Five times over: 42 new Angustopila species highlight Southeast Asia's rich biodiversity (Gastropoda, Stylommatophora, Hypselostomatidae).

The Southeast Asian genus Angustopila, currently comprising 13 nominal species, encompasses the world's tiniest land snails. This work shows that there are far more species than previously suspected, and that this genus is in fact, a very speciose group of tiny snails widely distributed in Southeast Asia. Angustopila is revised based on type material of known species as well as 211 samples newly collected in China, Laos, Myanmar, Thailand and Vietnam. Altogether, 53 species and one subspecies are recognised, of which 42 species and subspecies are new to science: A.akrodon Páll-Gergely & Hunyadi, sp. nov., A.apiaria Páll-Gergely & Hunyadi, sp. nov., A.apiostoma Páll-Gergely & Vermeulen, sp. nov., A.apokritodon Páll-Gergely & Hunyadi, sp. nov., A.antidomedon Páll-Gergely & Hunyadi, sp. nov., A.babel Páll-Gergely & Vermeulen, sp. nov., A.bathyodon Páll-Gergely & Hunyadi, sp. nov., A.bidentata Páll-Gergely & Jochum, sp. nov., A.cavicola Páll-Gergely & Dumrongrojwattana, sp. nov., A.cicatricosa Páll-Gergely & Vermeulen, sp. nov., A.coprologosuninodus Páll-Gergely & Grego, ssp. nov., A.erawanica Páll-Gergely & Dumrongrojwattana, sp. nov., A.fratermajor Páll-Gergely & Vermeulen, sp. nov., A.fraterminor Páll-Gergely & Vermeulen, sp. nov., A.gracilis Páll-Gergely & Hunyadi, sp. nov., A.halongensis Páll-Gergely & Vermeulen, sp. nov., A.hyron Páll-Gergely & Vermeulen, sp. nov., A.maasseni Páll-Gergely & Vermeulen, sp. nov., A.majuscula Páll-Gergely & Hunyadi, sp. nov., A.margaritarion Páll-Gergely & Hunyadi, sp. nov., A.megastoma Páll-Gergely & Vermeulen, sp. nov., A.occidentalis Páll-Gergely & Hunyadi, sp. nov., A.oostoma Páll-Gergely & Vermeulen, sp. nov., A.papaver Páll-Gergely & Hunyadi, sp. nov., A.parallela Páll-Gergely & Hunyadi, sp. nov., A.prolixa Páll-Gergely & Hunyadi, sp. nov., A.pusilla Páll-Gergely & Hunyadi, sp. nov., A.pustulata Páll-Gergely & Hunyadi, sp. nov., A.quadridens Páll-Gergely & Vermeulen, sp. nov., A.rara Páll-Gergely & Hunyadi, sp. nov., A.reticulata Páll-Gergely & Hunyadi, sp. nov., A.somsaki Páll-Gergely & Hunyadi, sp. nov., A.steffeki Páll-Gergely & Grego, sp. nov., A.tetradon Páll-Gergely & Hunyadi, sp. nov., A.thersites Páll-Gergely & Vermeulen, sp. nov., A.tonkinospiroides Páll-Gergely & Vermeulen, sp. nov., A.tridentata Páll-Gergely & Hunyadi, sp. nov., A.tweediei Páll-Gergely & Hunyadi, sp. nov., A.uvula Páll-Gergely & Hunyadi, sp. nov., A.vandevenderi Páll-Gergely & Jochum, sp. nov., A.vitrina Páll-Gergely & Hunyadi, sp. nov., A.vomer Páll-Gergely & Hunyadi, sp. nov., A.werneri Páll-Gergely & Hunyadi, sp. nov. Angustopilasubelevata Páll-Gergely & Hunyadi, 2015 is moved to the synonymy of Angustopilaelevata (F. G. Thompson & Upatham, 1997), and A.singuladentis Inkhavilay & Panha, 2016 is a junior synonym of A.fabella Páll-Gergely & Hunyadi, 2015. Three species, namely A.elevata, A.fabella and A.szekeresi, are widespread over several hundred kilometres while some other species (A.huoyani, A.parallelasp. nov., A.cavicolasp. nov.) are known from just two sites a few hundred kilometres apart. All others are small range or single-site endemics. The reproductive anatomy of A.erawanicasp. nov. is described.

Fossil Carychiidae (Eupulmonata, Ellobioidea) from the Lower Pleistocene Nashua Formation of Florida, with the description of a new species.

