We are indebted to the editorial board of the Journal of Forestry Research for encouraging and authorizing us to use the representative results in this protocol from the article published there. We also kindly thank two anonymous reviewers for their valuable comments, which have substantially improved the manuscript. The research was funded by the Ministry of Agriculture of the Czech Republic, institutional support MZE-RO0118 and the National Agency of Agricultural Research (Project No. QK1810126).

1. LAI estimated using litter traps
<ol>
	<li>First, perform a field survey, investigating the site conditions and structure of the studied stands (i.e., inclination and exposition of the slope, forest or vegetation type, forest or vegetation density, homogeneity of the canopy closure, the crown size, and the crown base height).</li>
	<li>Select a suitable litter trap type for positioning below the canopy by choosing the mesh size of the net based on the size of the assimilation apparatus of the studied stands...

The authors have nothing to disclose. The representative results were used from the article &#268;ern&#253; J, Haninec P, Pokorn&#253; R (2018) Leaf area index estimated by direct, semi-direct, and indirect methods in European beech and sycamore maple stands. Journal of Forest Research. doi: 10.1007/s11676-018-0809-0 (online version) based on the kind permission of the Journal of Forestry Research editorial board.

Litter traps are deemed as one of the most accurate methods for performing LAI estimation8, but they are more labor-intensive and time-consuming than the indirect methods35,64 which were incorporated into this protocol. Within the entire LAI estimation procedure using litter traps, a precise estimation of the SLA is the most critical point10 because the SLA can vary with plant species65, date...

