A new method to quantify macroalgae and a practical sampler for experimentation in lotic habitats

Experimental studies in rivers and streams are extremely difficult to run due to the fact that the conditions of these environments are very complex and provide a high level of heterogeneity, which hinders the precise control and standardization of variables. In this study, we present a practical sampler that was designed to make it easier to conduct research projects involving benthic communities of lotic environments, as well as a new nondestructive technique for quantification of the macroalgal communities typically found in these habitats. The sampler consists of an acrylic square tube in which water flows normally inside. This structure carries a removable glass plaque with a known area and can simulate various ecological situations by changing both biotic and abiotic conditions. Thus, it can mitigate the differences between environmental characteristics where each sampler is exposed. The new technique involves capturing digital images that can monitor a unique macroalgal community in development throughout time and a more precise quantification when compared with other techniques that are widely applied. The sampler is easy to build and the images simple to quantify, allowing the detection of spatial and temporal variations in richness and abundance of investigated communities.


Introduction
Among the various primary producers from the benthic community of lotic environments, the algal element has been described as one of the most important (Lamberti, 1996;Necchi Junior et al., 2003;Branco et al., 2008).The algae community can establish itself in different ways and arrangements and an important form of algal flora is constituted by species that show noticeable macroscopic development.In other words, they are recognizable by the naked eye (John and Moore, 1985;Sheath et al., 1989), which is a trait that explains why they have been traditionally reported as lotic macroalgae (Sheath and Cole, 1992).
However, the overlapping between macroalgae and periphytic communities is inevitable (Wetzel, 1983), which is explained by the noticeable coexistence of these organisms in the environment where they live, making the delimitation of limits between micro (periphyton) and macroscopic (macroalgae) elements extremely difficult.In this context, there is a fundamental point which is carefully worked out from this verification: the methodology, which needs to be coherent with peculiarities from the community.
Some studies involving autotrophic communities, such as periphytic or macroalgae, evaluate the responses from algal community by a series of accepted and useful methods (Wetzel, 1981(Wetzel, , 1983).Among the most useful methods, we may cite: chlorophyll a extraction (Marker et al., 1980;Sartory and Grobbellar, 1984) and the quantification of dry mass and ash free dry mass (Schwarzbold et al., 1990).Additionally, the distribution of macroalgae in a specific area or region is frequently analyzed in the field using a transect technique associated to a visual estimation of abundance which gives the relative percentage of an area covered by algae in the environment (Sheath and Cole, 1992).
However, these methods are relatively inaccurate.For example, dry mass and/or ash free dry mass evaluation of the macroalgal community could include other organisms such as microalgae and non-algae organisms associated with their stalks (Sheath and Cole, 1992).Thus, all associated organisms and accumulated organic material would be inevitably quantified.
Likewise, results from analysis involving photosynthetic pigments (e.g.chlorophyll a) can be significantly altered due to interference from associated algal elements.On the other hand, the visual estimative technique from percentage cover extensively used in fieldwork is a useful semi-quantitative technique in ecological studies, which compares parts of streams.However, this procedure has become problematic for refined experimental studies because it depends on the observer and does not give absolute numbers.
Studies on the effects of abiotic variables in lotic macroalgae, such as physical, chemical or temporal variables, have used some methodological strategies that involves the use and application of both natural (Sládedkowá, 1962;Wetzel, 1965;Soares, 1981) and artificial substrates (Cattaneo et al., 1975;Panitz, 1980).In 1916, Hentschel was the first to use microscope slides and other kinds of substrates in qualitative and quantitative studies on periphyton (Sládedkowá, 1962).Since then, various studies have been carried out using artificial substrates (Schwarzbold, 1990).
The non-natural substrates are widely used in ecological studies because they are user-friendly and can be compared with the natural environment.Ecological studies on producer communities associated with artificial substrates in lotic environments are common in the literature (Biggs, 1988;Clifford et al., 1992;Fernandes and Esteves, 2003;Lobo et al., 2004;Barbee, 2005;Salomoni et al., 2007;Canani et al., 2010).
Thus, in order to obtain more precise and specific data about such communities we developed a practical model for the use of artificial substrates and a new methodology for lotic macroalgae quantification.This novel methodological model will be able to make macroalgae assessments and standardization of these studies more precise.

