Molecular phylogeny of the genus Chondrina (Gastropoda, Panpulmonata, Chondrinidae) in the Iberian Peninsula.

Chondrina Reichenbach, 1828 is a highly diverse genus of terrestrial molluscs currently including 44 species with about 28 subspecific taxa. It is distributed through North Africa, central and southern Europe, from Portugal in the West to the Caucasus and Asia Minor in the East. Approximately 70% of the species are endemic to the Iberian Peninsula constituting its main center of speciation with 34 species. This genus includes many microendemic taxa, some of them not yet described, confined to limestone habitats (being strictly rock-dwelling species). They are distributed on rocky outcrops up to 2000 m.a.s.l. It is a genus of conical-fusiform snails that differ mainly in shell characters and in the number and position of teeth in their aperture. So far, molecular studies on Chondrina have been based exclusively on the mitochondrial Cytochrome Oxidase subunit I region (COI). These studies gave a first view of the phylogeny of the genus but many inner nodes were not statistically supported. The main objective of the study is to obtain a better understanding of the phylogeny and systematics of the genus Chondrina on the Iberian Peninsula, using multilocus molecular analysis. Partial sequences of the COI and 16S rRNA genes, as well as of the nuclear Internal Transcribed Spacer 1 (ITS1-5.8S) and Internal Transcribed Spacer 2 (5.8S-ITS2-28S) were obtained from individuals of all the extant Chondrina species known from the Iberian Peninsula. In addition to this, the newly obtained COI sequences were combined with those previously published in the GenBank. Phylogenetic relationships were inferred using maximum likelihood and Bayesian methods. The reconstructed phylogenies showed high values of support for more recent branches and basal nodes. Moreover, molecular species delimitation allowed to better definethe studied species and check the presence of new taxa.

De novo genome assembly of the land snail Candidula unifasciata (Mollusca: Gastropoda).

Among all molluscs, land snails are a scientifically and economically interesting group comprising edible species, alien species and agricultural pests. Yet, despite their high diversity, the number of genome drafts publicly available is still scarce. Here, we present the draft genome assembly of the land snail Candidula unifasciata, a widely distributed species along central Europe, belonging to the Geomitridae family, a highly diversified taxon in the Western-Palearctic region. We performed whole genome sequencing, assembly and annotation of an adult specimen based on PacBio and Oxford Nanopore long read sequences as well as Illumina data. A genome draft of about 1.29 Gb was generated with a N50 length of 246 kb. More than 60% of the assembled genome was identified as repetitive elements. In total, 22,464 protein-coding genes were identified in the genome, of which 62.27% were functionally annotated. This is the first assembled and annotated genome for a geometrid snail and will serve as reference for further evolutionary, genomic and population genetic studies of this important and interesting group.

Incorporating palaeogeography into ancestral area estimation can explain the disjunct distribution of land snails in Macaronesia and the Balearic Islands (Helicidae: Allognathini).

The systematics and biogeographical history of the Eastern Mediterranean and Macaronesian land snail tribe Allognathini (Helicidae: Helicinae) is investigated based on mitochondrial and nuclear DNA sequence data. Our molecular phylogenetic analyses indicate that the genus-group systematics of the tribe needs to be revised. We show for the first time that the narrow-range endemics Lampadia and Idiomela from the Madeira Archipelago belong to Allognathini and represent together the sister group of the diverse Canary Island Hemicycla radiation. We therefore suggest synonymising Lampadiini with Allognathini. Sister to these Macaronesian genera was the Balearic Island Allognathus radiation. Pseudotachea was not recovered as a monophyletic group and the two currently recognised species clustered in Iberus. Similarly, Adiverticula was not recovered as a monophyletic group and clustered in Hemicycla. We therefore suggest synonymising Pseudotachea with Iberus and Adiverticula with Hemicycla. The six genera in Allognathini, which we distinguish here (Cepaea, Iberus, Allognathus, Hemicycla, Idiomela and Lampadia), originated in Western to South-western Europe according to our ancestral area estimation and the fossil record. The disjunct distribution of the Balearic Islands and Macaronesian sister clades and the mainly Iberian Iberus clade that separated earlier can be explained by the separation of the Betic-Rif System from the Iberian Peninsula during the late Oligocene to early Miocene, along with independent Miocene dispersals to the Balearic Islands and Macaronesia from the Iberian Peninsula, where the ancestral lineage became extinct.

Whole-genome re-sequencing data to infer historical demography and speciation processes in land snails: the study of two Candidula sister species.