Recent fossil shell mining for a new rail line in the Orlando area of Orange County, Florida has uncovered two species of the ellobioid genus Carychium O. F. Müller, 1773 in a bed of freshwater marl from the Lower Pleistocene Nashua Formation. To taxonomically interpret these finds, the well-preserved shells were imaged via high-resolution X-ray tomography (micro-CT) to view significant internal diagnostic characters such as the columellar configuration and the degree of lamellar sinuosity and their relationship in context to the entire shell. The image data are compared to that of type material and extant and fossil Carychium species inhabiting the SE USA, Mexico, Central America, and Jamaica. Based on these results, the species Carychiumfloridanum G. H. Clapp, 1918 and Carychiumnashuaensesp. nov. are identified from fossil shells dating from the Early Pleistocene. This work documents the first fossil members of C.floridanum and the first fossil Carychium from the SE USA.

New genetic data reveals a new species of Zospeum in Bosnia (Gastropoda, Ellobioidea, Carychiinae).

Recent integrative investigations of the terrestrial ellobiid genus, Zospeum, have revealed significant findings concerning its Alpine-Dinaric evolution and taxonomy. Due to the expected discrepancy between the useful, but limited, 1970s' classification system based on shell data and the results of recent genetic analyses in the latest investigation, a revision of the entire radiation was undertaken, and a new classification system was devised by the present authors in an earlier paper. Concurrent to this work, molecular sequences from two Austrian caves were published independently of our revision by another research group. By incorporating these genetic data within our phylogenetic framework here, we show that the Austrian individuals are genetically most similar to Zospeumamoenum and consequently, classify them within that species. We additionally reveal two new genetic lineages from the largely under-sampled southern extension of Zospeum's known distributional range. The first lineage, deriving from the region of Dubrovnik, Croatia, is a potential candidate for genetically clarifying Zospeumtroglobalcanicum. The second lineage derives from the municipality of Tomislavgrad, Bosnia-Herzegovina and is herein, described a new species: Zospeumsimplex Inäbnit, Jochum & Neubert, sp. nov.

Land Snail with Periostracal Hairs Preserved in Burmese Amber.

Excellently preserved fossils often provide important insights into evolutionary histories and adaptations to environmental change in Earth's biogeologic record. Mid-Cretaceous Burmese amber, for example, is a proven reservoir for spectacular findings. Here we document the first record of a fossil land snail with periostracal hairs preserved in amber. We interpret the development of hairs as an adaptation to the tropical forest environment, serving as a mechanism to increase adhesion of the snail to plants during foraging while collecting and transporting seeds in the process. The present record coincides with a major global radiation of angiosperms, a main food resource for terrestrial snails. As such, the expansion of flowering plants likely triggered this evolutionary adaptation and, thus, the diversification of land snails in the Cretaceous.

Re-evaluation of Zospeumschaufussi von Frauenfeld, 1862 and Z.suarezi Gittenberger, 1980, including the description of two new Iberian species using Computer Tomography (CT) (Eupulmonata, Ellobioidea, Carychiidae).

The present study aims to clarify the confused taxonomy of Z.schaufussi von Frauenfeld, 1862 and Zospeumsuarezi Gittenberger, 1980. Revision of Iberian Zospeum micro snails is severely hindered by uncertainties regarding the identity of the oldest Iberian Zospeum species, Z.schaufussi von Frauenfeld, 1862. In this paper, we clarify its taxonomic status by designating a lectotype from the original syntype series and by describing its internal and external shell morphology. Using SEM-EDX, we attempt to identify the area of the type locality cave more precisely than "a cave in Spain". The shell described and illustrated by Gittenberger (1980) as Z.schaufussi appears not to be conspecific with the lectotype shell, and is considered a separate species, Z.gittenbergeri Jochum, Prieto & De Winter, sp. n. Zospeumsuarezi was described from various caves in NW Spain. Study of the type material reveals that these shells are not homogenous in shell morphology. The holotype shell of Z.suarezi is imaged here for the first time. The paratype shell, illustrated by Gittenberger (1980) from a distant, second cave, is described as Zospeumpraetermissum Jochum, Prieto & De Winter, sp. n. The shell selected here as lectotype of Z.schaufussi, was also considered a paratype of Z.suarezi by Gittenberger (1980). Since this specimen is morphologically very similar to topotypic shells of Z.suarezi, the latter species is considered a junior synonym of Z.schaufussi (syn. n.). The internal shell morphology of all these taxa is described and illustrated using X-ray Micro Computer Tomography (Micro-CT).

