Effects of fragmentation on vascular plant diversity in a Mediterranean forest archipelago

Abstract

We analysed the effects of patch size and isolation on vascular plants in Quercus cerris forest surrounding Rome (Italy). We randomly sampled 96 plots within 18 forest patches with homogeneous environmental variables; the patches ranged from 1.4 ha to 424.5 ha and were divided into four size classes. We performed the analyses at the patch level using linear regression. At the size class level, the analysis of species richness response to fragmentation (area effect) was performed with ANOVA, while the effect on community composition was analysed by means of PERMANOVA. We also investigated which species could be used as indicator species for each size class. Lastly, to evaluate the advantages of conserving several small patches as opposed to few large ones, we used a cumulative area approach ranking forest fragments. The correlation between species richness and patch area was positive, with a significant difference between the “large” and “small” size classes, while analysis on community composition showed that “large” versus “medium” and “large” versus “small” were significantly different. Nemoral species were recognised as indicators in the “large” class, and shrub and edge species in the “small” class. Our results indicate that 10 ha may be a suitable forest size threshold for planning and conservation.     

Ecology of Foliicolous Lichens at the “Botarrama” Trail (Costa Rica), a Neotropical Rain Forest. I. Species Composition and its Ecogeographical Implications1

A total of 217 species of typically foliicolous lichens were found at the “Botarrama” trail, a premontane rain forest in the Braulio Carrillo National Park, Costa Rica. The species composition confirmed the classification of this study sice as transitional between lowland wet and montane rain forest. In terms of species tichness, the foliicolous lichen flora was dominated by the families Gomphillaceae (Calenia, Echinoplaca, Gyalideopsis, and Tricharia), Trichotheliaceae (Porina and Tricbothelium), and Pilocarpaceae (Byssoloma and Fellhanera). The most frequent species were Porina mirabilis (including its supposed anamorph Phyllophiale alba), Gyalectidium filicinum, Porina rufula, and Strigula platypoda. A large proportion of the species was rare. Frequency distributions based on area cover corresponded well to general community models by closely fitting a log-normal approximation. Half of the species had narrow ecological amplitudes while more than 70 percent exhibited a wide geographical distribution, thus making che study site representative of a Neotropical rain forest. The use of foliicolous lichens as indicators of altitudinal zonation is discussed.     

Respiration‐induced weathering patterns of two endolithically growing lichens

The two endolithic lichen species Hymenelia prevostii and Hymenelia coerulea were investigated with regard to their thallus morphology and their effects on the surrounding substrate. The physiological processes responsible for the observed alterations of the rock were identified. Whereas the thallus surface of H. coerulea was level, H. prevostii formed small depressions that were deepest in the thallus center. In a cross‐section, both species revealed an algal zone consisting of algal cavities parallel to the substrate surface and a fungal zone below. However, H. prevostii revealed significantly larger cavities with more than twice the cell number and a denser pattern of cavities than H. coerulea, resulting in a biomass per surface area being more than twice as large. Below H. prevostii the layer of macroscopically visibly altered rock material was about twice as deep and within this layer, the depletion of calcium and manganese was considerably higher. In simultaneous measurements of the oxygen uptake/oxygen release and pH shift, the isolated algal strains of both lichens revealed respiration‐induced acidification of the medium in the dark. At higher light intensities, H. coerulea and to a lesser extent also H. prevostii alkalized the medium which may lessen the acidification effect somewhat under natural conditions. In a long‐term growth experiment, the isolated algal strains of both lichens revealed acidification of the medium to a similar extent. Neither acidic lichen substances nor oxalic acid was identified. The significant differences between the weathering patterns of both species are based on the same respiration‐induced acidification mechanism, with H. prevostii having a greater effect due to its higher biomass per area.     

Modern and fossil traces in terrestrial lithic substrates

A number of biogenic processes leads to the formation of distinctive traces in terrestrial lithic substrates. These include: burrowing by vertebrates in moderately lithified rocks; scraping by mammals; smoothing and polishing of limestone surfaces by the locomotion of mammals; excavation by bees, wasps, and ants producing nesting and dwelling tunnels; dissolution of limestone surfaces by terrestrial snails; endolithic activity of fungi, algae, and lichens on subaerial rock surfaces; root corrosion; etc. Processes of biochemical weathering, biophysical erosion, and enlargement of cracks and fissures by the pressure of plant roots do not leave distinctive traces and therefore lie outside the ichnological realm. The fossil preservation of terrestrial bioerosional traces is expected to be uncommon. Nevertheless, various possible means of preservation must be considered, such as by rapid burial by volcanic material, by fluvial sediments, by travertine or tufa, by loess, “conservation”; in caves, case hardening of surfaces of porous rocks, and preservation of subsoil traces below fossil soils.     

