Three main photosynthetic pathways operate in terrestrial plants, each with their particular biochemistry, including C3, C4 and CAM. These pathways have important consequences for optimum temperatures of photosynthesis (higher in C4 than in C3 plants), water- and nutrient-use efficiencies, and responsiveness to elevated CO2. Compared with C3 plants, C4 plants tend to perform well in warm, sunny and relatively dry and/or salty environments (e.g. in tropical savannah-like ecosystems), whereas CAM plants are generally very conservative with water and occur predominantly in dry and warm ecosystems. Some submerged aquatic plants have CAM too. There are obligate CAM species and also facultative ones, which may switch between C3 and CAM, depending on environmental factors (e.g. epiphytic orchids in high-elevation Australian rainforests). Two main identification methods are available, namely C-isotope composition and anatomical observations. CAM can be inexpensively confirmed by verifying that stomata are open at night and closed during the day, or by measuring diurnal patterns of organic acids or leaf pH values. Which method to choose (a combination would be the most reliable) depends on facilities or funding, as well as on the aim of the work (e.g. to contrast C4 v. C3, or CAM v. C3). Although C-isotope composition can be affected by environmental factors, intraspecific genetic differences and/or phenological conditions, intraspecific variability is small enough not to interfere with the distinction between C4 and C3 photosynthetic pathways. In many plant families, only C3 metabolism has been found. It is useful to know in which families C4 and CAM have been found, so that species from those families can be screened systematically as potential candidates for these pathways (see Material S4; Table 1). Below we describe two methods which in combination provide good contrast between pathway types.

What and how to collect?

Collect the fully expanded leaves or analogous photosynthetic structures of adult, healthy plants growing in full sunlight or as close to full sunlight as possible. We recommend sampling at least three leaves from each of three individual plants. If conducting anatomical analysis (see under (B) Anatomical analysis in the present Section), store at least part of the samples fresh (see Section 3.1).

(A) C-isotope analysis

Storing and processing

Dry the samples immediately after collecting. Once dry, the sample can be stored for long periods of time without affecting its isotope composition. If this is not possible, the sample should first be stored moist and cool (see under Section 3.1) or killed by using a micro-wave and then be dried as quickly as possible at 70–80°C, to avoid changes caused by loss of organic matter (through leaf respiration or microbial decomposition). Although not the preferred procedure, samples can also be collected from a portion of a herbarium specimen. Be aware that insecticides or other sprays that may have been used to preserve the specimen, can affect its isotope composition.

Bulk the replicate leaves or tissues for each plant, then grind the dried tissues thoroughly to pass through a 40-µm-mesh or finer screen. It is often easier with small samples to grind all of the material with mortar and pestle. Only small amounts of tissue are required for a C-isotope-ratio analysis. In most cases, less than 3 mg of dried organic material is used.


Carbon isotope ratios of organic material (δ13Cleaf) are measured using an isotope ratio mass spectrometer (IRMS, precision between 0.03 ‰ and 0.3 ‰, dependent on the IRMS used) and are traditionally expressed relative to the PDB (Pee Dee Belemnite) standard as δ13C in units of per mil (‰), i.e. parts per thousand. After isotopic analysis, the photosynthetic pathway of the species can be determined on the basis of the following (see graphic explanation in Material S4; Fig. 1):

C3 photosynthesis δ13C: –21‰ to –35‰,

C4 photosynthesis: –10‰ to –14‰,

Facultative CAM: –15‰ to –20‰ and

Obligate CAM: –10‰ to –15‰.

Separating C3 or C4 from CAM plants is difficult on the basis of δ13C alone (for facultative CAM plants, δ13C values have been found to range as widely as from –14‰ to –23‰). However, as a rule of thumb, if δ13C is between –10‰ and –23 ‰, and the photosynthetic tissue is succulent or organic acid concentrations are high during the night, but low during the day, then the plant is CAM. In such cases, anatomical observations and diurnal measurements of gas exchange or biochemical analysis would be decisive (see (B) Anatomical analysis in the present Section).