Despite the global biodiversity of terrestrial gastropods and their ecological and economic importance, the genomic basis of ecological adaptation and speciation in land snail taxa is still largely unknown. Here, we combined whole-genome re-sequencing with population genomics to evaluate the historical demography and the speciation process of two closely related species of land snails from western Europe, Candidula unifasciata and C. rugosiuscula. Historical demographic analysis indicated fluctuations in the size of ancestral populations, probably driven by Pleistocene climatic fluctuations. Although the current population distributions of both species do not overlap, our approximate Bayesian computation model selection approach on several speciation scenarios suggested that gene flow has occurred throughout the divergence process until recently. Positively selected genes diverging early in the process were associated with intragenomic and cyto-nuclear incompatibilities, respectively, potentially fostering reproductive isolation as well as ecological divergence. Our results suggested that the speciation between species entails complex processes involving both gene flow and ecological speciation, and that further research based on whole-genome data can provide valuable understanding on species divergence. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.

Experimental simulation of environmental warming selects against pigmented morphs of land snails.

In terrestrial snails, thermal selection acts on shell coloration. However, the biological relevance of small differences in the intensity of shell pigmentation and the associated thermodynamic, physiological, and evolutionary consequences for snail diversity within the course of environmental warming are still insufficiently understood. To relate temperature-driven internal heating, protein and membrane integrity impairment, escape behavior, place of residence selection, water loss, and mortality, we used experimentally warmed open-top chambers and field observations with a total of >11,000 naturally or experimentally colored individuals of the highly polymorphic species Theba pisana (O.F. MÜller, 1774). We show that solar radiation in their natural Mediterranean habitat in Southern France poses intensifying thermal stress on increasingly pigmented snails that cannot be compensated for by behavioral responses. Individuals of all morphs acted neither jointly nor actively competed in climbing behavior, but acted similarly regardless of neighbor pigmentation intensity. Consequently, dark morphs progressively suffered from high internal temperatures, oxidative stress, and a breakdown of the chaperone system. Concomitant with increasing water loss, mortality increased with more intense pigmentation under simulated global warming conditions. In parallel with an increase in mean ambient temperature of 1.34°C over the past 30 years, the mortality rate of pigmented individuals in the field is, currently, about 50% higher than that of white morphs. A further increase of 1.12°C, as experimentally simulated in our study, would elevate this rate by another 26%. For 34 T. pisana populations from locations that are up to 2.7°C warmer than our experimental site, we show that both the frequency of pigmented morphs and overall pigmentation intensity decrease with an increase in average summer temperatures. We therefore predict a continuing strong decline in the frequency of pigmented morphs and a decrease in overall pigmentation intensity with ongoing global change in areas with strong solar radiation.

Molecular phylogeny of Candidula (Geomitridae) land snails inferred from mitochondrial and nuclear markers reveals the polyphyly of the genus.

The genus Candidula (Geomitridae), consisting of 28 species in Western Europe as currently described, has a disjunct distribution in the Iberian Peninsula, Italy, the Balkans, the Aegean Islands, and one species on the Canary Islands. Although the genus is seemingly well defined by characters of the reproductive system, the relationships within the genus are still unclear and some authors have indicated a possible subgeneric division based on the internal morphology of the dart sac. Despite substantial phylogenetic incongruence, we present a well-resolved molecular phylogeny of Candidula based on two mitochondrial genes (COI and 16S rRNA), the nuclear rDNA region (5.8S rNRA + ITS2 + 28S rRNA) and seven additional nuclear DNA regions developed specifically for this genus (60SL13, 60SL17, 60SL7, RPL14, 40SS6, 60SL9, 60SL13a), in total 5595 bp. Six reciprocally monophyletic entities including Candidula species were recovered, grouping into two major clades. The incorporation of additional geomitrid genera allowed us to unequivocally demonstrate the polyphyly of the genus Candidula. One major clade grouped species from southern France and Italy with the widely distributed species C. unifasciata. The second major clade grouped all the species from the Iberian Peninsula, including C. intersecta and C. gigaxii. Candidula ultima from the Canary Islands was recovered as separated lineage within the latter clade and related to African taxa. The six monophyla were defined as six new genera belonging to different tribes within the Helicellinae. Thus, we could show that similar structures of the stimulatory apparatus of the genital system in different taxa do not necessarily indicate a close phylogenetic relationship in the Geomitridae. More genera of the family are needed to clarify their evolutionary relationships, and to fully understand the evolution of the stimulatory apparatus of the genital system within the Geomitridae.