Ultrastructural effects of trace elements and environmental pollution in Italian “Triangle of Death” on Pseudevernia furfuracea (L.) Zopf

Abstract

The lichen Pseudevernia furfuracea was exposed to environmental trace elements in the district of Acerra (province of Naples, southern Italy), one of the points forming Italy's “Triangle of Death”. P. furfuracea thalli were exposed in bags at different sites for 6 months, periodically collected and examined by transmission electron microscopy (TEM) to assess ultrastructural changes. An our earlier study demonstrated that these exposed lichens were strongly contaminated by trace elements (Sorbo S, Aprile G, Strumia S, Castaldo Cobianchi R, Leone A, et al. 2008. Trace element accumulation in P. furfuracea (L.) Zopf exposed in Italy's so-called Triangle of Death. Sci Total Environ 407: 647–654.). The algal cells were more affected than the fungal symbiont. Exposition at urban sites gave the most frequent changes. Four trace elements (Cd, Pb, Cu and Zn) were used to study the effects of ultrastructural trace element both in field and in vitro treatments. The lichen developed comparable ultrastructural changes when exposed to different trace elements and the changes were not specific to the treatment used (lichen bag exposition, in field and in vitro treatments). The in vitro treatment gave the highest frequency of damage at all time points. X-ray TEM microanalysis revealed trace elements inside the cell walls and the cytoplasmic vesicles of the lichens cultured with the trace elements; this localization is probably related to tolerance mechanisms.     

THE IMPACT OF STORM-FLOW ON RIVER BIOFILM ARCHITECTURE1

The impact of storm-flow on river biofilm architecture was investigated using transmission (TEM) and scanning (SEM) electron microscopy. TEM resin substrata were colonized under light-grown (LG) or dark-grown (DG) conditions for 33 weeks in the Clywedog River, North Wales, prior to exposure to ambient-flow (approx. 60 cm·s^?1) or storm-flow (approx. 235 cm·s^?1+ river sediment) in a laboratory flume. Line transect methodology was used to quantify information from TEM ultrathin sections of LG material. In the LG ambient-flow biofilm, bacteria were more abundant directly adjacent to the substratum and were noticeably denser directly under the adnate diatom Cocconeis. Higher in the biofilm, the bacteria were loosely dispersed in the matrix between other cells. Cyanobacteria occurred most frequently as single cells but were also found in large “palisade” formations adjacent to the substratum. Significant horizontal and vertical nearest-neighbor associations were noted for both bacteria and cyanobacteria. Cells of Cocconeis were common adjacent to the substratum, providing shelter to, and often elevated upon, an “organic pad” of bacteria, cyanobacteria, and densely staining exopolysaccharide. Cyanobacteria and Cocconeis were resistant to removal by storm-flow, but Cocconeis frustules were sometimes damaged. Bacteria in the LG storm-flow samples were less common adjacent to the substratum and were sometimes more dispersed higher in the biofilm than in ambient-flow samples. We suggest that storm-flow hydrodynamic forces may redistribute bacteria adjacent to the substratum into higher areas of the biofilm. In addition, bacteria and the exopolysaccharide matrix were sometimes removed down to the substratum by storm-flow, unless beneath Cocconeis. The DG biofilm consisted almost entirely of bacteria. Storm-flow only removed surface growth from DG biofilms, and SEM revealed peritrich stalk abrasion and “blow-down.” Pre-disturbance biofilm architecture appears to influence the form of destruction. We suggest that the “microcosms” of Cocconeis and their underlying cells not only serve as an inoculum to recolonize the surface when conditions permit but enhance immigration by interrupting flow patterns across the surface.     

