Molecular phylogeny and character evolution in the Western Palaearctic Helicidae s.l. (Gastropoda: Stylommatophora)

In this study, we present a molecular phylogeny for the west Palaearctic Helicidae sensu lato based on sequence data from two mitochondrial (COI, 16S rDNA) and two nuclear (ITS-1, 18S rDNA) genes. Maximum likelihood analysis and Bayesian inference revealed well supported monophyletic clades partly conflicting traditional classifications. Based on these results, we propose the following system. The Western Palaearctic Helicidae s.l. consist of two families, Helicidae and Hygromiidae. Within the Helicidae, three well supported subfamilies can be recognised: the Helicinae, Ariantinae, and Helicodontinae. The Hygromiidae consist of three clades: the Hygromiinae, the Helicellinae, and a yet unnamed clade comprising the genera Sphincterochila and Cochlicella. We then used the phylogeny to study the evolution of anatomical, and ecological characters traditionally used for systematic classification. In the Helicidae s.l., two independent evolutionary transitions to life in xeric environments occurred, which allowed the occupation of new niches with a subsequent radiation of the Helicellinae-Cochlicella/Sphincterochila clade and the Helicinae. Whereas, the multiplication of the Glandulae mucosae is a synapomorphy of the Hygromiidae, the lovedart sac apparatus is present in all groups and thus, the trait cannot provide a synapomorphy for either families or subfamilies. Additionally, we evaluated the use of structural molecular genetic characters for taxonomic assessment. The presence of an unique loop region of the 16S rDNA gene and a short tandem repeat in the ITS-1 region provide independent evidence for the monophyly of these major two groups, and can be used for preliminary classification.     

Molecular phylogeny of Australian Helicarionidae, Euconulidae and related groups (Gastropoda: Pulmonata: Stylommatophora) based on mitochondrial DNA

The delineation and phylogenetic relationships of the pulmonate family Helicarionidae are currently poorly understood. We have undertaken a phylogenetic analysis of Australian members of Helicarionidae. Three consecutive mitochondrial genes (COI, tRNA-val and 16S rRNA) were sequenced for 36 species from Helicarionidae and related groups. Helicarionidae grouped with Ariophantidae and Urocyclidae with good support in all trees, as did Microcystinae with Trochomorphidae, corresponding to the results of a previous morphological study, but the relationships of Euconulinae and Limacidae could not be resolved. Cystopeltidae never grouped with Helicarionidae and should be regarded as a separate family. The position of tRNA-val in the mitochondrial genome provided a new synapomorphy for Microcystinae. In addition, the COI and 16S rRNA sequences showed a high degree of compositional heterogeneity and incompatibility of phylogenetic signals, highlighting the importance of testing for the decay of the historical signal prior to the phylogenetic analysis.     

基于核基因和线粒体基因序列的中国Singhardina亚属系统发育（半翅目：叶蝉科：小叶蝉亚科：小叶蝉族）

测定了中国Singhardina亚属15个代表种的核糖体28S基因D2区和线粒体16S基因、COI基因部分序列,分别采用最大简约法、最大似然法和贝叶斯法构建分子系统树。系统发育树显示了相似的拓扑结构,明晰了各分支间的关系。首次探讨了中国Singhardina亚属4个种团间的系统发育关系。结果显示,Singhardina亚属构成了1个独立支系,代表1个单系群。本研究首次提出种团Eurhadina(Singhardina)rubra,并推测该种团可能是Singhardina亚属中最原始的种团。种团Eurhadina(Singhardina)robusta和E.(Singhardina)mamata为姐妹群。种团E.(Singhardina)vittata不能从种团E.(Singhardina)punjabensis中分出得到了分子数据的支持。     

Evidence for ecological speciation in the sister species Candidula unifasciata (Poiret, 1801) and C. rugosiuscula (Michaud, 1831) (Helicellinae, Gastropoda)

This paper studies the speciation of two land snail species in south-east France. By using two mitochondrial and one nuclear gene, the species are shown to be sister species within the larger clade of western European Candidula species. The species never occurred syntopically, but a narrow contact zone population was identified. Recent range expansions inferred from phylogeographical methods indicated that the present day distributions are not limited by the colonization capacity of the species. Analysis of environmental variables suggested distribution along an ecotone. The gastropod community is also correlated to this gradient. Morphometric shell analysis showed that the divergence between the sister species is a result of desiccation-resistant shell characters. We therefore conclude that the cause for the ecological speciation is most probably the establishment of the Mediterranean climate ≈ 3.2 mya. A model of ecological speciation with a moving ecotone is put forward.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79, 611–628.     