The Sampler
Our sampler is proposed for the application of artificial substrates (modified from Peterson et al., 1983).It is made of transparent acrylic pieces that were welded to form a square tube measuring approximately 15 cm length, 8 cm width and 8 cm height.There are slots to fit a removable rectangular slide inside the tubes, which work as artificial substrates for algal colonization (see Figure 1).
This model can simulate various environmental conditions, as follows: 1) different levels of light intensity by covering the tube walls with light reducing films (described by Tonetto, 2010); 2) various substrate types which can be made in different forms or with different 3) different chemical conditions (e.g.nutrients and polluters), using diffuser substrates that can gradually release substances, and; 4) herbivore levels, using mesh to selectively control herbivores.
The sampler can also be built in different sizes, with larger lengths and transverse areas depending on the research goals.Additionally, it has important inert characteristics and a defined area that can investigate the macroalgae biomass variation.The sampler is easy to be produced, it has a low cost of production and is made of renewable materials.However, certain precautions should be observed when installing and maintaining the samplers in streams.For the installation, the samplers should be fixed using some heavy material to ensure their stability and total submersion in the lotic environment.This procedure prevents the movement of the sampler due to the flow.We recommended that the samplers are attached, using stainless steel wire, to a plywood board associated with a concrete base of approximately 15 kg (as proposed by Biggs, 1988).To maintain the samplers throughout the experimental period, the biofilm should be removed periodically (using a soft brush) that might cover the external and internal walls of the samplers.This procedure avoids any interference in the availability of light within it.Regular maintenance is also important to remove any debris (manually) that may be retained on the samplers.
It is worth mentioning that the number of used in each study must be calculated considering the objective of the study and especially the high heterogeneity typically found in lotic environments.

The Technique
The presented technique has been proposed to improve the accuracy of quantitative assessment of benthic algal communities, with special emphasis on the stream macroalgae, by applying a technique never used for these communities.
The quantification offered by this study is based on the covering percentage; it means that this technique represents better the competition for establishment of macroalgae in a determined space.For this purpose, the procedure described here is based on similar techniques used in other areas of scientific research, which are based on analyzing digital pictures, such as the ones that evaluate the foliar area of plants (Godoy et al., 2007;Rico-Garcia et al., 2009) or the canopy cover of woody areas (Canham, 1988;Englund et al., 2000).
Hence, the removable slides, inserted into the sampler described above, can be removed from the tubes and some pictures can be taken.The pictures should have a high resolution to ensure that the algae which are colonizing the substrate can be clearly seen.For standardization purposes, photos must be taken with the camera 15 cm (in height) far from the colonized slides.To help analysis pictures in the laboratory, we recommend the researcher to map the slide in the field notes.This can be useful when looking for determined species on pictures.The process takes less than 5 minutes until the slides return to the samplers, preventing damage to algae communities due to desiccation.
To ensure this method is used to its full potential and also the quality of the results, specimens from the natural substrates should be collected.In the laboratory, based on morphological forms, the collected material can be linked with macroalgae presented in the pictures.
After photographic documentation, the pictures of the artificial substrates that had been colonized can be analyzed using software (AutoCAD ® or other software with similar functions), with the objective of quantifying the community and each species individually.Considering this, the area colonized by each species (in each photograph) should be marked and estimated in terms of the percentage of the total area of the substrate (see Figure 2).
This method is more accurate to determine the area occupied by the macroalgae flora, which represents an advance when compared with the estimative visual technique, which was applied earlier.In order to calibrate the quantification method, it is important to establish the same scale for all images.Although the analyses of covering percentage is a semi quantitative method, it is not destructive, which ensures absence of the algal biomass loss.Moreover, monitoring the temporal variation of the sampled macroalgae communities is more realistic, since the same community is periodically evaluated rather than different samples collected on separated dates.Therefore, our method can be the most appropriate to quantify macroalgae abundance through time.The sampler and the technique described were used in an experimental study, where the colonization of the macroalgal community was observed over a period of 91 days in four streams in southern Brazil (Tonetto, 2010).During this experimental period, 100% of the glass plaques remained intact, showing high durability of the sampler.The flow of water and light availability within the samplers were not affected, while the analysis technique proved to be easy to use and highly accurate in terms of quantifying the biomass of macroalgae.
In conclusion, using the samplers described in this paper and applying the quantification described here, have a powerful tool to obtain more precise results from the lotic macroalgal communities.By using our model, researchers will be able to enhance the practicability and versatility of their work and, at the same time, save time and financial resources.

Figure 1 .
Figure 1.a) Schematic drawing of the sampler made by acrylic plaques and with a removable glass slide inside.b) Photo illustrating the sampler.

Figure 2 .
Figure 2. a) Photograph of an artificial substrate colonized by macroalgae (in this case, Tetraspora lubrica).b) The same photograph with the demarcated areas occupied by macroalgae in computer software.