(B) Anatomical analysis

C3 and C4 plants typically show consistent differences in leaf anatomy, best seen in a cross-section. Using a razor blade or microtome, make cross-sections of leaf blades of at least three plants per species, making sure to include some regular veins (particularly thick and protruding veins, including the midrib and major laterals, are not relevant). C3 plants have leaves in which all chloroplasts are essentially similar in appearance and spread over the entire mesophyll (photosynthetic tissues). The mesophyll cells are not concentrated around the veins and are usually organised into ‘palisade’ and ‘spongy’ layers parallel to, and respectively adjacent to, the upper to lower epidermis (see Material S4; Fig. 2) (vertically held C3 leaves often have a palisade layer adjacent to each epidermis and a spongy layer between the two palisades). The cells directly surrounding the veins (transport structures with thin-walled phloem and generally thicker-walled xylem cells), called bundle sheath cells, normally contain no chloroplasts. C4 plants, in contrast, typically exhibit ‘Kranz anatomy’, viz., the veins are surrounded by a distinct layer of bundle-sheath cells (Material S4; Fig. 2) that are often thick-walled, and possess abundant, often enlarged chloroplasts that contain large starch granules. The mesophyll cells are usually concentrated around the bundle-sheath cells, often as a single layer whose cells are radially oriented relative to the centre of the vein, and contain smaller chloroplasts with no starch grains. These differences can usually be identified easily under an ordinary light microscope. Many plant physiology and anatomy textbooks give further illustrations of Kranz v. typical C3 leaf anatomy (see More on methods below in the present Section).

If Kranz anatomy is observed, the species is C4. If not, it is likely to be C3 unless the plant is particularly succulent and belongs to one of the families with CAM occurrence. In the latter case, it could be classified as (possible) CAM. Many CAM leaves do not have typical C3 palisade or spongy mesophyll layers, but only a thin layer of more or less isodiametric, chloroplast-containing cells just under their epidermis, with the entire centre of the leaf consisting of large, thin-walled, colourless parenchyma cells that store water and organic acids. If living plants are within easy reach, an additional check could be to determine the pH of the liquid obtained by crushing fresh leaf samples in the afternoon (see Section 3.5), and again (with new, fresh samples from the same leaf population) at pre-dawn. Because in a CAM plant, organic (mostly malic) acids build up during the night, and are broken down during the day to supply CO2 for the photosynthesis in the leaf, CAM species show a distinctly lower pH after the night than they do in the afternoon. In addition, C-isotope ratios can provide further evidence to distinguish between CAM and C3 or C4 metabolism (see (A) C-isotope analysis above in the present Section).

Special cases or extras

(1) Permanent slides or photographs and chloroplast visibility. A range of methods is available for making the microscope slides permanent; however, be aware that some may result in poorer visibility of the chloroplasts. One method for retaining the green colour of the chloroplasts is to soak the plant or leaves in a solution of 100 g CuSO4 in 25 mL of 40% formal alcohol (formaldehyde alcohol), 1000 mL distilled water and 0.3 mL 10% H2SO4 for 2 weeks, then in 4% formal alcohol for 1 week, subsequently rinse with tap water for 1–2 h and store in 4% formal alcohol until use. However, material thus treated can be sectioned only by using a microtome after embedding or freezing it, in contrast to many living, turgid leaves, which can be sectioned free-hand by using a suitable technique such as sectioning a rolled-up leaf or a stack of several leaves. Photomicrographs of freshly prepared sections are an alternative way to keep records for later assessment.

References on theory, significance and large datasets: O’Leary (1981); Farquhar et al. (1989); Earnshaw et al. (1990); Ehleringer et al. (1997); Lüttge (1997); Zotz and Ziegler (1997); Wand et al. (1999); Pyankov et al. (2000); Sage (2001); Hibberd and Quick (2002).

More on methods: Farquhar et al. (1989); Ehleringer (1991); Hattersley and Watson (1992); Mohr and Schopfer (1995); Belea et al. (1998); Pierce et al. (2002); Taiz and Zeiger (2010).