plants Article Do Bryophyte Elemental Concentrations Explain Their Morphological Traits? Marcos Fernández-Martínez 1,2,* , Jordi Corbera 2 , Oriol Cano-Rocabayera 2,3, Francesc Sabater 2,3 and Catherine Preece 1,2 1 Research Group PLECO (Plants and Ecosystems), Department of Biology, University of Antwerp, 2610 Wilrijk, Belgium; [email protected] 2 Delegació de la Serralada Litoral Central, ICHN, 08302 Mataró, Catalonia, Spain; [email protected] (J.C.); [email protected] (O.C.-R.); [email protected] (F.S.) 3 Department of Ecology, University of Barcelona, 08028 Barcelona, Catalonia, Spain * Correspondence: [email protected] Abstract: Differences in the elemental composition of plants, mainly C, N, and P, have been shown to be related to differences in their nutritional status, and their morphological and functional traits. The relationship between morphological traits and micronutrients and trace elements, however, has been much less studied. Additionally, in bryophytes, research devoted to investigating these relationships is still very scarce. Here, we analysed 80 samples from 29 aquatic and semi-aquatic (hygrophytic) moss species living in Mediterranean springs to investigate the relationship between moss nutrient concentrations and their micro- and macroscopic morphological traits and growth forms. We found that, across species, the elemental concentration of mosses was more tightly linked to macroscopic traits than to microscopic traits. Growth forms could also be successfully explained by Citation: Fernández-Martínez, M.; the concentration of elements in mosses. Apart from macronutrients and their stoichiometric ratios Corbera, J.; Cano-Rocabayera, O.; Sabater, F.; Preece, C. Do Bryophyte (C:N, C:P, and N:P), micronutrients and trace elements were also important variables predicting Elemental Concentrations Explain moss morphological traits and growth forms. Additionally, our results showed that microscopic Their Morphological Traits? Plants traits were well related to macroscopic traits. Overall, our results clearly indicate that the elemental 2021, 10, 1581. https://doi.org/ composition of mosses can be used to infer their morphological traits, and that elements other than 10.3390/plants10081581 macronutrients should be taken into account to achieve a good representation of their morphological and, potentially, functional traits when comparing the elemental composition across species. Academic Editors: Kristian Peters, Henrik Toft Simonsen and Keywords: stoichiometry; morphology; traits; elemental composition; moss; cell Marko Sabovljevic Received: 9 July 2021 Accepted: 29 July 2021 1. Introduction Published: 31 July 2021 Bryophytes are amongst the most fascinating organisms of the plant kingdom, given Publisher’s Note: MDPI stays neutral their ecology, physiology, and morphology. Their lack of true cuticles and roots make them with regard to jurisdictional claims in very sensitive to environmental conditions [1], and those features make them well suited published maps and institutional affil- to monitoring changes in environmental conditions. Particularly under environmental iations. pollution, bryophytes have been shown to accumulate heavy metals and change their elemental composition or, in the event that they cannot tolerate the new conditions, they disappear. Accordingly, bryophytes have been used to assess the ecological consequences of air and water pollution (e.g., nitrate or heavy metals) [2–7]. However, their elemental composition under normal conditions, and how it is related to their functioning, have been Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. seldom explored, despite a few studies focused on bryophyte C, N, and P or macronu- This article is an open access article trient concentrations [8–12]. Nonetheless, information on the elemental composition of distributed under the terms and bryophytes is particularly lacking for a large array of micronutrients and trace elements, conditions of the Creative Commons particularly from non-polluted areas. Attribution (CC BY) license (https:// The elemental composition of organisms has been repeatedly shown to be a very good creativecommons.org/licenses/by/ indicator of their morphological and functional traits (e.g., photosynthesis, respiration), 4.0/). the ecological strategies they follow, and their relationship with their environment [13–17]. Plants 2021, 10, 1581. https://doi.org/10.3390/plants10081581 https://www.mdpi.com/journal/plants Plants 2021, 10, 1581 2 of 13 Accordingly, the elemental composition of organisms has been repeatedly shown to present an important adaptive value, both in plants and animals [18,19]. Generally, plants pre- senting higher concentrations of N and P, and lower C:N and N:P