Nuñez et al. Helgol Mar Res (2018) 72:16 https://doi.org/10.1186/s10152-018-0519-1 Helgoland Marine Research ORIGINAL ARTICLE Open Access Genetic and morpho‑physiological diferentiation in a limpet population across an intertidal gradient Jesús Darío Nuñez*, Pedro Fernández Iriarte, Emiliano Hernán Ocampo, Enrique Madrid and Maximiliano Cledón Abstract Marine organism adaptive capacity to diferent environmental conditions is a research priority to understand what conditions are important in structuring the spatial distribution of natural populations. In this context, this study evalu- ates whether potential diferences in Siphonaria lessonii morphology (size and shell shape) and physiology (water loss regulation) at diferent shore heights are linked to genetically distinguishable individuals. To achieve this goal, we compared size-frequency distribution, morphometric, and genetic data (ISSR-PCR technique) of S. lessonii from the upper, middle and lower shore. We complemented these feld samplings with laboratory experiments on water loss and mortality. Genetic analysis showed diferent genetic composition for individuals from the upper and lower shore. This pattern was accompanied by morpho-physiological variations: the upper shore had fewer small limpets, lower shell shape dispersion (with a morphotype characterized by a higher shell), and less water loss and mortality related to air exposure than the lower shore. The results reported herein support the idea that the extreme and unpredict- able conditions of the upper shore may impose strong selection pressure on its inhabitants, leading to considerable morpho-physiological diferentiation consistent with diferent genetic composition. This probably plays an important role in structuring the spatial distribution of natural S. lessonii populations with a possible efect on size-structure distribution. Keywords: Siphonaria lessonii, Vertical distribution, Desiccation, Shell shape, ISSR Background modify their metabolic rates [7–10], and morphological Marine organism adaptive capacity to diferent environ- changes, such as body size and shape, that reduce water mental conditions is a research priority to understand loss [11]. In recent years, ecological researchers have what conditions are important in structuring the spa- integrated these adaptive changes with genetic studies, tial distribution of natural populations [1, 2]. Intertidal enabling the development of hypotheses with an eco- species generally are subject to extreme environmental genetic context. Tese studies have found evidence of fuctuations that afect their desiccation rates, among changes in the frequencies of some alternative alleles other efects. To cope, they have developed a variety of related to local adaptation across environmental gradi- adaptations for regulating body temperature and evapo- ents, such as in populations distributed along latitudinal ration [3]. For example, they have developed behaviors clines [12, 13] or intertidal gradients [14–16]. In the lat- that minimize stress [4–6], physiological regulations that ter case, some adaptive characteristics such as changes in body shape and color or physiological traits may result from the strong selection pressure on the inhabitants of *Correspondence: [email protected] the upper shore levels, leading to considerable physi- Instituto de Investigaciones Marinas y Costeras (IIMyC), Consejo Nacional de Investigaciones Científcas y Técnicas (CONICET), Universidad Nacional ological [17] and genetic diferentiation [16, 18–20] even de Mar del Plata, CC 1260 Correo Central, B7600WAG​ Mar del Plata, within a single species [14, 15]. In this context, and due Argentina to recent advances in the studies of species evolution, the © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Nuñez et al. Helgol Mar Res (2018) 72:16 Page 2 of 10 specialists emphasize the importance of gene fow and quadrats randomly placed along the three shore heights. divergent natural selection as key concepts in studies on Size-frequency distributions were developed for each the evolution of reproductive isolation (and subsequent shore height after sorting measured limpets into 1 mm speciation) [21]. size classes. A permutational multivariate analysis of var- In the southwest Atlantic, one of the most common iance (PERMANOVA) [33, 34] was performed to deter- limpet species is Siphonaria lessonii (Pulmonata, Sipho- mine diferences in population size-structure among nariidae) [22, 23]. Its distribution in South America shore heights. Prior to PERMANOVA, a test of multi- extends from Peru in the Pacifc