Methodology

The selection of a tree species, and of an individual representative of that species, is inevitably a matter of compromise. Of course, we looked for trees that were old, large and full of epiphytes among the tree species characteristic of the surrounding forest, but other choice criteria also had to be taken into account. In particular, accessibility, which determines the working time that can be devoted to collecting each day and therefore the time needed to complete the inventory of a tree.

After several reconnaissance missions in Peru and Colombia, our first choice was an enormous specimen of Dussia tessmannii (50m high) located at an altitude of 400m in a foothill forest in the Andes mountain range, in the Rio Abiseo National Park in Peru. After this study on a tree at low altitude, we wanted to obtain a picture of the biodiversity associated with a tree located at a higher altitude, where the diversity in epiphytic plants is considered to be maximum in the scientific literature. Following a meeting with Dr. Damien Catchpole, holder of the record for the number of epiphytes found in a tree, and a visit to Yanachaga-Chemillén National Park, we selected the tree inventoried by Catchpole in 2003 to serve as the second study subject for the Life On Trees programme. The tree, a 32m tall individual of Ficus americana subsp. andicola, is located 2450m above sea level in the San Alberto Valley in Peru. The third tree in the programme is situated at an intermediate altitude, 850m, in the forest of the La Isla Escondida private reserve in the department of Putumayo in Colombia. The tree is a 40m specimen of the Brosimum utile species.

The AccèsCimes non-profit organisation, which provides professional climbers for the programme, is responsible for installing and implementing the access system to the selected trees. A new system to facilitate ascents has been successfully tested. It consists of a set of pulleys and a boat winch coupled to an electric motor. This system makes it possible to be hauled effortlessly to a central point in the crown. Displacement ropes are laid on high branches to operate translations from one sector of the crown to another without having to come down to the ground.

Apart from the trunk, along which unskilled climbers can move vertically without any advanced technical skills, most of the tree is only accessible to experienced climbers, the only ones able to move in three dimensions. The vast majority of surveys and collections in the crown, from its centre to the ends of the branches, are therefore not carried out by scientists but by climbers. Their role is therefore vital in collecting target organisms in a complex environment. A circular platform 2.5m in diameter, installed at the heart of the crown, provides a safe and comfortable station and enables the scientists to guide the climbers in their collection.

Since most of the data collection is carried out by a small number of professional climbers, it is difficult to carry out several tasks at the same time in the crown of the tree, so there is no point in having too many people in the field at the same time. In addition, some protocols take longer to complete than others, and they are not all compatible with each other. The study of a tree therefore requires several work phases, carried out by small groups of experts. Spreading the collection campaigns over a year, at different seasons, also maximises the chances of encountering different species assemblages, or finding plants in flower, which are essential for their identification.

Within a tree, habitat conditions vary drastically from the ground to the canopy. It is therefore necessary to sample using a method that takes into account the variety of environments – in particular the presence of humus accumulated on the branches – and microclimatic conditions – relative air humidity, temperature, light intensity – which determine the distribution of species within the tree.

As part of the Life On Trees programme, based on experience gained during similar missions in tropical forests, we are carrying out stratified sampling of the fauna and flora so as to cover all the available habitats. We divided the trees into six height classes, using Johansson’s zonation scheme to guide the sampling.

While an exhaustive inventory of species is conceivable in the basal part of the tree, its height, structural complexity and the volume of its crown make a complete inventory impossible. Only the use of a standardised method can provide a representative picture of the species composition associated with the tree. Several sampling protocols are necessary, depending on the groups of organisms targeted, in order to estimate the total species richness in the tree.

For groups that are anchored to the tree and made up of a large number of small organisms, such as bryophytes or lichens, it is possible to carry out a quantitative sampling by taking samples of all the organisms from small areas of equivalent size distributed throughout the tree. Extrapolations can then be made to obtain a realistic picture of the number of species and their distribution.

