in a Neotropical rainforest. Also, although family-level identifications in Greenland provide useful insights into the local flora, this information most often does not have meaningful applications in rainforests. After deciding on the appropriate level of identification, the researcher then needs to determine whether multiple markers are necessary to ensure that all species can be distinguished.

What kind of a reference library will you use to identify the target barcodes? Query identification in a database that contains all plants is more challenging than with a tailored reference library. For example, identifying a sequence of Oxalis (Oxalidaceae) is easy in a database of Scandinavian plant sequences because there is only a single native Oxalis species. Any queried Oxalis sequence would match the Scandinavian Oxalis acetosella because it would be the only reference Oxalis sequence in a local database. In contrast, a database with South American Oxalis species has hundreds of taxa, and identification requires a marker with sufficient variation to discriminate between these species. Thus, for Scandinavia, one could use a marker with limited variation but universal primers, whereas for South America a specific marker or markers should be sought that can distinguish all Oxalis species present in a global database. It is therefore critical to pick your marker(s) based on the expected diversity in your reference library.

What is your source of reference sequences? If you want to identify species, which is common in studies aiming to authenticate herbal drugs and supplements, you need to include all putative species in your reference library. For example, if your goal is to identify a European wild collected Hypericum, your reference library should ideally include all European Hypericum species that could be confused or substituted for Hypericum perforatum. A reference library can be compiled from de novo sequenced amplicons from voucher accessions or from reference sequences mined from public repositories.

After choosing one or several markers, it is important to consider the following:

Are universal primers available? If yes, this facilitates your project. However, are these primers really universal? Check this by seeing whether the study publishing the primers gets cited by relevant studies and look for larger studies and reviews that might provide more information about (1) amplification success with these primers; (2) ability to amplify from degraded or poor DNA extracts, a common challenge when working with older herbarium vouchers or processed herbal products; and (3) the need to tweak amplification protocols to make these primers work. If no universal primers are available, try to find studies using this marker and see which primers were used, to find suitable primers that you can then test. If possible, use studies targeting the same target order, family or genus. If there are no previously published primers for your marker, then it is necessary to design your own. If your primers target a widely used marker, then the primer performance that is assessed based on matching these novel primers to multiple sequence alignments of published data (in-silico testing) is generally reliable. If only genomic data is available, however, the accuracy of in-silico testing will be highly dependent on the relatedness of the reference genomes.

Do the primers amplify the right part of the marker? Primers can target fragments of longer loci, i.e., parts of rbcL, matK, nrITS. It is thus important that the segment the primers amplify is useful for your study. It should generate sequences that are identifiable in your reference library and variable enough for your intended level of identification. For example, targeting trnL intron with the universal g-h primers will yield short amplicons, and these have less variation than the entire trnL-F region. Make sure you reassess your marker choice after selecting suitable primers.

How many primers per marker will you use? Long markers can be hard to amplify from degraded templates and can be split up into multiple primer pairs. Degraded DNA is a common challenge when working with a common challenge when working with older herbarium vouchers or processed herbal products. Different combinations of forward and reverse primers can also increase the chance of successful amplification as having multiple different primers can increase the chance that one of these has a good fit to the organism being tested. However, the primer pair with the best fit and targeting the shorter marker will amplify more effectively than other pairs or longer fragments, and can lead to amplification bias.

Once a suitable combination of markers has been found and suitable primers or primer panels have been selected, it is important to test the primers on a sufficient number of your samples. Template DNA quality, DNA concentration, and the effects of inhibiting secondary metabolites can all influence the efficacy of the PCR and might require optimization to obtain the best possible results for the largest number of samples. This is beyond the scope of this book, but sufficient online resources are available to help you with optimization. In addition, there are many online discussion forums to troubleshoot PCR optimization.

The subsequent chapters in section 2 describe different sequencing platforms and approaches to obtain DNA sequences for downstream analysis, and section 3 provides an overview of applications of molecular identification of plants. Depending on whether one chooses standard DNA barcoding using Sanger sequencing, DNA metabarcoding using Ion Torrent, Illumina, or other platforms, or a variety of whole or reduced library representation genome sequencing approaches, one will need to choose different wet lab steps to create the relevant sequencing libraries. Check out the relevant chapter for your application to find out more.

