Diana Delicado

Morphological and molecular evidence for cryptic species of springsnails [genus Pseudamnicola (Corrosella) (Mollusca, Caenogastropoda, Hydrobiidae)]

Abstract Several Pseudamnicola (Corrosella) populations of the central and eastern Iberian Peninsula have been ascribed to Pseudamnicola (Corrosella) astieri (Dupuy, 1851), though recent evidence demonstrates the species could be endemic to the departments of Var and Alpes-Maritimes in France. Through the identification of cryptic species using a combined morphological and phylogenetic approach, this paper provides a detailed morphological description of Pseudamnicola (Corrosella) astieri, clarifying its taxonomic boundaries and confirming it as a French endemic. In parallel, by comparing Pseudamnicola (Corrosella) populations from the provinces of Castellón and Valencia in Eastern Spain, it was observed that rather than Pseudamnicola (Corrosella) astieri they represented a new species here described as Pseudamnicola (Corrosella) hauffei sp. n. Among other characters, the two species show marked differences in shell shape, male and female genital systems, radular formula and concentration of the nervous system. Pseudamnicola (Corrosella) hauffei sp. n. was also compared morphologically to another two Pseudamnicola (Corrosella) species living in nearby areas [Pseudamnicola (Corrosella) hinzi Boeters, 1986 and Pseudamnicola (Corrosella) navasiana (Fagot, 1907)], molecularly to Pseudamnicola (Corrosella) falkneri (Boeters, 1970), the type species of the subgenus, and to the rest of the Pseudamnicola (Corrosella) species described so far. Morphological differentiation between the species is supported by a genetic divergence of 7.4% inferred from a partial sequence (658 bp) of the mitochondrial gene cytochrome c oxidase subunit I (COI). On the basis of an average 8% (5.39 to 11.15%) divergence estimated for the COI gene in other Pseudamnicola (Corrosella) species reported in GenBank, the existence of two specific entities is here proposed, which will have impact on conservation policies both in France and in Spain.

Effects of habitat transition on the evolutionary patterns of the microgastropod genus Pseudamnicola (Mollusca, Hydrobiidae)

Molecular phylogenies of extant species are considered effective tools to infer mechanisms of speciation. Here, we benefit from this utility to investigate the evolutionary history of an organismal group linked to different aquatic ecosystems, the microgastropod genus Pseudamnicola (family Hydrobiidae). Previous studies have found around 45 species of the nominal subgenus P. (Pseudamnicola), most of them in coastal stream localities of several Mediterranean islands and mainland territories, whereas only 12 species of the other subgenus, P. (Corrosella), have been collected from springs and headwaters of mountainous regions of the Iberian Peninsula and south of France. As springs often act as isolated habitats affecting dispersion and constraining gene flow, we supposed that the temporal history and mode of diversification of species from both subgenera should differ and therefore be reflected in their phylogenetic patterns. To assess this hypothesis, we performed a molecular phylogeny based on mitochondrial and nuclear DNA sequences and later conducted an independent analysis to examine the potential effect of certain geographic and ecological variables in the genetic divergences of the subgenera. Additionally, we estimated the ancestral area of diversification of both groups. Published anatomical revisions and our molecular analyses suggest that the genus Pseudamnicola should be divided into three genera: the two previous subgenera plus a new one described here. As postulated, the evolution of the spring organisms was strongly related to habitat fragmentation and isolation, whereas dispersal followed by divergence seem to have been the most common speciation processes for euryhaline species inhabiting coastal streams and low river stages in which waters remain connected. On the contrary, rather than habitat fragmentation or dispersion, environmental conditions have played a larger role during the deep divergent split leading to the three genera.

A rare case of stygophily in the Hydrobiidae (Gastropoda: Sadleriana)

