Phenotypically similar species coexisting in extreme environments like sulfidic water are subject to two opposing eco-evolutionary processes: those favoring similarity of environment-specific traits, and those promoting differences of traits related to resource use. Europe, Movile Cave in Romania, and the Frasassi cave system in Italy [21C22]. Here we used a recently developed ecomorphological framework for [23C24] to test whether processes such ecological filtering and interspecific competition have affected functional morphological traits that are likely to relate to different dimensions of the ecological niche. We predict that coexisting species will (1) resemble each other in traits that reduce the toxic effect of sulfide, and (2) differ in traits that correlate functionally to resource use. Furthermore, we reconstructed the morphologies 84687-43-4 of the inferred ancestors of coexisting species pairs in order to check whether functional morphological traits evolved over time. Materials and Methods Study caves and study species Movile Cave is situated in southeastern Romania on the Dobrogea Plateau (Fig 1). The surrounding of the cave is ecologically homogenous and covered with steppe vegetation. The fauna of the cave was extensively studied and includes 18 aquatic species. The macrofauna consist of two amphipods, an isopod, and two possible predatorsa leech and a water bug . Our two focal species are Danc?u 1970 and a yet undescribed species, provisionally named as cf. species consistently appears nested in the genus on molecular phylogenies (see  and the Results section of this paper), and its genus status is under revision (unpublished). Even though south-eastern Romania is poorly explored, the vicinity of Movile Cave up to a radius of 24 km has been a subject of thorough sampling. Apart from our focal species pair, five other species have been reported from this area making up a regional species pool. Karaman & Sarbu 1993 is another species thriving exclusively in sulfidic water but restricted to a spatially separated sulfidic aquifer. Three further species (Karaman 1932, Schellenberg 1935, Danc?u 1964) are apparently restricted to sulfide-free waters. Finally, Karaman & Sarbu 1995 is widely distributed in Dobrogea, mostly in sulfide-free water, but on rare occasions it was found in sulfidic waters as well. Fig 1 Geographic position of the analyzed sulfidic caves (F = Frasassi cave system, M = Movile Cave). The Frasassi cave system is located in central Italy, within the Adriatic part of the Apennine Mountains, about 40 km from your coast (Fig 1). This cave system lies in a geographically and geologically varied area alongside a 500-meter deep and two-kilometer Rtp3 long canyon formed from the Sentino River. The surface surroundings are covered by thermophilous Mediterranean vegetation, pine forests and deciduous forests . Although the fauna of this cave system has been less extensively analyzed than in Movile, amphipods are among the largest aquatic animals there. Out of the four known varieties, one (sp.4 in ) was collected only once and can be considered as an accidental visitor, whereas the other three are regular inhabitants of the cave system. The focal varieties, Karaman 1985 and Karaman, Borowski and Dattagupta 2010, dwell primarily in sulfidic water throughout the system and coexist in some places at a distance of a few centimeters . The third local varieties, Karaman, Borowski and Dattagupta 2010, has been found specifically in one sulfide-free pool. The area surrounding this cave system is definitely ecologically more heterogeneous than the Movile area, and the regional amphipod varieties pool is definitely less known. To accomplish robust estimations of the regional varieties pool despite this limitation, we regarded as for 84687-43-4 the analyses all varieties within a range three times larger than for Movile Cave (i.e., inside a radius of about 75 km); according to  these varieties are Schi?dte 1855, Garbini 1894, Costa 1851, Vigna-Taglianti 1966, agg., Karaman 1984, Ruffo & Vigna Taglianti 1967. Samples for phylogenetic and morphometric analyses The comparative analysis of morphological development of sulfide-dwelling varieties requires a wider phylogenetic context. For this purpose, we selected a subset of a large DNA dataset [23C26, 28C29] that includes representatives of all major lineages, and all close 84687-43-4 relatives of our focal varieties. Altogether 44 varieties were 84687-43-4 used to reconstruct phylogenetic human relationships and ancestral morphologies. Lists of varieties, sampling sites, 84687-43-4 sequence accession figures, and morphometric data are offered in the S1 and S2 Furniture). No specific permissions were required for these locations and the study does not include endangered or safeguarded varieties. Phylogenetic reconstruction We used two variable sections of the 28S rRNA gene adding.