Horned lizard (Phrynosoma) phylogeny inferred from mitochondrial genes and morphological characters: understanding conflicts using multiple approaches

The genus Phrynosoma includes 13 species of North American lizards characterized by unique and highly derived morphologies and ecologies. Understanding interspecific relationships within this genus is essential for testing hypotheses about character evolution in this group. We analyzed mitochondrial ND4 and cytochrome b gene sequence data from all species of Phrynosoma in conjunction with a previously published dataset including 12S and 16S rRNA gene sequences and morphological characters. We used multiple phylogenetic methods and diagnostic tests for data combinability and taxonomic congruence to investigate the data in separate and combined analyses. Separate data partitions resulted in several well-supported lineages, but taxonomic congruence was lacking between topologies from separate and combined analyses. Partitioned Bremer support analyses also reveals conflict between data partitions in certain tree regions. When taxa associated with well-supported clades were removed from analyses, phylogenetic signal was lost. Combined, our results initially suggest conflict between data partitions, but further tests show the data are only appropriate for phylogenetic reconstruction of those parts of the topology that were well resolved. Nonetheless, our data analyses reveal five well-supported clades: (1) Phrynosoma ditmarsi and Phrynosoma hernandesi, (2) P. ditmarsi, P. hernandesi, and Phrynosoma douglasii, (3) P. ditmarsi, P. hernandesi, P. douglasii, and Phrynosoma orbiculare, (4) Phrynosoma mcallii and Phrynosoma platyrhinos, and (5) Phrynosoma braconnieri and Phrynosoma taurus.     

New genera, species, and improved phylogeny of Glissomonadida (Cercozoa)

Glissomonadida is an important cercozoan order of predominantly biflagellate gliding bacterivores found largely in soil and freshwater. Their vast diversity is largely undescribed. We studied 23 mostly newly isolated strains by light microscopy and sequenced their 18S rDNA genes; nine represent new species. For two misidentified ATCC 'Heteromita triangularis' strains, we establish novel gliding genera and species: the sandonid Mollimonas lacrima, the only glissomonad forming anterior and posterior pseudopodia, and Dujardina stenomorpha, a strongly flattened member of the new family Dujardinidae. A new strain from Oxfordshire grassland soil is the first reliably identified isolate of the virtually uniflagellate, smooth-gliding glissomonad genus, AllantionSandon, 1924. Phylogenetic analysis and cytological features reveal Allantion to be a member of Allapsidae. Sandona limna and Bodomorpha prolixa from Lake Baikal and Sandona hexamutans from volcanic Costa Rican soil are described as new species. Fifteen glissomonad strains were from grassland beside Lake Baikal. We describe two as new species of Sandona (S. heptamutans and S. octamutans); the others included strains of Sandona and Allapsa species that have already been described; and three were new species of Sandona and Allapsa but these died before being described. We discuss the ecological and evolutionary significance of these new strains.     

Population Genetics and Identity of an Introduced Terrestrial Slug: Arion subfuscus s.l. in the North-east USA (Gastropoda, Pulmonata, Arionidae)

Several European species of the terrestrial slug genus Arion have been introduced into North America. A case in point is the species complex A. subfuscus s.l. which has become one of the most abundant slug taxa in North America. In Europe this complex consists of at least two cryptic species, viz. A. fuscus and A. subfuscus s.s., the latter of which is further subdivided in five strongly divergent mtDNA lineages (A. subfuscus S1–S5). In order to determine which of these A. subfsucus s.l. taxa are present in the NE USA and in order to assess their population genetic structure, we compared mtDNA, nDNA and allozyme variation between populations from the NE USA and Europe. Our results show that (1) at least A. subfuscus S1 has become successfully established in the NE USA, (2) founder effects are the most likely explanation for the loss of a large amount of molecular genetic variation in populations from the NE USA (i.e. a loss of 96% of the 16S rDNA haplotypes, 67% of the ITS1 alleles and 46% of the alleles at polymorphic allozyme loci), and (3) part of the remaining genetic variation in NE USA populations was probably due to multiple introductions from the British Isles and the European mainland, and the hybrid structure of most of these source populations. Apparently, the extreme loss of molecular genetic variation in this introduced species has not prevented it from successfully establishing and spreading in novel environments.     

