# TheUlva spp. Conundrum: What Does the Ecophysiology of Southern Atlantic Specimens Tell Us?

Authors: Vinícius Peruzzi Oliveira; Barbara Lage Ignacio; Nuno Tavares Martins; Leticia Dobler; Alex Enrich-Prast
Journal: Journal of Marine Biology
 (2019)

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## Abstract

Species of the genusUlva are common in anthropogenically disturbed areas and have been reported as the cause of green tides in many areas of the world. In addition, they rank among the main marine groups used in a wide range of commercial applications. By displaying few distinctive morphological characters, some taxonomical identifications are difficult and the genus is under a conundrum. Our aims were to provide ecophysiological information about threeUlva species in response to abiotic factors and to evaluate the proposal of ecophysiological information and the chlorophyll-a fluorescence technique as auxiliary tool to resolve the long-standing taxonomic confusion. We hypothesize that three cooccurring specimens (U. fasciataDelile,U. lactuca Linnaeus, andU. rigidaC. Agardh) have different ecophysiological responses (as measured by the effective quantum yield of photosystem II by pulse amplitude modulated fluorometers) under manipulated conditions of temperature and nutrient concentration.Ulva lactucaandU. rigidashowed different photosynthetic efficiencies related to temperature, whereas no difference was recorded forU. fasciata individuals. These results provide a reasonable explanation for the variability in spatial and temporal abundance of these species ofUlva on rocky shores. We proposed the use of ecophysiological information by chlorophyll-a fluorescence as an auxiliary tool to corroborate the taxonomic distinction ofUlva species. We reinforce the statement ofU. fasciata andU. lactuca as distinct valid species.


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## Body

## 1. Introduction

The genusUlva (Chlorophyta) comprises cosmopolitan macroalgae that inhabit a gradient from freshwater to fully saline shallow environments [1]. Species of this genus rank among the first species to be established in disturbed environments because of their morpho-physiological features, that allow tolerance to wide range of environmental conditions [2–4]. Some species ofUlva have been emphasized due to reports as the cause of green tides in many parts of the world [5]. Additionally, numerous studies have also demonstrated functions in bioremediation of eutrophic environments and uses of their biomass and carbohydrate content in the pharmaceutical, food, and energy chain [6–8].Ongoing advances in molecular biology have driven taxonomic revisions, and morphological features have lost credibility as the primary species delimitation criterion for many groups (see [9] for algae).Ulva is one of the genera involved in a taxonomic conundrum that entangles morphological or molecular data for species identification, mainly involving distinction aroundU. lactucaandU. fasciata(see [1, 10–13]). In spite of the undeniable contribution of molecular methods to taxonomy (see [14–17]), there is no easy way to determine the accurate match between the GenBank sequences and the morphological characterization of most studies, which has led to uncertainty and sometimes to misinterpretation (see [1, 11]).In the latter context, physiological markers that are associated with the photosynthesis process may be especially helpful. Photosynthetic organisms adjust the operation of their photosynthetic apparatus to optimize or preserve metabolism under stress, which can be detected by chlorophyll-a fluorescence variation [18, 19]. In this case, the evaluation of the effective quantum yield of PSII by pulse amplitude modulated (PAM) fluorometers has been proposed as an attractive measure of the ecophysiological condition [20–24]. This technique is notable for performing a nonintrusive real-time analysis under both laboratory and field conditions [25] and has been applied for establishing the differences among morphotypes of macroalgae [26].Under this background, the aim of this study was to provide comparative ecophysiological information about three cooccurring species ofUlvain the Southwestern Atlantic (Ulva rigidaC. Agardh,Ulva lactuca Linnaeus, andUlva fasciata Delile) that were morphologically identified. We assessed the effect of temperature and nutrient variation on the photosynthetic efficiency (by chlorophyll-a fluorescence measurements) of the three species. The general tested hypothesis was that species would have different responses to both of the tested factors. We subsequently expect to provide useful information for using PAM fluorometry ofUlva as a reliable tool for ecophysiological assessment and for the taxonomic debate aboutUlva.


## 2. Material and Methods

### 2.1. Sampling

The three species ofUlva (U. fasciata,U. lactuca, andU. rigida) were collected during summer (January 2010) in the intertidal zone at Prainha Beach (22° 57′S, 042° 01′W), Rio de Janeiro, Brazil. Immediately, the epibionts were removed, and the macroalgae were kept in cool boxes with local seawater (~30 μM NO3, 3 μM PO4, 19°C, and 35 PSU) filtered through 10-μm mesh filters.