Fulfilling the taxonomic consequence after DNA Barcoding: Carychiumpanamaense sp. n. (Eupulmonata, Ellobioidea, Carychiidae) from Panama is described using computed tomographic (CT) imaging.

Five years ago, the Panamanian evolutionary lineage (EL) C12 was uncovered along with four other ELs in an integrative phylogenetic investigation of worldwide Carychiidae. Since EL C12 lacked shell material post-molecular analysis to serve as a museum voucher, it remained undescribed. Now, after recent collection efforts of C12 and the congener, Carychiumzarzaae Jochum & Weigand, 2017 at their original Panamanian sites, C12 is morphologically described and formally assigned the name, Carychiumpanamaense Jochum, sp. n. In sync with recent taxonomic treatment of the genus, computed tomography (CT) is used in this work to differentiate shells of C.panamaense sp. n. from geographically-proximal, Caribbean, North and Central American congeners. Recent material of topotypic Carychiumjardineanum (Chitty, 1853) and undamaged C.zarzaae were additionally CT-scanned and assessed in the comparative analyses.

Resumen Hace cinco años, el linaje evolutivo (LE) panameño C12 fue descubierto junto con otros cuatro LEs en un estudio filogenético integrativo mundial de Carychiidae. El LE C12 permaneció sin ser descrito dado que, después de los análisis moleculares, no había conchas disponibles que sirvieran como material de referencia en museos.Ahora, después de esfuerzos recientes para colectar C12 y el congénere, Carychiumzarzaae Jochum & Weigand, 2017 en los sitios panameños originales, C12 es descrito morfológicamente y se le asigna formalmente un nombre, Carychiumpanamaense Jochum, sp. n. De acuerdo con el tratamiento taxonómico reciente del género, en este trabajo se emplea tomografía computarizada (TC) para diferenciar conchas de C.panamaense sp. n. de congéneres geográficamente cercanos del Caribe, Norte y Centro América. Además, en el análisis comparativo se escaneó con TC y se examinó material reciente del topotípico Carychiumjardineanum (Chitty, 1853) y C.zarzaae en buen estado.

Three new species of Carychium O.F. Müller, 1773 from the Southeastern USA, Belize and Panama are described using computer tomography (CT) (Eupulmonata, Ellobioidea, Carychiidae).

Three new species of the genus Carychium O.F. Müller, 1773, Carychium hardiei Jochum & Weigand, sp. n., Carychium belizeense Jochum & Weigand, sp. n. and Carychium zarzaae Jochum & Weigand, sp. n. are described from the Southeastern United States, Belize and Panama, respectively. In two consecutive molecular phylogenetic studies of worldwide members of Carychiidae, the North and Central American morphospecies Carychium mexicanum Pilsbry, 1891 and Carychium costaricanum E. von Martens, 1898 were found to consist of several evolutionary lineages. Although the related lineages were found to be molecularly distinct from the two nominal species, the consequential morphological and taxonomic assessment of these lineages is still lacking. In the present paper, the shells of these uncovered Carychium lineages are assessed by comparing them with those of related species, using computer tomography for the first time for this genus. The interior diagnostic characters are emphasized, such as columellar configuration in conjunction with the columellar lamella and their relationship in context of the entire shell. These taxa are morphologically described and formally assigned their own names.

Seven new hypselostomatid species from China, including some of the world's smallest land snails (Gastropoda, Pulmonata, Orthurethra).

Seven new species of Hypselostomatidae are described from the Chinese province Guangxi: Angustopila dominikae Páll-Gergely & Hunyadi, sp. n., Angustopila fabella Páll-Gergely & Hunyadi, sp. n., Angustopila subelevata Páll-Gergely & Hunyadi, sp. n., Angustopila szekeresi Páll-Gergely & Hunyadi, sp. n., Hypselostoma socialis Páll-Gergely & Hunyadi, sp. n., Hypselostoma lacrima Páll-Gergely & Hunyadi, sp. n. and Krobylos sinensis Páll-Gergely & Hunyadi, sp. n. The latter species is reported from three localities. All other new species are known only from the type locality. Specimens nearly identical to the type specimens of Angustopila huoyani Jochum, Slapnik & Páll-Gergely, 2014 were found in a cave in northern Guangxi, 500 km from the type locality. Adult individuals of Angustopila subelevata sp. n. (shell height = 0.83-0.91 mm, mean = 0.87 mm) and Angustopila dominikae sp. n. (shell height of the holotype = 0.86 mm) represent the smallest known members of the Hypselostomatidae, and thus are amongst the smallest land snails ever reported. We note that Pyramidula laosensis Saurin, 1953 might also belong to Krobylos. Paraboysidia neglecta van Benthem Jutting, 1961, which was previously included in Angustopila, is classified in Hypselostoma.