1. An author writes that she used DNA barcoding to identify Bellis perennis using rbcL. The generated query sequence matched 100% with the reference of Bellis perennis in GenBank. Can you think of two situations that would falsify this finding?
2. You are planning to use DNA barcoding to distinguish herbal medicines based on Paeonia. In the literature you find that five Paeonia species are commonly used in herbal medicines and that these can be distinguished using nrITS2 sequences. In your study of 37 herbals, you find that 15 contain species A, 7 B, 5 C, 3 D, and 2 E, whereas five samples fail to amplify nrITS2. 2A) How can you be sure that these 37 products contain only these five species? 2B) What does your experiment tell you about the five samples that failed to amplify?
3. You want to investigate if DNA barcoding can outperform morphology-based biodiversity assessments in terms of species identification. For what material do you expect DNA barcoding to be more useful than morphology-based identification?

matK – Plastid gene coding for maturase K. matK is one of the core plant DNA barcodes.

nrITS – Internal transcribed spacer (ITS) is a spacer situated between the small-subunit rDNA and large-subunit rDNA genes. In plants, it flanks the 18S and 26S rDNA genes. nrITS is split into two spacers, nrITS1 and nrITS2 with the 5.8S rDNA gene in between. nrITS is highly variable, and primers are designed in the conservated 18S, 5.8S, and 26S rDNA genes.

psbA-trnH – Plastid intergenic spacer region between the coding genes psbA and trnH. psbA-trnH has been advocated as a plant DNA barcoding marker.

Primer – Short DNA sequence used to amplify a marker.

rbcL – Plastid gene coding for ribulose-1,5-bisphosphate carboxylase-oxygenase. Most barcoding studies target the rbcLa region, but will refer to rbcL. rbcL is one of the core plant DNA barcodes. Plastids in plants are often incorrectly referred to as chloroplasts.

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1. Some things that might have been overlooked: (1) Does NCBI GenBank list more than one species of Bellis? If not, then it might be any other Bellis species not present in this database; (2) How much variation does rbcL have in Bellis? Does the query match 100% with more than one species of Bellis? If yes, then a more variable marker should be used.
2. Answer for 2A) The study can ascertain that only these five species are present if it includes a sequence reference database of all other Paeonia species (or those possibly present). If the sequence reference database contains only the five common species, then no such conclusion can be made. 2B) The failed samples could: not include Paeonia; contain degraded Paeonia DNA that is not amplifiable; or contain inhibitors that make the DNA nonamplifiable.
3. Answers could include: vegetative material such as roots, leaves, and seedlings, DNA extracts from bulk samples, soil DNA, faecal DNA, pollen DNA, or air-captured eDNA.

At the start of any (plant) metabarcoding study lies a clearly defined research question. A study design should furthermore encompass a clear sampling strategy, and identification of suitable DNA extraction techniques for the sample type used before carrying out downstream analysis (Zinger et al. 2019). As the chapters in Section 1 already details DNA extraction methods based on specific starting materials, this section will cover the subsequent steps, starting with selecting the plant barcodes to best answer the research question, choosing a nucleotide tagging strategy, sequencing and finally analysing the sequence output using bioinformatics pipelines.

Currently, metabarcoding is the dominant technique used in the identification of plants from mixed samples. Developments and improvements in addressing methodological challenges such as PCR bias may one day allow for unbiased quantitative inferences from metabarcoding datasets. This would be a huge step forward for the metabarcoding community since it is still controversial to use read counts as an indication for biomass (Deagle et al. 2019). With the continued advances in HTS technologies coupled with the inherent limitations of metabarcoding, there is also a possibility that alternative HTS techniques can be used in the future. For example, the development of more regional DNA reference databases based on whole organelle genomes instead of single barcode regions (Coissac et al. 2016) (see Chapter 10 DNA barcoding) would encourage the use of HTS techniques that rely on whole genomes or multiple non-standard barcode regions for taxonomic identification. Particularly, if sequencing becomes cheaper and if the limitations of metagenomics (see Chapter 12 Metagenomics) or target capture (see Chapter 14 Target capture) are addressed, we may see an increase in other types of methods used to identify plants in mixed templates. However, metabarcoding has the advantage of being a cheaper option, where large numbers of samples can be processed for meaningful statistical analysis. Bioinformatics pipelines are also well-established and better reference databases are available for mini barcodes as compared to whole organelles. This makes metabarcoding the preferred technique for many applications. In addition, ongoing efforts to build curated reference databases, design better primers, and detect potential plant-specific barcode regions might increase species resolution and circumvent many of the drawbacks associated with metabarcoding (Chua et al. 2021c).

Metabarcoding could potentially be used to determine plant composition in a landscape from bulk arthropod samples. Bulk arthropod samples have been used for biodiversity monitoring of vertebrates (Lynggaard et al. 2019), but it has not been used for any plant-related studies. Another potential application of metabarcoding is in forensic genetics (see Chapter 26 Forensic genetics, botany and palynology), where plants are used as evidence in criminal investigations (Bryant 2013). For example, morphological identification of pollen grains has been used to solve murders and determine marijuana distribution locations (Alotaibi et al. 2020; Bryant 2013). However, metabarcoding is underutilised in these applications where morphological identification is still the main technique. One possible limiting factor for this lack of utilisation could be that pollen DNA extraction destroys the samples and therefore cannot be stored as evidence (Bell et al. 2016). Metabarcoding could also potentially be used in meta-phylogeographic studies to simultaneously study the phylogeographic features and intraspecies patterns of many species (Turon et al. 2019).