### 2.2. The Species

The morphological taxonomy of the three studied species (U. fasciata,U. lactuca, andU. rigida) was based on botanical descriptions and inventories that considered in-depth confrontation among the species descriptions and the Southwestern Atlantic specimens [27–30]. Individuals were prepared as herbarium vouchers and deposited in the herbarium of the Universidade Federal do Rio de Janeiro/UFRJ (under the accession numbers RFA 42441; 42442; 42443).


### 2.3. Experimental Design

All the individuals were maintained in Erlenmeyer flasks in a proportion of 1 g of fresh mass L−1 pasteurized oligotrophic ocean water with added trace nutrients (<1 μM NH4; <1 μM NO3; <2 μM NO2; <1 μM PO4), at 20°C and 200 μmol photons m−2 s−1 (PAR, photosynthetically active radiation) for 15 days. This phase was established to set all the macroalgae to the same nutritional and physiological state before the manipulative experiment. This PAR was chosen because it is an intermediate value in relation to the saturation parameter that is found in the P-I curves (100–400 photons m−2 s−1).After the nutrient deprivation period, six treatments were applied using combinations of two temperatures and three concentrations of nutrients, with 5 replicates per treatment. The experimentper se lasted 10 hours. The factor “Temperature” was represented by 20°C and 30°C, and the factor “Nutrient” was represented by different additions of NaNO3- and Na2HPO4.12H2O of Von Stosch’s enriched seawater (VSE; 500 μM NO3- and 30 μM PO4-) [31]. The experimental setup values of temperatures are related to the temperature at the upper intertidal fringe at the sampling site, which ranges between 18°C (high tide) and 32°C (low tide) [29], and the nutrient concentrations were set as 0.1 VSE, 0.2 VSE, and 0.5 VSE. These concentrations refer to a gradient of nutrient concentrations that were recorded in Southwestern Atlantic [32, 33]. The treatment set as 0.1 VSE refers to natural eutrophicated waters (upwelling events at collecting site, Arraial do Cabo, Rio de Janeiro) [32], whereas 0.2 VSE and 0.5 VSE refer to the range of nutrient concentrations that were recorded in the human-disturbed Guanabara Bay, Rio de Janeiro [33].


### 2.4. Fluorescence Measurements

The photosynthetic efficiency of the three species ofUlvain every combination of temperature and nutrient concentration was characterized by chlorophyll-a fluorescence measurements using a submersible diving-PAM® fluorometer (Walz, Effeltrich, Germany). The measurements consisted of recording the effective quantum yield of PSII, which was calculated as Y = (F′m-Ft)/F′m, where F′m is the maximum fluorescence in the light (obtained by a saturating actinic light pulse – 8900 μmol photons m−2 s−1; 0.8 s) and Ft is the steady-state of fluorescence in the light [34, 35]. Considering the possibility of physiological differences along the thallus [36], the fluorescence was measured in a similar region of the thallus in each individual.


### 2.5. Data Analyses

To determine the effects, if any, of temperature and nutrients on these three species ofUlva, based on the Y measurements, a four-way analysis of variance was performed. The analysis included the (i) species (orthogonal, fixed, three levels); (ii) nutrients (orthogonal, fixed, three levels); (iii) temperature (orthogonal, fixed, two levels); and (iv) plot: Erlenmeyer flasks (random and nested in the interaction of factors 1 and 2, two levels). The SNKpost hoc test was used to examine the nature of the differences that were detected with ANOVA (0.05 significance level). Statistical analyses were performed using GMAV5 for Windows.


## 2.1. Sampling

The three species ofUlva (U. fasciata,U. lactuca, andU. rigida) were collected during summer (January 2010) in the intertidal zone at Prainha Beach (22° 57′S, 042° 01′W), Rio de Janeiro, Brazil. Immediately, the epibionts were removed, and the macroalgae were kept in cool boxes with local seawater (~30 μM NO3, 3 μM PO4, 19°C, and 35 PSU) filtered through 10-μm mesh filters.


## 2.2. The Species

The morphological taxonomy of the three studied species (U. fasciata,U. lactuca, andU. rigida) was based on botanical descriptions and inventories that considered in-depth confrontation among the species descriptions and the Southwestern Atlantic specimens [27–30]. Individuals were prepared as herbarium vouchers and deposited in the herbarium of the Universidade Federal do Rio de Janeiro/UFRJ (under the accession numbers RFA 42441; 42442; 42443).