For the larger plants, such as orchids and ferns, which are less numerous in terms of species and even less in terms of individuals, we preferred to take samples from small clumps of epiphytes with their substrate. All the plants, animals and decomposed organic matter from these patches were brought back to the field laboratory. These clumps have thus provided abundant material for different protocols and for the study of different target groups.

For endophytic fungi, which complete their entire cycle inside the tree, we take samples of wood and leaves from the base to the top of the tree. Endophytic fungi on the leaves and roots of epiphytic plants are also studied. The wood samples and pieces of leaves and roots are preserved in a solution that preserves the DNA or cultured to isolate fungi strains whose DNA will then be sequenced.

Among invertebrates, arthropods (mainly insects) form the most diverse group. To be effective and to have an overall picture of the fauna associated to the trees studied, each group of insects must be collected using techniques specific to that group.

For vertebrates, direct observations are made by bat and bird specialists at different times of the day in the tree crowns and in the immediate vicinity of the study trees. These observations are supplemented for other vertebrates by the study of environmental DNA from deposits of organic matter contained in bromeliads. The trees in the programme are home to numerous bromeliads that retain water between their leaves. These suspended mini-ponds form a small ecosystem within the tree ecosystem itself. The aquatic contents of these bromeliads are home to a wide variety of organisms and are of interest to several scientists in the Life On Trees programme. Their contents are collected, filtered and preserved for later study.

Even if the species they harbour are only secondarily associated with the supporting tree, we want to study all the biodiversity that a tree can harbour. It is also likely that the different microclimates generated by the tree influence the composition of these aquatic communities depending on where they are located in the crown.

As far as possible, and excluding opportunistic collections, specimens and samples are geolocated in the tree. In this way, we are able to keep track of their position, and we will be able to place some of the samples in a three-dimensional image obtained using the LIDAR (Laser Imaging Detection And Ranging) technique.

To accurately quantify the structure of our study trees, we produced three-dimensional digital twins using LiDAR technology.

LiDAR (Laser imaging Detection And Ranging) is a short-range remote sensing technology that can measure distances and create detailed 3D representations of objects and environments. When a laser pulse is emitted, the device measures the time between its emission and its rebound after hitting a surface. By analysing the return time of laser pulses, LiDAR systems can accurately calculate the distance to objects or surfaces.

Terrestrial laser scanning (TLS), in which a scanner is operated from the ground, is currently being used as a cutting-edge technology to capture the structure of tropical forests very accurately. TLS generally uses a laser scanner to scan trees from several angles from the ground. In tropical forests, tree canopies can reach heights of 50m or more. Because of the great distance between the scanner and the crown of the tree, even the best laser scanners have difficulty obtaining a clear image of the top of the canopy. For these large trees, the data is limited and suffers from numerous gaps, as their upper parts are often masked by obstacles (branches, foliage) located below.

To overcome this limitation of TLS, in the Life On Trees programme we are exploring the amount of additional 3D information that can be obtained by taking scans inside the crown of the tree. In this way, we can characterise the three-dimensional structure of the vegetation on the tree, including the leaves and epiphytic plants that grow there.

For more information on the methodology:

Leponce, M.; Basset, Y.; Aristizábal-Botero, Á.; Baïben, N.; Barbut, J.; Buyck, B.; Butterill, P.; Calders, K.; Cárdenas, G.; Carrias, J.- F.; Catchpole, D.; D’hont, B.; Delabie, J.; Drescher, J.; Ertz, D.; Heughebaert, A.; Hofstetter, V.; Leroy, C.; Melki, F.; Michaux, J.; Moreno, J.C.N.; Poirier, E.; Rougerie, R.; Rouhan, G.; Rufray, V.; Scheu, S. ; Schmidl, J.; Vanderpoorten, A.; Villemant, C.; Youdjou, N.; Pascal, O. (2024) Unveiling the above-ground eukaryotic diversity supported by individual large old trees: the ‘Life on Trees’ integrative protocol. Frontiers in Forests and Global Change 7 (https://doi.org/10.3389/ffgc.2024.1425492).