ratios, tend to be fast growing and more productive; reproduce more, and more frequently; and require fewer defence mechanisms; while a more conservative lifestyle is shown by nutrient-limited plants [15,20–22]. Similar patterns have also been shown in bryophytes [23,24] suggesting that a link between the elemental composition of plants and their morphological and functional traits should occur throughout the plant kingdom. Recent publications focused on bryophyte functional traits have considerably in- creased our knowledge regarding the relationship between the environment and bryophyte functional traits [25–29]. In particular, some of these studies identified water chemistry (e.g., pH, dissolved nutrients, and heavy metals) as an important determinant of moss traits and growth forms [2,25,28]. Other studies, focused on how several traits are related to each other, made very relevant contributions in order to emulate the leaf economic spectrum described for vascular plants [23,24]. These studies provided very relevant information on how bryophyte morphological traits, and N and P concentrations are related to their photosynthesis and dark respiration rates, indicating similar patterns to those observed in vascular plants [22]. However, to the best of our knowledge, no efforts have been devoted to investigating whether the elemental composition of bryophytes is related to their micro- and macroscopic morphological traits and growth forms across a large number of species. To fill in this gap, we here analysed 80 samples from 29 aquatic and semi-aquatic (hygrophytic) moss species to investigate the relationship between gametophyte moss nutrient concentrations and their morphological traits. We hypothesised that the elemental composition of moss species will be a very good indicator of their morphological traits, similar to that which has been reported in vascular plants [15,17,22]. Additionally, we hypothesise that moss species presenting higher concentrations of N and P, and low C:N, C:P, and N:P stoichiometric ratios, related to more productive organisms, will present larger and wider leaves and will be lighter (i.e., less dense and less mass per area). Our results will help us to further understand how the elemental composition of organisms determine their functional traits and ecological strategies in bryophytes. 2. Methods 2.1. Data Collection and Analyses of Elemental Composition and Morphological Traits We used previously published data in which we analysed the elemental composition of moss samples [30,31] from a large collection used in a previous study [25] for which their morphological traits were already estimated, including 29 different hygrophytic moss species. From the total collection of 100 samples with morphological traits, 80 samples had enough mass to analyse their elemental composition. Those 80 samples were used to perform this study. Moss samples were collected from springs distributed across Catalonia (north-eastern Iberian Peninsula, Figure S1) following a large climate and hydrochemical gradient [32]. In the subset of springs used here, water pH ranged from 5.17 to 8.34 (median: 7.20) and water conductivity ranged from 24 to 2094 (median: 498) µS cm−1. A detailed analysis of the water chemical composition can be found in the online material from references [25,30,31]. All mosses collected were in direct contact with the water of the springs throughout the year, with only occasional interruptions in water flow during winter and summer (when water freezes or during intense drought events, respectively). Given that these springs were draining water from aquifers, their water chemical composition was very stable in time. The climate of the area of study is Mediterranean, ranging from humid to sub-humid, and with large differences in mean annual temperature and precipitation (from 4.2 to 15.7 ◦C, and from 567.4 to 1202.4 mm y−1, respectively) [25,33]. Before the moss elemental analyses, we submerged all samples in a solution of acetic acid at pH 2.7 for 15 min in order to remove incrustations of CaCO3. A few samples required up to an hour to remove all CaCO3 incrustations. We then rinsed the samples Plants 2021, 10, 1581 3 of 13 with distilled water and dried them at 60 ◦C for 48 h. We ground the samples to a powder using liquid nitrogen and a mortar. Moss C and N concentration and δ13C and δ15N were determined by using a Flash EA1112 and TC/EA coupled to a stable isotope mass spectrometer Delta C through a Conflo III interface (ThermoFinnigan). We determined the concentration of 19 elements, including macro-, micronutrients and trace elements (P, K, S, Ca, Mg, Na, B, Fe, As, Al, Cd, Co, Cu, Cr, Hg, Mn, Ni, Pb, and Zn) by inductively coupled plasma mass/optical emission