## 2.3. Experimental Design

All the individuals were maintained in Erlenmeyer flasks in a proportion of 1 g of fresh mass L−1 pasteurized oligotrophic ocean water with added trace nutrients (<1 μM NH4; <1 μM NO3; <2 μM NO2; <1 μM PO4), at 20°C and 200 μmol photons m−2 s−1 (PAR, photosynthetically active radiation) for 15 days. This phase was established to set all the macroalgae to the same nutritional and physiological state before the manipulative experiment. This PAR was chosen because it is an intermediate value in relation to the saturation parameter that is found in the P-I curves (100–400 photons m−2 s−1).After the nutrient deprivation period, six treatments were applied using combinations of two temperatures and three concentrations of nutrients, with 5 replicates per treatment. The experimentper se lasted 10 hours. The factor “Temperature” was represented by 20°C and 30°C, and the factor “Nutrient” was represented by different additions of NaNO3- and Na2HPO4.12H2O of Von Stosch’s enriched seawater (VSE; 500 μM NO3- and 30 μM PO4-) [31]. The experimental setup values of temperatures are related to the temperature at the upper intertidal fringe at the sampling site, which ranges between 18°C (high tide) and 32°C (low tide) [29], and the nutrient concentrations were set as 0.1 VSE, 0.2 VSE, and 0.5 VSE. These concentrations refer to a gradient of nutrient concentrations that were recorded in Southwestern Atlantic [32, 33]. The treatment set as 0.1 VSE refers to natural eutrophicated waters (upwelling events at collecting site, Arraial do Cabo, Rio de Janeiro) [32], whereas 0.2 VSE and 0.5 VSE refer to the range of nutrient concentrations that were recorded in the human-disturbed Guanabara Bay, Rio de Janeiro [33].


## 2.4. Fluorescence Measurements

The photosynthetic efficiency of the three species ofUlvain every combination of temperature and nutrient concentration was characterized by chlorophyll-a fluorescence measurements using a submersible diving-PAM® fluorometer (Walz, Effeltrich, Germany). The measurements consisted of recording the effective quantum yield of PSII, which was calculated as Y = (F′m-Ft)/F′m, where F′m is the maximum fluorescence in the light (obtained by a saturating actinic light pulse – 8900 μmol photons m−2 s−1; 0.8 s) and Ft is the steady-state of fluorescence in the light [34, 35]. Considering the possibility of physiological differences along the thallus [36], the fluorescence was measured in a similar region of the thallus in each individual.


## 2.5. Data Analyses

To determine the effects, if any, of temperature and nutrients on these three species ofUlva, based on the Y measurements, a four-way analysis of variance was performed. The analysis included the (i) species (orthogonal, fixed, three levels); (ii) nutrients (orthogonal, fixed, three levels); (iii) temperature (orthogonal, fixed, two levels); and (iv) plot: Erlenmeyer flasks (random and nested in the interaction of factors 1 and 2, two levels). The SNKpost hoc test was used to examine the nature of the differences that were detected with ANOVA (0.05 significance level). Statistical analyses were performed using GMAV5 for Windows.


## 3. Results

Differences in the Y were found for the interaction of species and temperature (Table1). For bothU. lactuca andU. rigida, the individuals that were incubated at 30°C showed a higher photosynthetic efficiency than did those incubated at 20°C, whereas no difference in photosynthetic efficiency was recorded forU. fasciata individuals (Figure 1). Even when an ANOVA was performed for onlyU. fasciata, no difference in photosynthetic efficiency was found.Table 1
Four-way analyses of variance of the photosynthetic capacities (based on the effective quantum yield of the PSII measurements) of three species ofUlva (orthogonal, fixed) that were incubated under different nutrient (orthogonal, fixed, three levels) and temperature (orthogonal, fixed, two levels) conditions. Plot: Erlenmeyer flasks where individuals were incubated (random and nested in the interaction of factors 1 and 2, two levels).SS is sums of squares; DF is degrees freedom; MS is mean of square and P-value <0.05.
Source SS DF MS F P Species (Sp) 0.0003 2 0.0001 0.18 0.84 Temperature (Te) 0.0129 1 0.0129 9.40 0.02 Nutrient (Nu) 0.0018 2 0.0009 0.64 0.56 Plot (Te×Nu) 0.0082 6 0.0014 2.29 0.04∗ Sp×Te 0.0072 2 0.0036 4.67 0.03∗ Sp×Nu 0.0082 4 0.0020 2.64 0.09 Sp×Plot (Te×Nu) 0.0093 12 0.0008 1.29 0.23 Te×Nu 0.0032 2 0.0016 1.15 0.38 Sp×Te×Nu 0.0081 4 0.0020 2.63 0.09 residual 0.0861 144 0.0006 total 0.1452 179Figure 1
Photosynthetic capacities of the three species ofUlvaunder two temperature conditions (based on the effective quantum yield of PSII measurements). Data from different nutrient conditions and plots (Erlenmeyer flasks where individuals were incubated) were pooled. n=30. Standard deviation values are not shown because of scale-bar limitation (left to right: 0.021, 0.023, 0.032, 0.019, 0.026, and 0.035).