Molecular phylogeny of the speciose vole genus Microtus (Arvicolinae, Rodentia) inferred from mitochondrial DNA sequences

Voles of the genus Microtus represent one of the most speciose mammalian genera in the Holarctic. We established a molecular phylogeny for Microtus to resolve contentious issues of systematic relationships and evolutionary history in this genus. A total of 81 specimens representing ten Microtus species endemic to Europe as well as eight Eurasian, six Asian and one Holarctic species were sequenced for the entire cytochrome b gene (1140 bp). A further 25 sequences were retrieved from GenBank, providing data on an additional 23, mainly Nearctic, Microtus species. Phylogenetic analysis of these 48 species generated four well-supported monophyletic lineages. The genus Chionomys, snow voles, formed a distinct and well-supported lineage separate from the genus Microtus. The subgenus Microtus formed the strongest supported lineage with two sublineages displaying a close relationship between the arvalis species group (common voles) and the socialis species group (social voles). Monophyly of the Palearctic pitymyid voles, subgenus Terricola, was supported, and this subgenus was also subdivided into two monophyletic species groups. Together, these groupings clarify long-standing taxonomic uncertainties in Microtus. In addition, the "Asian" and the Nearctic lineages reported previously were identified although the latter group was not supported. However, relationships among the main Microtus branches were not resolved, suggesting a rapid and potentially simultaneous radiation of a widespread ancestor early in the history of the genus. This and subsequent radiations discernible in the cytochrome b phylogeny, show the considerable potential of Microtus for analysis of historical and ecological determinants of speciation in small mammals. It is evident that speciation is an ongoing process in the genus and that the molecular data provides a vital insight into current species limits as well as cladogenic events of the past.     

Towards the phylogeny of the Curculionoidea (Coleoptera): Reconstructions from mitochondrial and nuclear ribosomal DNA sequences

With about 60,000 described species, Curculionoidea represent the most species-rich superfamily in the animal kingdom. The immense diversity apparently creates difficulties in the reconstruction of the phylogenetic relationships. Independent morphological studies have led to very different classifications. This study is based on molecular data from two independent molecular sources, the 16S and 18S rDNA. Sensitivity analyses were conducted for the sequence alignment (gap costs were varied) as well as the phylogenetic reconstruction algorithms and some of their parameters. The higher-level relationships reconstructed within Curculionoidea are sensitive to alignment and reconstruction method. Nemonychidae or Oxycorynidae+Belidae were found to be sister to all remaining Curculionoidea in many analyses. The 16S rDNA sequence data (obtained from 157 species) corroborate many tribes and genera as monophyletic. It is observed that the phylogenetic reconstruction of genera with specific genetic features such as polyploidy and parthenogenetic reproduction is difficult in weevils. The curculionid subfamily Lixinae appears monophyletic. A new monophylum consisting of Entiminae, Hyperinae, Cyclominae, Myllorhinus plus possibly the Cossoninae is distinguished and we call it Entiminae s.l. For most other subfamilies and families homoplasy concealed the phylogenetic signal (due to saturation of the 16S sequences), or the species sampling was insufficient, although our sampling scheme was rather broad. We observed that although data from one source can easily be misleading (16S) or hardly informative (18S), the combination of the two independent data sets can result in useful information for such a speciose group of organisms. Our study represents the most thorough analysis of molecular sequence data of the Curculionoidea to date and although the phylogenetic results appear less stable than expected, they reflect the information content of these sequence data realistically and thus contribute to the total knowledge about the phylogeny of the Curculionoidea.     