A Fourier Transform-Raman Spectroscopic Study of Lichen Strategies on Granite Monuments

The biodeterioration of granite by Lecidea fuscoatra (L.) Ach., Porpidia cinereoatra (Ach.) Hertel & Knoph, and P. macrocarpa (DC.) Hertel & Schwab growing in the same environmental conditions has been studied by using Fourier transform-Raman spectroscopy. Results were significantly different for the three species, with P. cinereoatra being the most active biodeteriorative lichen. This lichen was also the only one in which calcium oxalate and gypsum were identified spectroscopically. Physical disturbances to the substratum were evidenced in all lichens studied by the incoporation of material, such as quartz and feldspar, into their thalli, but this phenomenon varied considerably from species to species. The results indicate that lichen species can adopt different strategies and can have different biodeteriorative effects on granite, independently of environmental conditions and substrate.     

Caloplaca erodens [sect. Pyrenodesmia], a new lichen species from Italy with an unusual thallus type

Caloplaca erodens is a new species of sect. Pyrenodesmia, characterised by an orbicular, sorediate, bluish-grey thallus which is endolithic but emerges at the periphery with a white, K−, obscurely lobate prothallus. The species is frequent on calcareous outcrops and walls of isolated churches and ruins of the Central Apennines (Sibillini, Gran Sasso), where it may occur in large monospecific populations, from 1000 to 2500 m asl., and is also known from dry sites of the southern Alps. It has been found with apothecia only in the type locality, being predominantly sterile. The reproduction of this lichen is evidently linked to the release of fragments of clusters of photobiont cells and mycobiont hyphae which are continuously exposed with the dissolution of the substratum. Readily distinguished from the apparently similar endolithic C. alociza (which is characterised by numerous apothecia, black, K+ purple prothallus, and esorediate thallus), C. erodens probably belongs to the C. circumalbata complex, whose taxa are always epilithic but have a white, K− prothallus. The phylogenetic position of the new species within sect. Pyrenodesmia as inferred by ITS sequences of the nuclear ribosomal DNA is shortly discussed.     

Tree and stand level variables influencing diversity of lichens on temperate broad-leaved trees in boreo-nemoral floodplain forests

Tree and stand level variables affecting the species richness, cover and composition of epiphytic lichens on temperate broad-leaved trees (Fraxinus excelsior, Quercus robur, Tilia cordata, Ulmus glabra, and U. laevis) were analysed in floodplain forest stands in Estonia. The effect of tree species, substrate characteristics, and stand and regional variables were tested by partial canonical correspondence analysis (pCCA) and by general linear mixed models (GLMM). The most pronounced factors affecting the species richness, cover and composition of epiphytic lichens are acidity of tree bark, bryophyte cover and circumference of tree stems. Stand level characteristics have less effects on the species richness of epiphytic lichens, however, lichen cover and composition was influenced by stand age and light availability. The boreo-nemoral floodplain forests represent valuable habitats for epiphytic lichens. As substrate-related factors influence the species diversity of lichens on temperate broad-leaved trees differently, it is important to consider the effect of each tree species in biodiversity and conservation studies of lichens. Nomenclature Randlane et al. (2007) for lichens; Leht (2007) for vascular plants.     

Occurrence and possible roles of melanic pigments in lichenized ascomycetes

Many species of lichenized ascomycetes are capable of synthesizing darkly colored melanin pigments by polymerizing phenolic compounds. Available data suggest that different clades of lichens produce various kinds of melanins, with N₂-fixing Peltigeralean lichens producing N-rich DOPA or “eumelanins” and lichens from other orders producing N-poor “allomelanins.” In general, melanic lichens seem to be more common in environments with high levels of abiotic stress such as polar and montane regions, but they are by no means restricted to these habitats. Here, we review the occurrence of melanins in lichens, their chemical structure, methods of quantification and biological roles. Good evidence exists that melanins in lichens protect the mycobiont from high UV, and the photobiont against high PAR. However, given their many unique properties, it seems likely that melanins help lichens to survive a range of biotic and abiotic stresses.     

