DNA damage in storage cells of anhydrobiotic tardigrades

In order to recover without any apparent damage, tardigrades have evolved effective adaptations to preserve the integrity of cells and tissues in the anhydrobiotic state. Despite those adaptations and the fact that the process of biological ageing comes to a stop during anhydrobiosis, the time animals can persist in this state is limited; after exceedingly long anhydrobiotic periods tardigrades fail to recover. Using the single cell gel electrophoresis (comet assay) technique to study the effect of anhydrobiosis on the integrity of deoxyribonucleic acid, we showed that the DNA in storage cells of the tardigrade Milnesium tardigradum was well protected during transition from the active into the anhydrobiotic state. Specimens of M. tardigradum that had been desiccated for two days had only accumulated minor DNA damage (2.09 ± 1.98% DNA in tail, compared to 0.44 ± 0.74% DNA in tail for the negative control with active, hydrated animals). Yet the longer the anhydrobiotic phase lasted, the more damage was inflicted on the DNA. After six weeks in anhydrobiosis, 13.63 ± 6.41% of DNA was found in the comet tail. After ten months, 23.66 ± 7.56% of DNA was detected in the comet tail. The cause for this deterioration is unknown, but oxidative processes mediated by reactive oxygen species are a possible explanation.     

Tardigrade Distribution in a Medium-sized City of Central Argentina

The distribution and abundance of tardigrades in the city of General Pico (Argentina) are analyzed from samples collected during autumn and spring 2001. Sample sites included paved urban locations with different traffic intensities, non-paved periurban places, and places with peculiar conditions such as the city industrial area and the bus station. Trees of the same species were selected in each area and from each of them nine subsamples of lichens and/or moss, 11 mm in diameter, were taken with steel corers. The diversity, density and relative abundance of tardigrades was recorded and analyzed. Sampling sites were ordinated and classified with PCA and clustering. The number total of species collected in the research was 5. The maximum number of species per site and per tree was 4. The species recorded were: Echiniscus rufoviridis, Milnesium cf. tardigradum, Ramazzottius oberhaeuseri, Macrobiotus areolatus and an undescribed species of the genus Macrobiotus. The average density was approximately 10 specimens per cm² and the maximum values reach over 50 specimens per cm². R. oberhaeuseri and M. cf. tardigradum were the most frequent species. R. oberhaeuseri dominates in periurban areas with high suspension dust and very exposed to the sun. M. cf. tardigradum dominates on paved streets with intense vehicle traffic. Results supported the hypothesis of the relationship between air quality and tardigrade diversity.     

Genetic diversity of Riptortus pedestris (Hemiptera: Alydidae) populations in the Republic of Korea based on COI sequence

Riptortus pedestris (Fabricius) has caused severe agricultural damage in Asian regions. To clarify genetic relationships among different populations of R. pedestris in Korea, 294 COI sequences were generated from 294 individual samples collected in 42 local regions. In total, 36 haplotypes were detected from the 294 COI sequences, and the genetic distances among the 42 local populations ranged from 0.00 % to 1.50 %. In the AMOVA results, a variability of more than 98 % was observed within populations, and the median joining (MJ) networks revealed that R. pedestris has expanded by crossing the nine geographical groups. Most of the samples shared one haplotype, H2; however, some samples obtained from the same regions displayed slight genetic differences. These results indicate that R. pedestris have undergone a series of genetic variations.     

Towards the understanding of the origin of the Polish remote population of Polyommatus (Agrodiaetus) ripartii (Lepidoptera: Lycaenidae) based on karyology and molecular phylogeny

Many species of the subgenus Agrodiaetus have dotlike distribution ranges, and the delimitation of the majority of species is only possible on the basis of chromosomal and/or molecular data. In our research, we used a combination of chromosomal and molecular mitochondrial and nuclear markers to analyse the taxonomic identity and to study the phylogeographic history of an enigmatic Agrodiaetus population from South Poland. We discovered this population to be chromosomally and genetically indistinguishable from the widely distributed West Palaearctic species Polyommatus ripartii (Freyer, 1830). Moreover, this population was found to be genetically homogenous and to share the single identified COI+COII haplotype with populations from remote localities in Spain, Bulgaria and Ukraine. Coalescence‐based dating with COI+COII marker estimated that the Polish population originated most likely 10 600–14 300 years ago. This estimation corresponds well to the age (11 700–12 000 years) of palaeontological remnant of Onobrychis arenaria, a food plant of P. ripartii, found in Poland. Generally, the data obtained support the hypotheses that (1) the common ancestor of the Central European populations originated in a refugium in the North Balkan, (2) after the last glacial maximum, this ancestor became broadly distributed in Europe and (3) the Nida population in Poland represents a relict of this ancient distribution.     