Complete DNA sequence of the mitochondrial genome ofCepaea nemoralis (Gastropoda: Pulmonata)

The nucleotide sequence of a mitochondrial genome of the pulmonate gastropod molluscCepaea nemoralis has been determined. Contained within the 14,100 basepairs (bp) are the two ribosomal RNA genes and 13 protein coding genes typical of metazoan mitochondrial genomes. TheCepaea mtDNA does contain a gene for ATPase subunit 8, like the clausiliid pulmonate,Albinaria, and the chiton,Katharina, but unlike the bivalve mollusc,Mytilus. The mitochondrial genetic code ofCepaea is proposed to be the same as that ofMytilus, Katharina, andDrosophila. Only 14 putative tRNA genes are presented, although there is sufficient unassigned sequence to encode the remainder of the expected total of 22 tRNA genes. These 14 tRNA genes are a mixture of standard cloverleaf structures and nonstandard structures containing TV replacement loops as seen in nematode and mosquito mitochondrial genomes. If the eight unidentified tRNA genes are indeed present, very little unassigned sequence would remain to serve as a control region. Genes are transcribed from both strands of the molecule. Base composition is the least biased for any reported animal mitochondrial genome and is also very little skewed between strands using measures independent of base composition. TheCepaea mitochondrial gene order is quite unlike that of any other reported metazoan mtDNA, with the exception of the recently reported partial sequences ofAlbinaria. No gene bound-aries are shared among all the reported molluscan taxa, demonstrating a complete lack of conservation of mitochondrial gene order across the phylum Mollusca.     

Immunocytochemical Localisation of Glycosaminoglycan-Like Activity in the Mucus and Associated Tissues of Terrestrial Slug Species (Gastropoda: Pulmonata)

Indirect immunofluorescence assays were conducted on cryotome sections of four terrestrial slug species from three distinct phylogenetic groups, Arion ater (L.), Arion hortensis (Férussac), Tandonia (Milax) budapestensis (Hazay), and Deroceras reticulatum (Müller) using monoclonal antibodies for two glycosaminoglycans (GAGs), heparan sulphate, and chondroitin sulphate. Specific staining for a heparan sulphate-like component was demonstrated in the foot and tail regions of each species and was particularly intense in A. ater and A. hortensis, notably in the epidermis and associated mucus-like material, and in mucus-like material from the pedal gland region of the latter species. Subsequent studies with A. ater confirmed the presence of heparan-sulphate–like activity in the caudal gland duct region. No evidence of specific staining for chondroitin sulphate-like activity was found in any of the slug species. This work suggests that a specific GAG, or a group of closely related GAGs, is a common component of mucus in a range of slug species and of different types of mucus, including trail (pedal) mucus and the more viscous mucus produced by the caudal gland.     

Genotype versus phenotype variability in Chlorella and Micractinium (Chlorophyta, Trebouxiophyceae)

The most recent revision of the genus Chlorella, based on biochemical and SSU rDNA analyses, suggested a reduction to a set of four "true" spherical Chlorella species, while a growing number of morphologically different species such as Micractinium (formerly Micractiniaceae) were found to cluster within the clade of "true"Chlorella. In this study, the generic concept in Chlorellaceae to Chlorella and Micractinium was evaluated by means of combined SSU and ITS-2 rDNA sequence analyses and biotests to induce development of bristles on the cell wall. Molecular phylogenetic analyses of Chlorella and Micractinium strains confirmed their separation into two different genera. In addition, non-homoplasious synapomorphies (NHS) and compensatory base changes (CBC) in the secondary structures of SSU and ITS-2 rDNA sequences were found for both genera using this approach. The Micractinium clade can be differentiated into three different genotypes. Using culture medium of the rotifer Brachionus calyciflorus, phenotypic plasticity in Chlorella and Micractinium was studied. Non-bristled Micractinium cells developed bristles during incubation with Brachionus culture medium, whereas Chlorella did not produce bristles. Grazing experiments with Brachionus showed the rotifer preferred to feed on non-bristled cells. The dominance of colonies versus solitary cells in the Micractinium culture was not correlated with the "Brachionus factor". These results suggest that morphological characteristics like formation of bristles represent phenotypic adaptations to the conditions in the ecosystem.     