DIFFERING ONTOGENETIC ORIGINS OF PCR (“KRANZ”) SHEATHS IN LEAF BLADES OF C4 GRASSES (POACEAE)

The origin and early development of procambium and associated ground meristem of major and minor veins have been examined in the leaf blades of seven C₄ grass species, representing different taxonomic groups and the three recognized biochemical C₄ types (NAD-ME, PCK, and NADP-ME). Comparisons were made with the C₃ species, Festuca arundinacea. In “double sheath” (XyMS+) species (Panicum effusum, Eleusine coracana, and Sporoboìus elongatus), the procambium of major veins gives rise to xylem, phloem, and a mestome sheath; associated ground meristem differentiates into PCA (“C₄ mesophyll”) tissue and the PCR (“Kranz”) sheath. Development in the C₃ species parallels this pattern, except that associated ground meristem differentiates into mesophyll and a parenchymatous bundle sheath. In contrast, major vein procambium of “single sheath” (XyMS–) species (Panicum bulbosum, Digitaria brownii, and Cymbopogon procerus) differentiates into xylem, phloem and a PCR sheath; associated ground meristem gives rise to PCA tissue. These observations of major vein development support W. V. Brown's hypothesis that the PCR sheaths of “double sheath” (XyMS+) C₄ grasses are homologous with the parenchymatous bundle sheaths of C₃ grasses, while in “single sheath” (XyMS–) C₄ species they are homologous with the mestome sheath. Although there are some similarities in the development of the major and minor vascular bundle procambium in the C₄ species examined, the ontogeny of the smaller minor veins is characterized by a precocious delineation of the PCR sheath layer that may even precede the appearance of the distinctive cytological features of ground meristem and procambium. This contracted development in minor veins appears to be related to their close spacing in mature leaves and to their comparatively late appearance during leaf ontogeny.     

CELL SIZE DIFFERENTIATION IN THE BLOOM-FORMING DINOFLAGELLATE GYMNODINIUM CF. NAGASAKIENSE1

Two subpopulations differing essentially by their mean cell size were observed regularly in cultures and natural samples of the naked dinoflagellate Gymnodinium cf. nagasakiense Takayama et Adachi (currently known as Gyrodinium aureolum Hulburt), a species which frequently forms red tides in North European seas. “Large” cells represented the typical forms; they were morphologically similar to cells of the closely related Japanese species G. nagasakiense, which did not form any subpopulation of reduced size. “Small” and “large” cells of G. cf. nagasakiense had the same DNA content, but the nucleus of the former appeared to be much more condensed during interphase. Each cell type was able to divide and had its own growth dynamics; therefore, any intermediary between pure populations of “small” and of “large” cells were observed in culture. The “large” form generated a “small” cell by an atypical budding-like division, whereas the “small” form gave back a “large” form, once it ceased to divide, by simple enlargement of its cell body. Factory inducing cell size differentiation are yet unclear. Neither nitrogen nor phosphorus starvation induced a significant increase in the relative proportion of “small” and budding cells. Although cell size differentiation is associated with the formation of gametes in a variety of dinoflagellates, we demonstrated that “small” cells of G. cf. nagasakiense are able to divide asexually, in contrast to gametes of most other species. The high proliferative power of “small” cells as compared with normal cells suggests that they could play a significant role during red tides of G. cf. nagasakiense; in contrast, cells of the Japanese species G. Nagasakiense could sustain high growth rates with larger cell size because this species generally blooms in waters much warmer than those found in northern Europe.     

In vitro receptivity of carbonate rocks to endolithic lichen-forming aposymbionts

Sterile cultured isolates of lichen-forming aposymbionts have not yet been used to investigate lichen-rock interactions under controlled conditions. In this study mycobionts and photobiont of the endolithic lichens Bagliettoa baldensis and Bagliettoa marmorea were isolated and inoculated with coupons of one limestone and four marbles commonly employed in the Cultural Heritage framework. After one year of incubation, microscopic observations of polished cross-sections were performed to verify if the typical colonization patterns observed in the field may be reproduced in vitro and to evaluate the receptivity of the five lithotypes to endolithic lichens. The mycobionts of the two species developed both on the surface of and within all the lithotypes, showing different penetration pathways which depend on mineralogical and structural features and highlight different receptivity. By contrast, algae inoculated with the coupons did not penetrate them. Observations suggest that the hyphal penetration along intrinsic discontinuities of rocks is a relatively fast phenomenon when these organisms are generally considered as slow-growing. Samples from limestone outcrops and abandoned marble quarries, colonized by the same species or other representatives of Verrucariaceae, showed penetration pathways intriguingly similar to those reproduced in vitro and highlighted that lichen-driven erosion processes only increase the availability of hyphal passageways after a long-term colonization. These results show that in vitro incubation of sterile cultured lichen-forming ascomycetes with rock coupons is a practicable experimental system to investigate the lichen-rock interactions under controlled conditions and, together with analysis in situ, may support decisions on conservative treatments of historical and cultural significant stone substrata.     