LAI, defined as half of the total leaf surface area per unit of horizontal ground surface area1, is a key variable used in many bio-geophysical and chemical exchange models focused on carbon and water fluxes2,3,4. LAI is directly proportional to the active surface of leaves where it drives primary production (photosynthesis), transpiration, energy exchange, and other physiological attributes connected with a range of ecosystem processes in plant communities5.
Numerous approaches and instruments for performing LAI estimation have been developed, and they are currently available on the market6,7,8,9. Ground-based methods for performing LAI estimation can be grouped into two main categories: (i) direct, and (ii) indirect methods10,11,12. The first group includes methods measuring leaf area directly, while the indirect methods infer LAI from measurements of more readily measurable parameters, using radiative transfer theory (in terms of time, labour-intensiveness, and technology)13,14.
This protocol deals with the practical use of litter traps and the needle technique, as non-destructive semi-direct methods10; and the optical device plant canopy analyzer as an indirect method6,7 for performing LAI estimation on a chosen sample from temperate deciduous forest stands in Central Europe (see its structural and dendrometric characteristics in Appendix A and Appendix B).
In deciduous forests and crops, it is possible to perform non-destructive semi-direct LAI estimation using litter traps11 distributed below the canopy layer15. Litter traps provide precise LAI values for deciduous species in which LAI reaches a plateau within the growing season. However, for species that can replace leaves during the growing season, such as poplar, the method overestimates LAI11. This method assumes that the content of the traps represents the average amount of leaves that fall during a leaf-fall period in the stand16, especially during the autumn months. Traps are opened boxes or nets (Figure 1) with a predetermined sufficient size (minimum 0.18 m2, but preferably over 0.25 m2)10,17, lateral sides preventing the wind from blowing leaves into/out of the traps, and with a perforated bottom avoiding decomposition of the leaves; which are located below the canopy layer of the studied stand, however, above the ground surface11. The distribution of the traps can be either random18 or systematic in transects19 or a regular spacing grid20. The number and distribution of traps are a crucial methodological step for performing an accurate LAI estimation reflecting the unique stand structure, spatial homogeneity, expected wind speed and direction, especially in the case of sparse stands (or alleys and orchards), and the work capacity for evaluating data. The precision of LAI estimation increases with the rising frequency of traps within studied stands11,21 (see Figure 2).
The recommended frequency of collecting samples of the litter-fall from each trap is at least monthly10 and even twice per week in periods of heavy fall, which may coincide with heavy rainfall. It is necessary to prevent decomposition of the litter in the traps and the leaching of nutrients from the material during rain episodes in the case of chemical analysis. After collecting leaves in a field, a mixed sub-sample is used for estimating the specific leaf area (SLA, cm2 g-1)22, defined as the fresh projected area of leaves to its dry mass weight ratio. The rest of the collected litter is dried to a constant weight and used for calculating the dry mass of the litter as g cm-2 in the lab. Leaf dry mass on each collection date is converted into the leaf area by multiplying the collected biomass by SLA or leaf dry mass per area (LMA, g cm-2) as the inverse parameter to SLA23,24. A fresh projected area of particular leaves can be determined using a planimetric approach. The planimetric method is based on the dependency between the area of a specific leaf and the area covered by the leaf in the horizontal surface. The leaf is horizontally fixed to the scan screen, and its average is measured using a leaf area meter. Then, its area is calculated. Many leaf area meters based on different measurement principles are available on the market. Some of them include, for instance, the LI-3000C Portable Leaf Area Meter, which uses the orthogonal projection method, and the LI-3100C Area Meter, which measures leaf average using a fluorescent light source and a semi-conducted scanning camera. The next device, the CI-202 portable laser leaf area meter, codes a leaf length using a code reader. Besides them, the AM350 and BSLM101 Portable Leaf Area Meters are also commonly used for performing accurate leaf area estimation.