Ostreopsis cf. ovata in the Gulf of Gabès (south-eastern Mediterranean Sea): morphological,molecular and ecological characterization

In the last few decades, the frequency of the toxic benthic dinoflagellate Ostreopsis cf. ovata proliferation has increased in the Mediterranean Sea. These blooms are associated with harmful effects on human health and the environment. The present work provides the first long term study on the spatio-temporal distribution of O. cf. ovata in relation to physical parameters in the Gulf of Gabès coastal waters (south-eastern Mediterranean Sea), as well as its morphological, molecular and physiological features. The strains of O. cf. ovata were identified morphologically by light and epifluorescence microscopy. The morphology and the size range of cultured strains were similar to those described regarding O. cf. ovata isolated from the Mediterranean Sea. The ultrastructural analysis of O. cf. ovata cells using the transmission electron microscopy showed the presence of numerous vesicles (VE) containing spirally coiled fibers (SCFs) connected to the mucus canal (CH). The phylogenetic tree based on the internal transcribed spacer region containing the 5.8S rDNA (ITS-5.8S rDNA) revealed that O. cf. ovata strains were placed into the Mediterranean/Atlantic clade. In addition, O. cf. ovata toxicity was evaluated by the mouse bioassay and a dose level ≥ 4 × 10⁴ cells was found to be lethal to mice. The examination of the O. cf. ovata occurrence in the Gulf of Gabès at a large temporal scale (1997–2012) revealed a clear seasonal pattern with dominance from midsummer (July) to late autumn (November). Furthermore, a positive correlation was found between the abundance of O. cf. ovata and salinity, whereas no correlation was found as regards temperature. The occurrence of O. cf. ovata was only detected at salinity above 35 and the highest concentrations were observed at 45. Laboratory experiments confirmed such a result and showed that isolated O. cf. ovata strains had optimal growth at salinity ranging between 35 and 45, with its peak at 40.     

Relationships of Afroablepharus Greer, 1974 skinks from the Gulf of Guinea islands based on mitochondrial and nuclear DNA: patterns of colonization and comments on taxonomy

Partial sequences of three mitochondrial DNA genes, 12S rDNA, 16S rDNA and cytochrome b, and one nuclear gene, c-mos, were used to assess the phylogenetic relationships of species belonging to the genus Afroablepharus from the volcanic islands of the Gulf of Guinea (West Africa) and neighboring continental Africa. Additionally, partial sequences of cytochrome b were used to compare levels of sequence divergence within populations. The three forms from S?o Tomé, Príncipe and Annobon (one per island) are genetically distinct, with high levels of divergence, supporting the recognition of a distinct species in each island. Populations within each island contain very low levels of genetic diversity. These three forms form a monophyletic group suggesting a single initial colonization followed by radiation to the other islands, possibly from S?o Tomé to Príncipe and Annobon. This is contrary to what was found in other reptiles from these islands such as Mabuya (sensu lato) and Hemidactylus, which colonized the islands multiple times. Assuming a molecular clock for cytochrome b of about 2% divergence per million years (usually applied to Sauria), the lineage on Annobon island exceeds the age of the island, thus casting further doubt on this widely used divergence estimate. Partial sequences of c-mos showed no variation within islands. Five to seven sites were variable among islands, which is a high value further supporting the treatment of each island form as a distinct species.     

In vivo efficacy of praziquantel against Echinoparyphium aconiatum (Trematoda: Echinostomatidae) parasitizing the great pond snails Lymnaea stagnalis (Gastropoda: Lymnaeidae)

The present study had a practical goal. I aimed to determine whether praziquantel could reduce the production of Echinoparyphium aconiatum (Trematoda: Echinostomatidae) cercariae in infected snails Lymnaea stagnalis (Gastropoda: Lymnaeidae) without killing the hosts. Praziquantel is a broad-spectrum antihelminth agent. It caused a total cessation of cercaria shedding when the praziquantel concentration in the treatment bath was 10 mg/L and the treatment time was 30 h or longer. A next research step which has to be taken before giving detailed recommendations about using praziquantel for ceasing production of E. aconiatum cercariae in parasitized snails is to follow the survivorship and performance of treated snails after a praziquantel exposure for longer than in this medium-term (3 days) experiment.     