Different effects of rain and artificial watering on element zonation patterns in lichen thalli and bark: A study on Physcia biziana (Massal.) Zahlbr. v. leptophylla Ve˘zda

Translocation processes between the inner and outer rings of lichen thalli, as well as between lichens and bark substratum, induce zonation patterns, with a dynamic stratification of pollution history memory which is often affected by the intensity of the atmospheric events. Two experiments of passive biomonitoring are presented, aimed to measure metal concentrations and zonation patterns in lichens and bark, before and after raining events or after a water sprinkling treatment. Ten elements were monitored in the lichen Physcia biziana (Massal.) Zahlbr. v. leptophylla Ve?zda in a moderately polluted area of Campania region (Italy). Element concentrations changed with lichen age, with generally higher concentrations in the older than the younger tissues. Rain and sprinkling had differential effects on lichen and bark: a significant decrease of the overall concentrations was observed after rain in bark, whereas a slight increase was observed in lichen thalli; however, when lichen thalli were just rehydrated with a sprinkling treatment, some elements were found to increase in the younger portions of the thalli and decrease in the older portions. The extent to which zonation patterns account for site “pollution memory” or are affected by the short-term local atmospheric variability is discussed.     

中国橙衣属和果衣属地衣的新记录种

通过对采自云南省景东彝族自治县无量山地衣标本的研究,发现了橙衣属的一个中国新记录Caloplaca indica(三室类型孢子)和果衣属的一个中国新记录种Ramboldia haematites。R. haematites在福建和广西同样存在。该文详细描述了其形态学、解剖学和化学方面的特征,并且提供了显微结构照片。本研究丰富了云南、福建和广西地衣的物种多样性,为橙衣属和果衣属地衣分类学研究提供基础数据和可靠资料。所用标本存放在山东师范大学植物标本室(SDNU)。     

SEAWEED ANATOMY AND PHOTOSYNTHETIC PERFORMANCE: THE ECOLOGICAL SIGNIFICANCE OF LIGHT GUIGES,HETEROGENEOUS ABSORPTION AND MULTIPLE SCATTER12

Light absorption by two green seaweeds with similar photophysiology but different anatomies are compared: i) Ulva lactuca var. rigida (C. Ag.) Le Jolis, an optically translucent species of two cell layers both bearing chloroplasts; and, ii) Codium fragile subsp. tomentosoides (van Goor) Silva, an optically opaque species with a colorlelss medulla surrounded by a cortex of choloroplast-bearing utriclels. Thallus absorptance (fraction of incident light absorbed) was measured for various pigment contents. Absorptance by U. lactuca was dependent on pigment concentration in an exponential manner and never exceeded 0.6, whereas absorptance by C. fragile was independent of pigment concentration and always approached a balue of 1.0. Water in the medullary tissue of C. fragile is often of the utricles. The utricles appear to be “integrating spheres” enhancing the capture of incident light, aided by the wave-guide function of the thin peripheral layer of cytoplasm and a reflector function at their base. Photosynthitic performance for U. lactuca saturates at high light intensities and attenuates rapidly with decreasing intensities. In contrast, photosynthetic performance for C. fragile saturates at low light intensities and attenuates slowly with diminishing radiation. Extrapolated diel variation in photosynthesis shows that U. lactuca's anatomy is adaptive for high light intensity environments, whereas C. fragile's anatomy is adaptive for low light intensity environments. Both seaweeds fit into the ecological category of “fugitive” species, and compete in the Long Island Sound (Atlantic Ocean) rocky intertidal for free-space. Predictions are presented for relative species abundances along a monotonic gradient of light intensity.     