Furthermore, leaf area meters based on systems that analyze video exist. These leaf area meters consist of a video camera, a digitalisation frame, a screen, and a PC, including suitable software for making the data analysis such as WD3 WinDIAS Leaf Image Analysis System11. Currently, conventional scanners connected to a PC can be used for an estimating leaf area. Afterwards, the leaf area is calculated as a multiple of the number of black pixels and its size depends on the selected resolution (dots per inch &#8211; dpi), or the leaf area is measured through specific software, for instance, WinFOLIA. Finally, the total dry mass of leaves collected within a known ground surface area is converted into the LAI by multiplying by SLA and a shrinkage coefficient25 which reflects the changes in the area of fresh and dried leaves. Shrinkage depends on the tree species, water content and leaf softness. The shrinkage of leaves in length and width (what affect the projected area) is usually up to 10%26, for instance, it ranges from 2.6 to 6.8% for oak27. Sorting leaves by species for weighing and establishing the specific leaf area ratio is necessary to determine the contribution of each species to the total LAI28.
LAI determination by the needle technique is an inexpensive method derived from the inclined point quadrat method29,30,31,32. In deciduous stands, it is an alternative for performing LAI estimation without using traps10 based on the assumption that the total leaf number and their area in a tree are equal to what is collected on the soil surface after a complete leaf-fall20. A thin sharp needle is pierced vertically into the litter lying on the ground immediately after the leaf-fall10. After the complete leaf-fall, the leaves are collected from the ground onto a needle of a vertical probe, are related to the contact number and equal the actual LAI value. An intensive sampling (100-300 sampling points per studied stand per field probe) by the needle technique is required to quantify a mean contact number and to derive the LAI value correctly10,20,33.
The plant canopy analyzer (e.g., LAI-2000 or LAI-2200 PCA) is a commonly used portable instrument for performing an indirect LAI estimation by taking a measurement of the light transmission throughout the canopy7 within the filtered blue portion of the light spectrum (320-490 nm)34,35 to minimize the contribution of the light which has passed through the leaves, was scattered by the canopy and is passing through the foliage7,34. In the blue part of the light spectrum, the maximum contrast between the leaf and sky is achieved, and the foliage appears black against the sky34. Therefore, it is based on the canopy gap fraction analysis7. The instrument has been widely used for making eco-physiological studies in plant communities such as crops36, grasslands37, coniferous stands8, and deciduous stands38. The plant canopy analyzer uses a fisheye optical sensor with a FOV of 148&#176; 35 to project a hemispherical image of the canopy onto silicon detectors to arrange them into five concentric rings39 with central zenith angles of 7&#176;, 23&#176;, 38&#176;, 53&#176;, and 68&#176; 9,40,41. Five view caps (i.e., 270&#176;, 180&#176;, 90&#176;, 45&#176;, and 10&#176;) can be used to restrict the azimuth view of the optical sensor27 to avoid shading by obstacles in an open area (for the above-referenced reading) or the operator in the sensor&#8217;s FOV during LAI estimation can adjust the FOV sensor to an open area for above-canopy readings. Measurements using the plant canopy analyzer are taken above (or in a sufficiently extended open area) and below the studied canopy7. The same view caps must be used for both above and below readings to avoid biases of gap fraction estimation34. The LAI-2000 PCA produces an effective leaf area index (LAIe) as introduced by Chen et al.42, or rather an effective plant area index (PAIe) as woody elements are included in the sensor reading value. In deciduous stands with flat leaves, the LAIe is the same as the hemi-surface LAI. In the case of evergreen forest stands, the LAIe is necessary to correct for the clumping effect at the shoot level (SPAR, STAR)43, the clumping index at scales larger than the shoot (&#937;E)44, and the contribution of woody elements including stems and branches (i.e., woody-to-total area ratio),45 which cause a systematic LAI underestimation20. The clumping index on a higher spatial scale than the shoot or leaf could be quantified as an apparent clumping index (ACF), which can be estimated using the plant canopy analyzer when more restrictive view caps are used27. As these authors state that this ACF is deduced from a ratio of LAI values calculated from transmittance by different procedures for homogeneous and non-homogeneous canopies according to Lang46, we presume that this clumping index describes rather canopy homogeneity. Besides the ACF calculation, new diffuser caps that enable a more extensive application of LAI-2200 PCA in respect of weather conditions, a user menu instead of Fct codes, and the possibility to take many more measurements per file session are among the main technological upgrades compared to the former LAI-2000 PCA34,47. Measurements and subsequent internal software calculations are based on four assumptions: (1) light blocking plant elements including leaves, branches, and stems, are randomly distributed in the canopy, (2) foliage is an optically black body that absorbs all the light it receives, (3) all plant elements are the same projection to the horizontal ground surface as a simple geometric convex shape, (4) plant elements are small compared to the area covered by each ring11.