Waves of dispersal in island-hopping Chondrina species (Gastropoda, Pulmonata, Chondrinidae)

The aim of this study was to unravel the historical biogeography of the speciose land snail genus Chondrina. To this end phylogenetic hypotheses were tested using mitochondrial DNA sequence data.Mitochondrial DNA sequences of the Cytochrome Oxidase subunit I region were obtained for 89 individuals, representing just over 70% of the extant Chondrina species. The extent of molecular genetic diversity and phylogeographical patterns were investigated by using neighbour joining, parsimony and bayesian methods for phylogeny reconstructions. The resulting data were used to infer historical biogeographical patterns for the genus Chondrina.The three phylogenetic methods yielded congruent topologies for the phylogeny reconstruction. Six clades were identified, each of which with at least one taxon that is known from the Iberian peninsula. The most parsimonious scenario indicates at least three waves of dispersal out of the Iberian peninsula into the North and East of Europe and Northern Africa.The phylogenetic relationships combined with the distributional patterns of the various species, indicate that only vicariance events cannot explain the actual situation. Apparently, separate waves of dispersal and subsequent speciation occurred, each time starting from the southwestern part of the present generic range. Until recently, this was obscured by repetitive cases of parallel or convergent evolution in shell characters, as became evident with the use of molecular methods.     

The first record and occurrence of Stagnicola turricula (Held, 1836) (Gastropoda: Pulmonata: Lymnaeidae) in Upper Silesia (Southern Poland) in relation to different environmental factors

The degradation of freshwater environments, e.g.: eutrophication, drainage, water pollution, has led to the decline of lymnaeid species distribution. Some of them are recorded in the Red Book and Red List of species as rare, vulnerable or legally protected. The survey was carried out in Upper Silesia (Southern Poland), which is one of the biggest coal basins in the world. This region is devoid of natural water bodies; only reservoirs of an anthropogenic origin are common. Anthropogenic reservoirs, which are not very degraded, constitute important lentic habitats in Upper Silesia and they provide refuges for wildlife including rare and vulnerable molluscan species. This survey uncovered the first occurrence of Stagnicola turricula in the anthropogenic reservoirs in this area. S. turricula has been subdominant in molluscan communities. Sixteen molluscan species, including 11 gastropod and 5 bivalve species, were recorded at the sampling sites. Based on a redundancy analysis (RDA), the organic matter content in the bottom sediments and pH were the parameters most associated (statistically significant) with the distribution of molluscan species including S. turricula.     

The Genus Obelus Hartmann, 1842 (Gastropoda, Pulmonata, Helicoidea) and its Phylogenetic Relationships

The genus Obelus, so far known from shells only, is re-described on the basis of the anatomy of its genital system. Its range is here restricted to north-western Africa and the Canary Islands. Four species are traditionally listed for the Canary Islands: Obelus despreauxii, O. moderatus, O. mirandae and O. cyclodon. The presence of the last species on the Canary Islands is doubtful, whereas the fossil/subfossil presence of O. pumilio is documented. The species Helix morata also belongs to the genus, as well as a new species, O. discogranulatus sp. nov. The genital systems of all confirmed extant Canarian species are shown. Obelus has a peculiar vaginal stimulator appendix (“appendicula”) which is homologous to the penial appendix of the Orthurethra and to the stimulatory organ of other Stylommatophora, with the exception that the A₃ portion is missing. The genus is diagnosed by characters of the vaginal stimulator appendix, such as a curved, finger-like A₂ portion of the appendicula ending proximally in a blind, well-developed muscular sac, and by the slender tubular A₄ portion arising laterally from the muscular sac duct. We conclude that Obelus belongs to the Cochlicella group because it shares an appendicula with the other genera of the group. However, it differs in anatomical details of this structure. The Cochlicella group should be recognized as a separate family Cochlicellidae Schileyko, 1972, with close relationships to the Helicellidae and the Hygromiidae. A new diagnosis for Cochlicellidae is proposed and its phylogenetic relationships are discussed.This is: Notes on the Malacofauna of the Canary Islands, No. 46.