Cyanobacteria and Cyanobacterial Lichens from Inselbergs of the Ivory Coast,Africa

This study focuses on the saxicolous lichens and cyanobacteria of the open, exposed rock surface of inselbergs. Twenty-three species of cyanobacteria and 17 cyanobacterial lichen species (“cyanolichens”) from several inselbergs and other rocky outcrops of three major climatic regions, savanna, transition zone and rain forest, are reported from the Ivory Coast. Inselbergs are isolated and frequently mountains consisting of Precambrian granites or gneisses that abruptly rise from the surrounding plains. Cyanobacteria were found to be the dominating organisms on all rock surfaces. The lichens found mainly belong to the family Peltulaceae and a few were present from the family Lichinaceae. Nine species of the cyanolichens and most of the cyanobacteria are new for the Ivory Coast. A gradient in total species number (cyanolichens and cyanobacteria) occurs from the savanna to the rain forest, with a decrease in species number towards the rain forest. Saxicolous cyanobacterial lichens reached a higher species number in the savanna type ecosystem (11) than on inselbergs in the rain forest (7). The cyanolichens and cyanobacteria found are characteristic for larger, light-exposed rock surfaces and species like P. congregate, P. lingulata, P. tortuosa and P. umbilicata preferentially occur on the granite or sandstone of inselbergs.     

Analysis of waving and sound‐production display in the ghost crab,Ocypode stimpsoni

The ghost crab Ocypode stimpsoni displays waving and sound production. Sounds are produced by thumping the sand substratum with the major cheliped, and two types of sounds can be discriminated; one with a low frequency of about 12 Hz, called rapping, and another with a higher frequency (about double), called quivering. In our observations, a sequence of waving and sound emission would sometimes terminate abruptly, or appear as independent components but the component order never changed. The most frequently observed patterns were “waving with rapping and quivering”;, “waving with quivering”; and “quivering only”; quivering sounds being involved in more than 80% displays. Quivering sometimes occurred immediately after crabs emerged from the burrow, or when they returned to the entrance after discarding an excavated sand mass. The occurrence frequency of waving and sounds, the wave amplitude, and the frequency of the sound increased when other crabs approached.     

Stand characteristics and biodiversity indicators along the productivity gradient in boreal forests: Defining a critical set of indicators for the monitoring of habitat nature quality

Abstract

We quantified the effects of anthropogenic disturbances on the structure and biodiversity of boreal forests on acidic soils and created a statistically supported rational set of indicators to monitor the stand “naturalness”. For that, we surveyed various traits of tree layer, understory, herb layer, forest floor and several widely accepted biodiversity epiphytic indicators in 252 old‐aged boreal stands in Estonia, mostly dominated by Scots pine or Norway spruce. Multifactorial general linear model analyses showed that many forest characteristics and potential indicators were confounded by the gradient of soil productivity (reflected by the forest site type), local biogeographic gradients and also by stand age. Considering confounding effects, boreal forests in a near‐natural state have more large‐diameter trees (diameter at breast height >40 cm) and larger variety of diameter classes, higher proportion of spruce or deciduous trees, a larger amount of coarse woody debris in various stages, a more closed tree canopy and denser understory than managed mature forests. By increasing light availability above the field layer, forest management indirectly increases the coverage of herbs and lichens on the forest floor but reduces the alpha‐ and beta‐diversity of herbs and the proportion of graminoids. Human disturbances reduce the relative incidence of many commonly accepted biodiversity indicators such as indicator lichens, woodpeckers, wood‐dwelling insects or fungi on trees. The test for the predictive power of characteristics reacting on disturbance revealed that only a fraction of them appeared to be included in a diagnostic easy‐to‐apply set of indicators to assess the nature quality of boreal forest: the amount of dead wood, the proportion of deciduous trees, the presence of specially shaped trees and woodpeckers and, as an indicator of disturbances, the forest herb Melampyrum pratensis. Many of these indicators have already been implemented in practice.     

Assessment of bacterial endosymbiont diversity in Otiorhynchusspp. (Coleoptera: Curculionidae) larvae using a multitag 454 pyrosequencing approach

Background

Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species.

Results

High-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of “Candidatus Blochmannia” and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and “Candidatus Blochmannia”, we identified a new phylogenetic clade of Rickettsia as well as “Candidatus Nardonella” endosymbionts in Otiorhynchus spp. which are closely related to “Candidatus Blochmannia” bacteria.

Conclusions

Here, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and “Candidatus Nardonella” endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.