1. How can overamplification of the most represented taxa in a single sequencing run of multiple complex mixtures be avoided?
2. Which DNA barcode region is most suitable for dealing with plant DNA from samples where DNA is expected to be degraded?
3. The nuclear ribosomal ITS region is shared between plants and fungi. How can undesirable fungal DNA amplification be avoided?

Adapters – Specific nucleotide sequences unique to different types of sequencing platforms that are added to amplicon libraries to allow for the attachment of library fragments to the flow cell for sequencing.

Amplicons – Products of PCR amplification.

ASVs – Amplicon sequence variants are also known as exact sequence variants or zero-radius OTUs. Although sometimes considered synonymous to OTUs, they correspond to all the unique reads in a dataset and do not require clustering used in creating OTUs.

Barcode – Targeted gene region, see Locus.

Demultiplexing – Bioinformatics step of assigning sequences to samples based on assigned nucleotide tags and/or library indexes.

Epilithic – Plant growing on surfaces of rocks, e.g., seaweeds.

Homopolymers – Nucleotide repetition, usually in tandem of more than 7 nucleotides.

Indel errors – Insertions or deletions in sequences resulting from mutations.

ITS – The internal transcribed spacer is a nuclear ribosomal region found between the small subunit ribosomal RNA (rRNA) and large-subunit rRNA genes.

Library indexes – Nucleotide index added to amplicon libraries to allow for the parallel sequencing of multiple libraries, which can be used bioinformatically to assign reads to the correct amplicon libraries.

Locus – Section and position in a chromosome where a particular DNA sequence is located. It can also be referred to as a barcode.

Macrofossils – Preserved plant remains large enough to be seen without a microscope.

matK – Maturase K is a gene found in the chloroplast genome.

Meta-phylogeography – Study of phylogeographic features and intraspecies variation.

Multiplexing – Parallel amplification of barcodes in one PCR reaction.

OTU – Operational taxonomic unit. The term is used to categorise clusters of similar sequences.

Overhangs – Stretch of unpaired nucleotides at the end of DNA fragments.

PCR – Polymerase chain reaction.

PCR stochasticity – Uneven amplification of molecules during PCR that can be a result of some sequences being present in lower copy numbers than others.

Phylogeography – Investigate the origin of genetic variation within closely related species across a landscape.

Primers – A short single-stranded nucleic acid sequence that serves as a starting point for the DNA replication in the PCR.

Primer set – Nucleic acid sequences explained above complementary to the 5’ end and 3’ end of the flanking regions of a loci.

Primer bias – Differences in DNA amplification due to a primer inefficiently binding to the target template. This can result from sequence divergence in the primer binding sites.

qPCR – Polymerase chain reaction used for quantifying DNA.

rbcL – The ribulose-1,5-bisphosphate carboxylase large subunit gene is found in the chloroplast genome.

Singletons – A sequence only present in one copy.

Nucleotide tags – Short nucleotide sequences added at the 5’ end of the primer in metabarcoding studies.

Tag jumps – Generation of amplicons with different tags than originally used, resulting in false positives in the data. For more detail see (Schnell et al. 2015).

Taxa – Plural of taxon. A taxon is a group of organisms that form a taxonomic group.

Taxonomic assignment – Matching the obtained sequences to taxa names.

trnH-psbA – An intergenic spacer region found in the chloroplast genome.

trnL – The trnL gene is part of the trnL-F region of the chloroplast genome.

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1. By using equimolar pooling of individual samples.
2. The highly stable P6 loop can best be targeted in this case, using trnL primers.
3. By using plant-specific ITS primers that minimise the amplification of fungal DNA.

Metagenomics is the study of genetic material recovered directly from environmental samples such as air, water, soil, or sediments (Bashir et al. 2014). It is also referred to as environmental genomics, ecogenomics, or community genomics (Guazzaroni et al. 2009). The DNA found in environmental samples are usually a mixture of genetic materials from multiple organisms. Typically, genomic DNA extracted from environmental samples is shotgun sequenced to identify organisms and/or make metabolic and other protein predictions (Porter and Hajibabaei 2018). Prior to sequencing, DNA molecules are first fragmented into smaller pieces. DNA molecules from samples are first randomly fragmented into size-controlled fragments. These fragments are then subsequently converted into libraries which consist of the DNA fragments attached to adapters specific to the sequencing platform used. Each library is then sequenced using the shotgun sequencing approach, often without targeted PCR amplification (Noonan et al. 2005). This provides a distinct advantage over PCR-based methods by enabling a less biassed investigation of a community, and the detection of all genes in a sample.