A new member of troglobitic Carychiidae, Koreozospeum nodongense gen. et sp. n. (Gastropoda, Eupulmonata, Ellobioidea) is described from Korea.

A new genus of troglobitic Carychiidae Jeffreys, 1830 is designated from Nodong Cave, North Chungcheong Province, Danyang, South Korea. This remarkable find represents a great range extension and thus, a highly distant distribution of troglobitic Carychiidae in Asia. The Zospeum-like, carychiid snails were recently included, without a formal description, in records documenting Korean malacofauna. The present paper describes Koreozospeum Jochum & Prozorova, gen. n. and illustrates the type species, Koreozospeum nodongense Lee, Prozorova & Jochum, sp. n. using novel Nano-CT images, including a video, internal shell morphology, SEM and SEM-EDX elemental compositional analysis of the shell.

Two new species of Zospeum Bourguignat, 1856 from the Basque-Cantabrian Mountains, Northern Spain (Eupulmonata, Ellobioidea, Carychiidae).

Two new species of the genus Zospeum Bourguignat, 1856 are described from caves in the Sierra de Aitzgorri (Gipuzkoa) and the Sierra Salvada (Burgos) in Northern Spain. The taxa Zospeumvasconicum sp. n. and Zospeumzaldivarae sp. n. have recently, without a formal name, been included in a molecular study of worldwide members of the Carychiidae. In the present paper, the shell morphology and variation of these species is described and illustrated.

A review of the microgastropod genus Systenostoma Bavay & Dautzenberg, 1908 and a new subterranean species from China (Gastropoda, Pulmonata, Hypselostomatidae).

A review of the microgastropod genus Systenostoma is provided. Thai and Malaysian species are transferred to a new genus, Angustopila (type species: Systenostoma tamlod Panha & Burch, 1999). A new subterranean Angustopila species is described here. Conchologically, the new species is most similar to the cave-dwelling, Thai A. tamlod (Panha & Burch, 1999). One Thai species (Systenostoma edentata) is transferred to the genus Hypselostoma. Vietnamese members remain in the genus Tonkinospira (nomen novum) for Systenostoma Bavay & Dautzenberg, 1908 (non Systenostoma Marsson, 1887). A comprehensive map of former Systenostoma species is presented. SEM and NanoCT images, including a video of A. huoyani sp. n. internal shell morphology, provide novel perspectives of the shells of Angustopila and of the scarcely known Vietnamese Tonkinospira species. The biology of these snails is not yet known but collection localities suggest a troglophilic ecology.

Evolution of microgastropods (Ellobioidea, Carychiidae): integrating taxonomic, phylogenetic and evolutionary hypotheses.

BACKGROUND: Current biodiversity patterns are considered largely the result of past climatic and tectonic changes. In an integrative approach, we combine taxonomic and phylogenetic hypotheses to analyze temporal and geographic diversification of epigean (Carychium) and subterranean (Zospeum) evolutionary lineages in Carychiidae (Eupulmonata, Ellobioidea). We explicitly test three hypotheses: 1) morphospecies encompass unrecognized evolutionary lineages, 2) limited dispersal results in a close genetic relationship of geographical proximally distributed taxa and 3) major climatic and tectonic events had an impact on lineage diversification within Carychiidae. RESULTS: Initial morphospecies assignments were investigated by different molecular delimitation approaches (threshold, ABGD, GMYC and SP). Despite a conservative delimitation strategy, carychiid morphospecies comprise a great number of unrecognized evolutionary lineages. We attribute this phenomenon to historic underestimation of morphological stasis and phenotypic variability amongst lineages. The first molecular phylogenetic hypothesis for the Carychiidae (based on COI, 16S and H3) reveals Carychium and Zospeum to be reciprocally monophyletic. Geographical proximally distributed lineages are often closely related. The temporal diversification of Carychiidae is best described by a constant rate model of diversification. The evolution of Carychiidae is characterized by relatively few (long distance) colonization events. We find support for an Asian origin of Carychium. Zospeum may have arrived in Europe before extant members of Carychium. Distantly related Carychium clades inhabit a wide spectrum of the available bioclimatic niche and demonstrate considerable niche overlap. CONCLUSIONS: Carychiid taxonomy is in dire need of revision. An inferred wide distribution and variable phenotype suggest underestimated diversity in Zospeum. Several Carychium morphospecies are results of past taxonomic lumping. By collecting populations at their type locality, molecular investigations are able to link historic morphospecies assignments to their respective evolutionary lineage. We propose that rare founder populations initially colonized a continent or cave system. Subsequent passive dispersal into adjacent areas led to in situ pan-continental or mountain range diversifications. Major environmental changes did not influence carychiid diversification. However, certain molecular delimitation methods indicated a recent decrease in diversification rate. We attribute this decrease to protracted speciation.