Stygobionts (i.e. obligately subterranean aquatic species) represent about 350 (Culver, 2012) or 9% of the total freshwater gastropod diversity of around 4,000 species (Strong et al., 2008). Hydrobioids (= Hydrobiidae s. l.; Davis, 1979) make up the majority of stygobiont snails (Bole & Velkovrh, 1986). Compared with their epigean (surface-dwelling) relatives, stygobiont gastropods are often characterized by pale, thin shells, by loss of body and eye pigmentation, and by reduction in body size and anatomical structures such as gills and reproductive organs (Hershler & Holsinger, 1990; Culver, 2012). These morphological correlates of subterranean habitat are often used to differentiate stygobiont and epigean hydrobioids (e.g. Simone & Moracchioli, 1994; Bodon, Ghamizi & Giusti, 1999b; Georgiev, 2011). However, a few species that live in both subterranean and epigean habitats (referred to as stygophiles) are variable in these characters (e.g. Fontigens nickliniana, Hershler, Holsinger & Hubricht, 1990; Litthabitella chilodia, Bodon et al., 1999a; Nanocochlea exigua, Ponder et al., 2005). Thus, the use of both morphological and molecular evidence is critical for confident delineation of species limits in these organisms. During the course of a field survey in Croatia, I collected Sadleriana cavernosa Radoman, 1978 (Hydrobiidae s. s., sensu Wilke et al., 2013) from its single known locality, Tounjčica Cave, and also found morphologically similar snails in two nearby springs. Radoman (1978) described S. cavernosa based on shell features and the lack of body and eye pigmentation, and later illustrated the genitalia and radula of this species (Radoman, 1983). The purpose of this study is to determine whether the newly discovered epigean populations are conspecific with S. cavernosa. Sadleriana cavernosa was collected from last 50 m of the small stream in Tounjčica Cave (45.24896°N, 15.31868°E) in June 2016. Snails were also collected from two springs along the river Tounjčica (Fig. 1A), one near the cave (herein referred to as spring 1; 45.2489°N, 15.32151°E) and the other about 7 km distant the cave, feeding a downstream-situated inflow of the Tounjčica River (spring 2; 45.21743°N, 15.38928°E). The subterranean specimens were found in sympatry with the hydrobiid species Belgrandiella pageti Schütt, 1970 and Hauffenia tovunica Radoman, 1978; the epigean specimens co-occurred with S. sadleriana (Frauenfeld, 1863). Snails and DNA vouchers were deposited in the biodiversity collection at Justus Liebig University Giessen (UGSB; Diehl et al., 2018); lot numbers UGSB18554 (Tounjčica Cave), UGSB 18552 (spring 1) and UGSB 18558 (spring 2). As only a few individuals (7–15) were collected at each locality, I extracted the total DNA of 1–2 specimens from each population and analysed a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. This fragment provides sufficient resolution to detect intraand interspecific variation in springsnails (Liu & Hershler, 2005; Delicado, Machordom & Ramos, 2013) and is the most frequent gene fragment available for Sadleriana species in GenBank. Total DNA was extracted from entire snails using the CTAB protocol described by Wilke et al. (2006). A fragment of COI was PCR-amplified with the primers LCO1490 and HCO2198 (Folmer et al., 1994), using the cycling conditions described by Schreiber et al. (2012). Amplified products were cleaned using ExoSAP-IT (Affymetrix, München, Germany) and sequenced with the Big Dye Terminator Kit in an ABI 3730 XL sequencer (Life Technologies, Carlsbad, CA). Sequences were edited using SEQUENCHER v. 4.6 (Gene Codes, Ann Arbor, MI) and aligned manually with COI sequences of four other spring species of the genus Sadleriana and two outgroups (Table 1) in MEGA7 (Kumar, Stecher & Tamura, 2016). Using the p-distance method, I calculated pairwise distances among 27 Sadleriana sequences for COI (658 bp) in MEGA7. The phylogenetic relationships of the newly discovered epigean populations were evaluated using maximum-likelihood (ML) and Bayesian-inference (BI) analyses. ML was undertaken in RAxMLHPC8 (Stamatakis, 2014) on the RAxML BlackBox (Stamatakis, Hoover & Rougemont, 2008) using the default substitution model GTR (Tavaré, 1986) + I (invariable positions) +G (including variation among sites). BI was run through two independent runs of four Metropolis-coupled chains of 2.5 million generations each and a sampling frequency of 1,000 in MrBayes v. 3.1.2 (Huelsenbeck, 2000; Huelsenbeck & Ronquist, 2001), using the evolutionary model TPM3uf (Kimura, 1981) + G selected in jModelTest v. 2.1.10 (Darriba et al., 2012) under corrected Akaike’s information criterion. Convergence between runs was determined upon the standard deviation of split frequencies reaching values of less than 0.01. The initial 10% of the sampled trees was discarded as burn-in. Clade robustness was assessed by nonparametric bootstrapping (BS; Felsenstein, 1985) using 100 pseudoreplicates in ML and by posterior probabilities (PP) in BI. The final topologies were visualized and edited in FigTree v. 1.4.3 (Rambaut, 2016). I also dissected 4–6 large adults, most females, from each locality after dissolving the shell in ethylenediaminetetraacetic acid (EDTA). Soft tissues were dissected under an Olympus SZX12 binocular microscope. Shells and organs were imaged with a

Ecological opportunity may facilitate diversification in Palearctic freshwater organisms: a case study on hydrobiid gastropods

BackgroundDifferences in species richness among phylogenetic clades are attributed to clade age and/or variation in diversification rates. Access to ecological opportunity may trigger a temporary increase in diversification rates and ecomorphological variation. In addition, lower body temperatures in poikilothermic animals may result in decreasing speciation rates as proposed by the metabolic theory of ecology. For strictly freshwater organisms, environmental gradients within a river continuum, linked to elevation and temperature, might promote access to ecological opportunity and alter metabolic rates, eventually influencing speciation and extinction processes. To test these hypotheses, we investigated the influence of environmental temperature and elevation, as proxies for body temperature and ecological opportunity, respectively, on speciation rates and ecomorphological divergence. As model systems served two closely related gastropod genera with unequal species richness and habitat preferences – Pseudamnicola and Corrosella.ResultsLineage-through-time plots and Bayesian macroevolutionary modeling evidenced that Pseudamnicola species, which typically live in lower reaches of rivers, displayed significantly elevated speciation rates in comparison to the ‘headwater genus’ Corrosella. Moreover, state-dependent speciation models suggested that the speciation rate increased with decreasing elevation, supporting the ecological opportunity hypothesis. In contrast, a significant effect of environmental temperature, as proposed by the metabolic theory of ecology, could not be observed. Disparity-through-time plots, models of ecomorphological evolution, and ancestral habitat estimation showed for Pseudamnicola species rapid morphological divergence shortly after periods of elevational and habitat divergence. In contrast, Corrosella species did not deviate from null models of drift-like evolution.ConclusionOur finding that speciation rates are correlated with elevation and ecomorphological disparity but not with environmental temperatures suggests that differences in ecological opportunity may have played a key role in Corrosella and Pseudamnicola diversifications. We propose that Pseudamnicola lineages experienced higher ecological opportunity through dispersal to new locations or habitats in lowlands, which may explain the increase in speciation rates and morphological change. In contrast, the evolution of Corrosella in headwaters is likely less facilitated by the environment and more by non-ecological processes.