<table>
	<tbody>
		<tr>
			<td>Area Meter</td>
			<td>LI-COR Biosciences Inc., NE, USA</td>
			<td>LI-3100C</td>
			<td>https://www.licor.com/env/products/leaf_area/LI-3100C/</td>
		</tr>
		<tr>
			<td>Computer Image Analysis System</td>
			<td>Regent Instruments Inc., CA</td>
			<td>WinFOLIA</td>
			<td>http://www.regentinstruments.com/assets/images_winfolia2/WinFOLIA2018-s.pdf</td>
		</tr>
		<tr>
			<td>File Viewer</td>
			<td>LI-COR Biosciences Inc., NE, USA</td>
			<td>FV2200C Software</td>
			<td>https://www.licor.com/env/products/leaf_area/LAI-2200C/software.html</td>
		</tr>
		<tr>
			<td>Laboratory oven</td>
			<td>Amerex Instruments Inc., CA, USA</td>
			<td>CV150</td>
			<td>https://www.labcompare.com/4-Drying-Ovens/2887-IncuMax-Convection-Oven-250L/?pda=4|2887_2_0|||</td>
		</tr>
		<tr>
			<td>Leaf Image Analysis System</td>
			<td>Delta-T Devices, UK</td>
			<td>WD3 WinDIAS</td>
			<td>https://www.delta-t.co.uk/product/wd3/</td>
		</tr>
		<tr>
			<td>Litter traps</td>
			<td>Any</td>
			<td>NA</td>
			<td>See Fig. 2</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Any</td>
			<td>NA</td>
			<td>Maximum diameter of 2 mm</td>
		</tr>
		<tr>
			<td>Plant Canopy Analyser</td>
			<td>LI-COR Biosciences Inc., NE, USA</td>
			<td>LAI-2000 PCA</td>
			<td>LAI-2200 PCA or LAI-2200C as improved versions of LAI-2000 PCA can be used, see: https://www.licor.com/env/products/leaf_area/LAI-2200C/</td>
		</tr>
		<tr>
			<td>Portable Laser Leaf Area Meter</td>
			<td>CID Bio-Science, WA, USA</td>
			<td>CI-202</td>
			<td>https://cid-inc.com/plant-science-tools/leaf-area-measurement/ci-202-portable-laser-leaf-area-meter/</td>
		</tr>
		<tr>
			<td>Portable Leaf Area Meter</td>
			<td>ADC, BioScientic Ltd., UK</td>
			<td>AM350</td>
			<td>https://www.adc.co.uk/products/am350-portable-leaf-area-meter/</td>
		</tr>
		<tr>
			<td>Portable Leaf Area Meter</td>
			<td>Bionics Scientific Technogies (P). Ltd., India</td>
			<td>BSLM101</td>
			<td>http://www.bionicsscientific.com/measuring-meters/leaf-area-index-meter.html</td>
		</tr>
		<tr>
			<td>Portable Leaf Area Meter</td>
			<td>LI-COR Biosciences Inc., NE, USA</td>
			<td>LI-3000C</td>
			<td>https://www.licor.com/env/products/leaf_area/LI-3000C/</td>
		</tr>
	</tbody>
</table>