Alpine crossroads or origin of genetic diversity? Comparative phylogeography of two sympatric microgastropod species.

The Alpine Region, constituting the Alps and the Dinaric Alps, has played a major role in the formation of current patterns of biodiversity either as a contact zone of postglacial expanding lineages or as the origin of genetic diversity. In our study, we tested these hypotheses for two widespread, sympatric microgastropod taxa--Carychium minimum O.F. Müller, 1774 and Carychium tridentatum (Risso, 1826) (Gastropoda, Eupulmonata, Carychiidae)--by using COI sequence data and species potential distribution models analyzed in a statistical phylogeographical framework. Additionally, we examined disjunct transatlantic populations of those taxa from the Azores and North America. In general, both Carychium taxa demonstrate a genetic structure composed of several differentiated haplotype lineages most likely resulting from allopatric diversification in isolated refugial areas during the Pleistocene glacial periods. However, the genetic structure of Carychium minimum is more pronounced, which can be attributed to ecological constraints relating to habitat proximity to permanent bodies of water. For most of the Carychium lineages, the broader Alpine Region was identified as the likely origin of genetic diversity. Several lineages are endemic to the broader Alpine Region whereas a single lineage per species underwent a postglacial expansion to (re)colonize previously unsuitable habitats, e.g. in Northern Europe. The source populations of those expanding lineages can be traced back to the Eastern and Western Alps. Consequently, we identify the Alpine Region as a significant 'hot-spot' for the formation of genetic diversity within European Carychium lineages. Passive dispersal via anthropogenic means best explains the presence of transatlantic European Carychium populations on the Azores and in North America. We conclude that passive (anthropogenic) transport could mislead the interpretation of observed phylogeographical patterns in general.

A new approach to an old conundrum--DNA barcoding sheds new light on phenotypic plasticity and morphological stasis in microsnails (Gastropoda, Pulmonata, Carychiidae).

The identification of microsnail taxa based on morphological characters is often a time-consuming and inconclusive process. Aspects such as morphological stasis and phenotypic plasticity further complicate their taxonomic designation. In this study, we demonstrate that the application of DNA barcoding can alleviate these problems within the Carychiidae (Gastropoda, Pulmonata). These microsnails are a taxon of the pulmonate lineage and most likely migrated onto land independently of the Stylommatophora clade. Their taxonomical classification is currently based on conchological and anatomical characters only. Despite much confusion about historic species assignments, the Carychiidae can be unambiguously subdivided into two taxa: (i) Zospeum species, which are restricted to karst caves, and (ii) Carychium species, which occur in a broad range of environmental conditions. The implementation of discrete molecular data (COI marker) enabled us to correctly designate 90% of the carychiid microsnails. The remaining cases were probably cryptic Zospeum and Carychium taxa and incipient species, which require further investigation into their species status. Because conventional reliance upon mostly continuous (i.e. nondiscrete) conchological characters is subject to fallibility for many gastropod species assignments, we highly recommend the use of DNA barcoding as a taxonomic, cutting-edge method for delimiting microsnail taxa.

Combined effects of chemical and temperature stress on Chironomus riparius populations with differing genetic variability.

Exposure to pollutants under multiple environmental stressors (e.g., climate change and global warming) and the genetic diversity of populations are suspected to have serious impacts on populations and ecosystems but have only rarely been analysed. In the present study, we investigated the effects of the biocide tributyltin (TBT) within a temperature gradient (17, 20 and 23 degrees C) on life history parameters of a genetically diverse (GEN+) and a highly inbred population (GEN-) of the midge Chironomus riparius. While endpoints, mortality and reproduction parameters were considered, the population growth rate as an integrative endpoint was determined. We found severe effects for GEN-, indicating that populations with lower genetic diversity are more endangered by combined stressors such as increasing temperature and chemical pollution compared to genetically diverse populations.