Revisiting the phylogeography of Asellus aquaticus in Europe: insights into cryptic diversity and spatiotemporal diversification

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From Asia to Europe across Siberia: phylogeography of the Siberian spined loach (Teleostei,Cobitidae)

We assessed the genetic variability of the Siberian spined loach Cobitis melanoleuca across its unusually broad distribution that encompasses areas greatly affected by Pleistocene glaciations. Due to extensive morphological variation among their populations, the taxonomic status of C. melanoleuca is complicated. It is unclear whether C. melanoleuca represents a single taxonomic unit or contains several species or subspecies. Our analyses showed low genetic variability in all populations without any phylogenetic structure. The absence of molecular distinctiveness indicates the conspecificity of all C. melanoleuca populations. Only a few common haplotypes shared by East Asian, Siberian and European populations were found at high frequency in the nuclear genes analysed. At the mitochondrial level, Siberian populations shared haplotypes with populations located at both extremes of the species’ range suggesting central populations as a source of current mitochondrial variability. Unimodal mismatch distributions and significant values from neutrality tests support a recent expansion of C. melanoleuca. Our time estimates suggest a postglacial colonisation of European waters around 1.0 MYA, indicating that C. melanoleuca may represent the last cobitid immigrant in Europe that used the northern route across Siberia to expand its range.     

Integrated morphometric and molecular analyses indicate three male morphotypes in the freshwater prawn Macrobrachium olfersii (Decapoda,Palaemonidae) along the Brazilian neotropical region

Males of Macrobrachium olfersii have a large cheliped (second pereiopod) with individual variations in shape and ornamentation, and they vary in size within and between populations. Some misidentification or doubts about taxonomic validity occur due to this morphological variability throughout their geographic distribution. We carried out a morphometric analysis to detect specific patterns and the potential occurrence of morphotypes in M. olfersii from different populations. Our data set included 52 females and 109 males collected in the Neotropical region, between the north and south limits of Brazilian territory, in coastal rivers from the states of Rio Grande do Norte, Bahia, Espírito Santo, Rio de Janeiro, São Paulo, Paraná and Santa Catarina. Body measurements were size standardized by allometric methods and explored by the principal component analysis (PCA) and non-hierarchical analysis of K-means clustering and canonical variate analysis (CVA). Our results suggested three distinct morphological groups (morphotypes) only for males in M. olfersii. Furthermore, the confirmation of monophyly of the different populations of M. olfersii was accomplished by sequences of 16S rDNA. Additional studies about relative growth in other populations and the analysis of the entire ontogenetic development of males in the laboratory will be important to complement the knowledge about morphotypes in this species.     

Genetic analysis of Turkish martens: Do two species of the genus Martes occur in Anatolia?

Stone marten (Martes foina) and European pine marten (M. martes) occur in western Eurasia. Current distributions of martens within Turkey and phylogenetic relationships among the Turkish and other populations of the two species within Eurasia remain relatively unknown. In this study, we aimed to determine genetic diversity within Martes populations inhabiting Turkey and to reveal the phylogenetic relationships among the Turkish and conspecific populations of the two marten species, using mitochondrial cytochrome b (CytB) sequences. Twenty‐four (24) haplotypes were identified among 86 marten samples collected across Turkey, including 23 novel haplotypes. Genetic distances among the Turkish haplotypes ranged from 0.1% to 0.8%, with an average of 0.3%. The 24 Turkish haplotypes were analysed together with those of conspecific populations deposited in GenBank. Phylogenetic (Bayesian Inference, maximum likelihood, neighbor‐joining) and network analyses revealed that all of the Turkish samples belonged to M. foina and that samples of M. martes were not encountered. Haplotypes of M. foina were divided into five haplogroups. The haplogroup including the two Chinese samples differed markedly from other the haplogroups. The remaining haplogroups contained samples from both the Turkish and European populations. We found that there was a genetically close relationship between the Turkish and the European stone marten populations. As a result of this study, M. martes may not be distributed in the Anatolian part of Turkey, possibly due to a barrier effect of two straits (Dardanelles and Bosporus) and the Caucasus Mountains. On the other hand, M. foina is distributed in both the Anatolian and Thracian parts of Turkey. Our results suggest that Turkey was likely one of the refuges for M. foina during Pleistocene glacial periods and is one of the centres of distribution of stone marten for Europe and the surrounding regions.     