leaf area index estimation using three distinct methods in pure deciduous stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

Average LAI values at the stand level of all studied stands in the 2013 growing season are presented in Figure 8. On all plots except A, the highest values were measured by litter traps, which serve as the reference level. Contrarily, the highest mean LAI value was estimated through the needle technique on plot A. All differences between LAI values estimated using litter traps and a plant canopy analyzer were not significant (p &#62; 0.05; Figure 8, left). On pl...

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Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

The leaf area index serves as the interface between leaves and the atmosphere in which many physiological processes are implemented, especially photosynthetic uptake. The main advantages of this method for leaf area index estimation are the straightforward applicability, short measurement time requirements, and the precision of the acquired data. Given that these methods have already been used for ecophysiological studies in crops, grasslands, conifers, and hardwood forest stands, they should be widely applicable to most other plant ecosystems.All of the described methods for leaf area index estimation are straightforward and simple to use. Researchers new to the methods should not encounter difficulties with implementing the measurements. Visual demonstration of these leaf area index estimation methods is essential for their comprehension, design, and implementation.Demonstrating the procedure will be Pavel Haninec, a PhD student from the Department of Forest Botany, Dendrology, and Geobiocoenology. For leaf area index estimation using litter traps, place 15 to 25 traps per investigated stand with a capturing area ranging from 0.18 meters squared up to 0.5 meters squared or more at the beginning of the growing season. Place the traps at regular spacing throughout the studied stand within one or two mutually perpendicular transects or a regular grid, a minimum of 0.1 meter above the ground surface to enable air to blow beneath the collecting part of the traps.Firmly fix each of the traps above the ground surface and below the stand canopy so that there are no changes in the capturing area. At each measurement time point, place the collected litter from each of the traps into appropriately labeled paper bags for transport to the lab. Back at the lab, separate the assimilation apparatus from the other litter components.To perform a specific leaf area estimation, thoroughly mix the sample from each trap before separating a subsample of at least 100 to 200 leaves from all of the used traps. After briefly soaking any dried leaves in 60 to 70 degrees Celsius water to keep the leaves from folding or curling, place the leaves from the subsample in a flat, straight manner onto a scan board without overlapping. Dry the subsample for 48 hours at 80 or 105 degrees Celsius to attain a constant weight in a ventilated oven with a thermostat to homogenize and maintain the internal temperature.At the end of the drying period, weigh the dry mass of the subsample on a lab scale with a minimum degree of accuracy of one gram and calculate the specific leaf area as the fresh projected area of leaves from the subsample designated for the specific leaf area estimation divided by the dry mass weight. Then, oven dry the rest of the sample from each trap for 48 hours at the same temperature that was used for the specific leaf area estimation and multiply the dry mass weight of the rest of the sample for each particular litter trap by the correct specific leaf area value to reach the total projected leaf area index per trap. To estimate the leaf area index using the needle technique, immediately after a complete leaf fall use a sufficiently long, sharp, metallic needle with no more than a two-millimeter diameter to puncture leaves at a more or less similar angle through the layer of freshly fallen leaves lying on the ground surface at each of at least 100 probed sampling points.Check to make sure that only freshly fallen leaves are present on the needle, removing any partially decomposed leaves from the previous year as necessary. Then, count the number of leaves pierced by the needle from each stab at each sampling point before repeating the needle technique leaf collection at subsequent sampling points. For leaf area index estimation using a plant canopy analyzer optical device, locate a suitable open clearing with identical sky conditions as above the observed plot, a maximum distance of one kilometer.Apply the same cap and orientation for both above-and below-canopy readings and slowly move with the sensor below the canopy in transect, watching the variability of the most upper ring readings. Using the same view cap for the both above-and below-canopy readings is necessary for correct leaf area index estimation using a plant canopy analyzer. The number and spacing of the transects depends upon the particular canopy structure of the stand.Perform the above-canopy readings as the first measurement of each stand transect or grid in a sufficient open area and perform the below-canopy readings in one to three transects and between five to 36 sampling points from 0.5 to two meters above the ground. Use the restriction view caps if the sensor is held below two meters to exclude the operator from the field of view and use a minimum distance between the sensor and the nearest element of the aboveground parts of the plant of at least four times the diameter or width of the component. Estimate the woody area index during the leaf-off period, both before bud breaking in early spring and after complete leaf fall in late autumn.To calculate the woody area index values from the field-measured raw data, use the the leaf area index 2200 File Viewer freeware and estimate the plant area index using the same procedure as for making the woody area index estimation. Then calculate the actual leaf area index value at the stand level as the difference between the mean plant area index and woody area index values. In this representative experiment, on Plots B, C, and D, the needle technique significantly underestimated the leaf area index obtained from the litter traps.Conversely, on Plot A, this technique overestimated the leaf area index measured using the litter traps at a not-significant level. Further, significant differences among the leaf area index values estimated by the plant canopy analyzer and the needle technique were found in all cases. The woody area index can be readily measured using a plant canopy analyzer after the complete leaf fall and before bud break.In this representative experiment, the most rapid leaf area index development was noted during the period from bud break occurring in April until the beginning of May. From May until the end of June, a continuation of the fast leaf area index development of leaves was observed, although with a lower intensity compared to the previous period. From the second half of June until the end of July, the leaf area index value declined in Plot B and the stagnation of the leaf area index was more evident during this period on Plot A.In all of the studied forest stands, the leaves started to fall at the end of September, as illustrated by the decrease in the leaf area index curve.The most critical part of the litter trap method is the accurate estimation of the specific leaf area from a sufficiently large subsample of leaves. For the plant canopy analyzer, it is essential to perform the measurements under standard overcast and windless conditions. Digital hemispherical photographs and laser scanning technology can also be used to determine the leaf area index and the spatial distribution of the leaves within the canopy.Currently, the most significant challenge in leaf area index estimation is using remote sensing to determine the foliage quality.

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13.2K Views.  Research Station at Opočno. The leaf area index serves as the interface between leaves and the atmosphere in which many physiological processes are implemented, especially photosynthetic uptake. The main advantages of this method for leaf area index estimation are the straightforward applicability, short measurement time requirements, and the precision of the acquired data. Given that these methods have already been used for ecophysiological studies in crops, grasslands, conifers, and hardwood forest stands, they should...