## 4. Discussion

There are currently 403 species ofUlva in the literature, of which 131 have been flagged as accepted [37]. Even with the development of new tools for taxonomic precision, the identification in the practice may differ from author to author. In this sense, fast and real-time physiological assessment can be helpful to distinguish species and morphotypes of macroalgae.In this study, two of the three species ofUlva (U. lactuca andU. rigida) showed differences in the Y as related to temperature. Although these species present large morphological distinctions, both showed a higher photosynthetic efficiency at the highest experimental temperature (30°C), denoting the sensitivity of these species to this temperature value. The physiological differences can be linked to the hydrodynamic environment, once they are under submergence for long periods of time. In this case, this effect of temperature on photosynthetic metabolism is well known for macroalgae [38, 39], and several positive effects on chemical reactions and molecular structures have been reported due to the changes in protein compounds and enzymatic activities [40, 41].In contrast,Ulva fasciata did not show differences in the photosynthetic efficiency related to the temperature variation. Specifically, this is the most abundant of the three species in the sampling place, dominant at the upper limit of the intertidal zone, as also reported by Guimarães and Coutinho [42]. Its photosynthetic apparatus tolerates changes of at least 10°C with no loss of efficiency, as corroborated by our study, which can confer an ecological advantage and seems to support its relative spatial and temporal dominance on rocky shores [38, 43].Despite their unequal abundances, the three species are usually recorded throughout the year, although this cooccurrence is most evident during the summer [29]. The summer period is characterized by high irradiance (i.e., high temperatures on rocky shores), cold waters, and a high input of nutrients from the upwelling and rainfall runoff [44, 45]. Taken together, these results suggest that the ecophysiological differences that were pointed in this study provide a reasonable explanation for the variability in the spatial and temporal abundance of these species ofUlva on rocky shores.It is important to highlight that despite the absence of nutrient effects on the photosynthetic response ofUlva species in our study, we are not fully disregarding the importance of this factor as a driver of the spatial and temporal variability ofUlva species on rocky shores. Nutrient enrichment favors ephemeral foliose macroalgae (see [3]) by increasing the photosynthetic response [46]. In fact, the genusUlva is recognized by the affinity for nitrogen [47–49], and differences in photosynthetic efficiency have been predicted (if not among species, at least among nutrient concentrations), in contrast to the present results. Considering the proper experimental design and applied manipulative measurements, we attribute these findings to a saturated rate of nutrient availability: light supply in our experiment.One of the most vexing taxonomic concerns refers to theU. fasciata andU. lactucaspecies, which cooccur withU. rigida. RegardingU. fasciataandU. lactuca, there is a long-standing taxonomic debate (see [1, 10–13, 50, 51]). Meanwhile, several studies confirmU. rigidaas a consistent valid species [52–54]. We corroborate the previous claim of Hiraoka et al. [10], Shimada et al. [50], and Kirkendale et al. [1]. Our study considered ecophysiological and morphological (based on [28–30]) data for specimens from Southwestern Atlantic. Therefore, we propose the use of ecophysiology as a complementary tool for taxonomic distinction among macroalgae based on chlorophyll-a fluorescence to enlighten this debate with additional taxonomic evidence. Similar morphophysiological studies, based on classic morphometry and photosynthetic responses (maximum electron transport rates, ETRmax), suggested different species ofDurvillaea(Phaeophyceae) for two morphotypes cooccurring in southern Chile [26].However, how this ecophysiology method would respond on other marine macroalgal taxa remains to be tested. We understand that it is hard to apply the methodology to identification of freshly collected specimens or species in the field without further investigation, either by morphology or molecular methods. We also recognize the undeniable contribution of morphology and molecular methods to taxonomy, and our proposal aims to enlighten the debate with additional evidence.



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