Milnesium berladnicorum sp. n. (Eutardigrada,Apochela, Milnesiidae), a new species of water bear from Romania

In a lichen sample collected from a tree in Bârlad town (Vaslui County, Romania), a new tardigrade species belonging to the genus Milnesium (granulatum group) was found. Milnesium berladnicorum sp. n. is most similar (in the type of dorsal sculpture) to Milnesium beasleyi Kaczmarek et al., 2012 but differs from it mainly by having a different claw configuration and some morphometric characters. Additionally, the new species differs from other congeners of the granulatum group by the different type of dorsal sculpture, claw configuration and some morphometric characters.     

Morphological versus genetic diversity of Viola reichenbachiana and V. riviniana (sect. Viola,Violaceae) from soils differing in heavy metal content

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non‐metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore‐bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non‐polluted sites, high levels of intra‐ and inter‐population variability, and the occurrence of inter‐specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter‐specific hybrids from non‐polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal‐polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.     

Soil tardigrade biodiversity with the description of a new eutardigrade genus and its phylogenetic position

Sixteen tardigrade species have been identified from a total of 943 specimens isolated from 69 positive soil samples collected in southern Spain (Andalusia, Huelva). Three genera (Hexapodibius, Xerobiotus and the new genus Sarascon) and eight species are new records for the Iberian Peninsula, increasing Iberian tardigrade biodiversity by 6%, and 10 species are new records for soil habitats, increasing soil tardigrade biodiversity by 9% (from 96 to 108 species). A newly discovered species and genus are described, followed by an analysis and discussion of the species' phylogenetic position and synapomorphies, based on morphological and total evidence (morphology and molecular – 18S and 28S rRNA – data combined) phylogenetic analyses. The new genus is distinguished from other Itaquasconinae genera, to which it is phylogenetically related, by having a Parascon buccopharyngeal apparatus and Ramajendas claws. In contrast to general eutardigrade evolution, which is primarily determined by claw morphology, itaquasconid evolution is more related to buccopharyngeal apparatus morphology, which shows homoplastic evolution at superfamily, family and subfamily levels, and also defines functional trophic groups in soil-related environments.

http://zoobank.org/urn:lsid:zoobank.org:pub:068B9D87-27E2-4B26-8920-FF03CEF9869A     

Phylogeny of European Pyrochroa (Coleoptera,Pyrochroidae) reveals cryptic taxa and different glacial histories

Only three saproxylic species of Pyrochroinae (Coleoptera: Pyrochroidae) are distributed in Europe, two of which belonging to Pyrochroa: P. coccinea and P. serraticornis. However, P. serraticornis is polytypic, for the presence of the endemic subspecies P. s. kiesenwetteri in southern Italy. Using both molecular and morphological data, we explored the phylogeny of the European Pyrochroa species. A multilocus (COI, CAD, 28S) phylogenetic analysis helped highlight different evolutionary histories for the two examined species. First, P. coccinea, distributed throughout Europe, showed a high differentiation among Italian and European populations. Furthermore, three different taxonomic entities were identified within P. serraticornis, among which the cryptic species Pyrochroa bifoveata sp. n. from central Europe is described and illustrated. A comprehensive identification key to the European Pyrochroinae is also provided. Our results also suggested an historical survival of P. coccinea and P. s. kiesenwetteri in glacial refugia in Italy, and a subsequent post-glacial spread of the former species throughout the Peninsula. In contrast, the current distribution of P. s. serraticornis likely originated from a post-glacial colonization of western European relict populations, while the survival of P. bifoveata plausibly occurred in more eastern glacial refugia (e.g. Carpathian or Balkan regions). Similarly, the European populations of P. coccinea could have originated from relict populations in glacial refugia out from the Italian Peninsula. More comprehensive data on the taxonomy, ecology and biogeography of Pyrochroa are needed to learn more about these species and to help preserve the European saproxylic fauna.     