Ocean to the north of variate homogeneity of variances (PERMDISP) [35] was Uruguay in the Atlantic Ocean [24]. Te way in which run to fnd diferences in within-group dispersion based some of the characteristics of this species vary accord- on the Bray–Curtis index applied to fourth-root trans- ing to the environmental conditions it lives in has been formed data. Te signifcance of the factor shore heights well studied. For example, the shape and size of the shell in both analyses was tested with 999 permutations. An can change along the shore height depending on environ- analysis of similarity percentages (SIMPER) [36] was mental humidity [25–29]. Its behavior patterns can also performed for data recorded for each size class present change with the environmental evaporation rates: in the at each shore height. Te SIMPER analysis identifed the lowest latitudes (such as 38°) of the Argentinean coast, size ranges that contribute most to the observed difer- the environmental evaporation is lower than middle lati- ences for shore height by the Bray–Curtis similarities tude (40°), S. lessonii does not exhibit site fdelity [6, 30, between samples. 31], while in the mean latitudes of the Argentinean and Chilean coasts, it exhibits moderate site fdelity [6, 32]. Tus, we hypothesize that due to the highly stressful Morphometric analyses environment where S. lessonii lives, which is translated To analyze shell morphology among diferent shore into morphological and physiological diferences, indi- heights, a set of 20 S. lessonii adult individuals were ran- viduals of the diferent intertidal heights will have genetic domly collected at each shore height. We used adult diferences. specimens (shell length > 6 mm) because smaller limpet Te aim of our study is to evaluate whether diferences shells are easily broken by manipulation. in S. lessonii morphology (size and shell shape) and physi- Te shells were photographed with a digital cam- ology (water loss regulation) at the diferent shore heights era (Canon Power Shot A580) from the right side. are linked to genetically distinguishable individuals. Digital images were taken against a white illuminated background in order to maximize the contrast of shell Methods outlines. All the images were binarized (i.e. transformed Study sites and sampling into white for the shell outline and black for the back- Tis study was carried out in the Waikiki rocky shore, ground, in pixels) so the outlines of each continuous con- (hereinafter WRS) (38°04′50S, 57°30′08W), Buenos Aires, tour (interface between the black and the white pixels) Argentina. Average WRS tidal amplitude is 0.80 m (maxi- were automatically obtained and digitalized using the mum 1.69 m) and during low tide, the beach is 10–20 m SHAPE software [37]. Te shell shape of these limpets wide. is rather simple with very few homologous points to be Field sampling for this study was performed during used as landmarks. Moreover, the landmarks are difcult April 2010 (mid-fall) at three selected shore heights. Te to locate, being classifed as type 2 landmarks (maximum lower shore was defned as the minimum sea level at curvature along the boundary or outline of the specimen) spring tides, which was dominated by mussel beds. Te [38]. Shell shape variation among S. lessonii individu- middle shore was defned as the maximum sea level at als from the three shore heights was therefore measured neap tides, which was predominantly covered by diverse using outline analyses based on the Elliptic Fourier analy- species of native algae. Te upper shore was defned as sis on the outline coordinates [39]. Elliptic Fourier analy- the maximum sea level at spring tides, dominated by bare sis are preferred over classical morphometric analyses rock with mussels and barnacles in cracks and crevices in cases landmarks are difcult to determine, and this [29]. During sample collection, patchy micro-habitats analysis have been used before in S. lessonii [25]. Elliptic such as crevices were avoided in order to avoid con- Fourier coefcients were mathematically normalized in founding habitat variation with shore height variation. order to avoid biased outcomes resulting from diferent sizes, locations, rotations and starting position of shells Population size structure [39]. Te closed curve of each shell was broken down into To determine feld distribution patterns of limpets, 15 harmonically related ellipses. Tese 15 harmonics rep- resent 99.99% of the total Fourier power spectrum [40]. we sampled all individuals present in ten (50 × 50 cm) Nuñez et al. Helgol Mar Res (2018) 72:16 Page 3 of 10