Intrathalline phycobiont diversity was investigated in a rosette-forming lichen, Parmotrema pseudotinctorum, using a combination of Sanger sequencing, 454-pyrosequencing, conventional light and confocal microscopy, and transmission electron microscopy. A total of 39 thalli sampled in five Canary Island populations were investigated. Three novel lineages of lichen phycobionts were detected, all being inferred within the Trebouxia clade G. The most abundant phycobiont lineage, occurring in all lichen populations investigated, is described here as Trebouxia crespoana sp. nov. This species produces spherical to pyriform cells possessing a crenulate chloroplast with lobes elongated at their ends, and one corticola-type pyrenoid with very thin, unbranched tubules of curved profile. Trebouxia crespoana is clearly distinguished from all other Trebouxia species by a characteristic cap-like cell wall thickening produced on one side of vegetative cells, and the larger size of vegetative cells that reach 21(–26) µm in diameter.

Lichen thalli represent the most conspicuous examples of fungal-algal interactions. Studies that describe phycobiont diversity within entire thalli are based mainly on Sanger sequencing. In some lichen species, this technique could underestimate the intrathalline coexistence of multiple microalgae. In this study different multi-tool approaches were applied to two lichen taxa, Circinaria hispida and Flavoparmelia soredians, to detect algal coexistence. Here, we combined Sanger sequencing, a specific polymerase chain reaction (PCR) primer, 454-pyrosequencing, phycobiont isolation and ultrastructural characterization. Furthermore, we compared pyrenoid ultrastructural features of lichenized phycobionts with microalgae isolated in culture. An improved methodology was used to isolate and propagate phycobionts which, in combination with fast genetic identification, resulted in a considerable reduction in time and cost to complete the process. This isolation method, coupled with a specific PCR primer, allowed for the detection of coexisting algae in C. hispida (four Trebouxia lineages). 454-pyrosequencing detected only a fraction of such diversity, while Sanger sequencing identified only the primary phycobiont. Ultrastructural features of the isolated algae were observed by transmission electron microscopy; the maintenance of the pyrenoid characteristics suggested the existence of different Trebouxia lineages. In F. soredians a single Trebouxia lineage was identified using all these approaches.

In cases of lichens with algal coexistence, a combination of different molecular and ultrastructural approaches may be required to reveal the underlying algal diversity within a single thallus. The approach proposed in this study provides information about the relationship between molecular and ultrastructural data, and represents an improvement in the delimitation of taxonomic features which is needed to recognize intrathalline Trebouxia diversity.

The diet of Antarctic salps was elucidated by investigating their gut content using automated ribosomal intergenic spacer analysis (ARISA) and 454-pyrosequencing. Salp samples were collected during the Lazarev Sea Krill Study in the western Weddell Sea (summer 2005–06 and 2007–08, autumn 2004 and winter 2006). Two salp species, Salpa thompsoni and Ihlea racovitzai, both occur in the Southern Ocean and can overlap geographically and seasonally. We provide evidence that, despite the non-selective feeding mechanism, the two co-occurring salp species might have different niches within a habitat. ARISA-patterns of 93 gut content samples revealed strong differences between the two salp species, even at the same sampling site. These differences were confirmed by 454-pyrosequencing of the V4-18S rDNA of ten salps. The pyrosequencing data indicate that flagellates, in particular dinophyceae, constitute a high proportion of the sequence reads identified in the gut content of both salp species. However, within the dinophyceae, differences in the read composition were detected between the two salp species. This supports the findings of a previous study where fatty acid signatures indicate a flagellate-based diet, even though microscopic analyses identified diatoms as the dominant component of salp gut contents.

Initial neonatal gut colonisation is a crucial stage for developing a healthy physiology, beneficially influenced by breast-feeding. Breast milk has been shown not only to provide nutrients and bioactive/immunological compounds, but also commensal bacteria, including gut-associated anaerobic Bifidobacterium spp. The aim of the present study was to investigate bacterial diversity in breast milk, with emphasis on identifying gut-associated obligate anaerobes. Breast milk collected from seven mothers at three sampling points (days 3–6, 9–14 and 25–30 postpartum) was analysed by combined culture-dependent and state-of-the-art, culture-independent methods (Sanger sequencing and 454-pyrosequencing). In addition to the predominance of facultative anaerobes such as Staphylococcus, Streptococcus and Propionibacterium (>90 % of isolated strains and 23·7 % relative abundance using pyrosequencing), significant populations of obligate anaerobes, including Bifidobacterium and Veillonella, were detected using pyrosequencing and confirmed by the isolation of viable strains (3·4 % of isolates and 1·4 % relative abundance). Pyrosequencing also revealed the presence of DNA of multiple major gut-associated obligate anaerobes (6·2 % relative abundance) such as Bacteroides and, for the first time, several members of the Clostridia, including butyrate producers, such as Faecalibacterium and Roseburia, which are important for colonic health. The present study suggests that breast milk may be a major source of bacterial diversity to the neonatal gut, including gut-associated obligate anaerobes, and may thus significantly influence gut colonisation and maturation of the immune system.