Proteomic Analysis of Tardigrades: Towards a Better Understanding of Molecular Mechanisms by Anhydrobiotic Organisms

Background

Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.

Principal Findings

Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions.

Conclusions

The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.     

A Comparative and Phylogenetic Study of the Ditylenchus dipsaci,Ditylenchus destructor and Ditylenchus gigas Populations Occurring in Poland

The genus Ditylenchus contains more than 80 recognized nematode species with a very wide host range. The most serious species are Ditylenchus dipsaci and Ditylenchus destructor. Populations of D. dipsaci species complex were collected from Allium cepa, Cichorium endivia and Phlox paniculata in Poland. The Ditylenchus gigas population was collected from Vicia faba minor, and populations of D. destructor, from Solanum tuberosum spp. tuberosum. Analyses of the rDNA sequences spanning both ITS1 and ITS2 fragment regions were carried out on the collected populations. The obtained DNA sequences were compared with those DNA sequences deposited in GenBank of populations isolated in other countries. Phylogenetic analysis was performed using the data obtained from the DNA sequence comparisons. The results indicated that there is no clear distinction between European and non‐European populations within D. dipsaci. The results also showed no clear distinction between populations isolated from different host plant species, including populations found in Poland. The populations of D. destructor described here constitute a common group together with American and Chinese populations belonging to the haplotype C of the D. destructor species. On the other hand, the D. gigas population was localized separately from those populations that have been described up until now, from Europe and Africa. This is also the first report on the occurrence of D. gigas in Poland.     

The potential for using red claw crayfish and hybrid African catfish as biological control agents for Schistosoma host snails

The potential of red claw crayfish and hybrid African catfish (Clarias gariepinus and Clarias ngamensis) as predators for Schistosoma host snails was evaluated in 2014 by monitoring the consumption of snails by crayfish and catfish in experimental tanks over time under laboratory conditions. After 15 days, both crayfish and catfish had significantly reduced the populations of Bulinus globosus. Crayfish consumed 6.9 snails d^?1, whereas catfish consumed 5.9 snails d^?1. However, when supplied with an alternative prey, Melanoides tuberculata (100 individuals per tank), crayfish clearly preferred M. tuberculata (100% consumed over seven days) to B. globosus (100 individuals per tank) (54% consumed over the same period). Catfish, conversely, did not have a clear preference for either prey species, consuming 77% and 88% of M. tuberculata and B. globosus, respectively. It was also observed that young catfish were more efficient predators than older ones, because of ontogenetic shifts in their diet with age. Hybrid catfish retain the molluscivorous characteristics of their parent stock and red claw crayfish also preys on Schistosoma host snails. However, the effectiveness of both predators is affected by the presence of an alternative prey. Therefore, under suitable conditions, these species can be considered for biological control of schistosomiasis transmission.     

High levels of intraspecific genetic divergences revealed for Antarctic springtails: evidence for small‐scale isolation during Pleistocene glaciation

We examined levels of genetic variability within and among populations of three Antarctic springtail species (Arthropoda: Collembola) and tested the hypothesis that genetic divergences occur among glacially‐isolated habitats. The study was conducted in southern Victoria Land, Ross Dependency, Antarctica, and samples were collected from locations in the vicinity of the Mackay Glacier. We analyzed mtDNA (cytochrome c oxidase subunit I; COI) sequence variability for 97 individuals representing three species (Gomphiocephalus hodgsoni, N = 67; Cryptopygus nivicolus, N = 20; and Antarcticinella monoculata, N = 8). Haplotype diversity and genetic divergences were calculated and used to indicate population variability and also to infer divergence times of isolated populations using molecular clock estimates. Two of the three species showed high levels of genetic divergence. Gomphiocephalus hodgsoni, a widespread and common species, showed 7.6% sequence divergence on opposite sides of the Mackay Glacier. The more range restricted C. nivicolus showed 4.0% divergence among populations. The third species, A. monoculata, was found in only one location. Molecular clock estimates based on sequence divergences suggest that populations separated within the last 4 Mya. We conclude that habitat fragmentation resulting from Pliocene (5 Mya) and Pleistocene (2 Mya to 10 Kya) glaciations has promoted and maintained high levels of diversity among isolated springtail populations on relatively small spatial scales. The region surrounding the Mackay Glacier is likely to have provided refugia for springtail populations during glacial maxima and remains an area of high genetic and species diversity for Collembola within the Ross Sea region.     

Invasion genetics of American cherry fruit fly in Europe and signals of hybridization with the European cherry fruit fly

The American cherry fruit fly is an invasive pest species in Europe, of serious concern in tart cherry production as well as for the potential to hybridize with the European cherry fruit fly, Rhagoletis cerasi L. (Diptera: Tephritidae), which might induce new pest dynamics. In the first European reports, the question arose whether only the eastern American cherry fruit fly, Rhagoletis cingulata (Loew) (Diptera: Tephritidae), is present, or also the closely related western American cherry fruit fly, Rhagoletis indifferens Curran. In this study, we investigate the species status of European populations by comparing these with populations of both American species from their native ranges, the invasion dynamics in German (first report in 1993) and Hungarian (first report in 2006) populations, and we test for signals of hybridization with the European cherry fruit fly. Although mtDNA sequence genealogy could not separate the two American species, cross‐species amplification of 14 microsatellite loci separated them with high probabilities (0.99–1.0) and provided evidence for R. cingulata in Europe. German and Hungarian R. cingulata populations differed significantly in microsatellite allele frequencies, mtDNA haplotype and wing pattern distributions, and both were genetically depauperate relative to North American populations. The diversity suggests independent founding events in Germany and Hungary. Within each country, R. cingulata displayed little or no structure in any trait, which agrees with rapid local range expansions. In cross‐species amplifications, signals of hybridization between R. cerasi and R. cingulata were found in 2% of R. cingulata individuals and in 3% of R. cerasi. All putative hybrids had R. cerasi mtDNA indicating that the original between‐species mating involved R. cerasi females and R. cingulata males.     

Cryptic species and colonization processes in Ophryotrocha (Annelida,Dorvilleidae) inhabiting vertebrate remains in the shallow‐water Mediterranean

Over the past several years, there has been growing interest in how bones of decaying mammals are colonized in the marine seabed. One of the most common opportunistic taxa occurring worldwide on bones is dorvilleid polychaetes of the genus Ophryotrocha. In a recent study in the Mediterranean, Ophryotrocha puerilis and Ophryotrocha alborana were two of the most abundant species occurring in experimentally deployed bones. These species have direct development and this makes them a suitable model to study the mechanisms and processes allowing organisms lacking a dispersive larval phase to colonize new substrates. Here, we address the colonization processes at the molecular level for populations of O. puerilis and O. alborana on experimentally deployed mammal bones in the shallow‐water Mediterranean collected over a year at 3‐month intervals. High genetic distances between some of the O. puerilis organisms collected indicated the occurrence of at least two cryptic sibling species (O. puerilis ‘Shallow’ and O. puerilis ‘Deep’) apart from O. puerilis sensu stricto. This was corroborated with phylogenetic analyses using an alignment of three concatenated genes (COI, 16S, H3) and with species delimitation analyses using COI. The haplotype network inferred from COI sequences for O. puerilis ‘Shallow’ showed a few common haplotypes shared between the two trimesters analysed and several other less represented haplotypes only present in one trimester. Thus, colonization of these experimental bones may have been achieved by a few organisms that arrived to the bones and were able to reseed, and by several individuals arriving to the experimental bones and not persisting across time. In contrast, the haplotype network for O. alborana revealed that none of the haplotypes present in three different trimesters were shared, suggesting that the populations arriving at the bones during each trimester were totally replaced by new individuals during the subsequent trimesters. Our study suggests that different species of shallow‐water Ophryotrocha occurring in the Mediterranean may have different patterns of substrate colonization despite sharing similar life histories.