Gut microbiome of the critically endangered New Zealand parrot, the kakapo (Strigops habroptilus)

Abstract

The kakapo, a parrot endemic to New Zealand, is currently the focus of intense research and conservation efforts with the aim of boosting its population above the current 'critically endangered' status. While virtually nothing is known about the microbiology of the kakapo, given the acknowledged importance of gut-associated microbes in vertebrate nutrition and pathogen defense, it should be of great conservation value to analyze the microbes associated with kakapo. Here we describe the first study of the bacterial communities that reside within the gastrointestinal tract (GIT) of both juvenile and adult kakapo. Samples from along the GIT, taken from the choana (≈ throat), crop and faeces, were subjected to 16 S rRNA gene library analysis. Phylogenetic analysis of >1000 16 S rRNA gene clones, derived from six birds, revealed low phylum-level diversity, consisting almost exclusively of Firmicutes (including lactic acid bacteria) and Gammaproteobacteria. The relative proportions of Firmicutes and Gammaproteobacteria were highly consistent among individual juveniles, irrespective of sampling location, but differed markedly among adult birds. Diversity at a finer phylogenetic resolution (i.e. operational taxonomic units (OTUs) of 99% sequence identity) was also low in all samples, with only one or two OTUs dominating each sample. These data represent the first analysis of the bacterial communities associated with the kakapo GIT, providing a baseline for further microbiological study, and facilitating conservation efforts for this unique bird.

Figure 1. Phylum-level distribution of kakapo-derived 16 S rRNA gene sequences.

Phylum-level affiliation of 16 S rRNA gene sequences obtained from the kakapo GIT. Samples to the left of the dotted line represent clone libraries derived from juveniles, and samples on the right represent adult-derived sequences.

Figure 2. OTU-level distribution of kakapo-derived 16 S rRNA gene sequences.

OTU-level affiliation of 16 S rRNA gene sequences obtained from the kakapo GIT. Values in this heatmap are scaled as a proportion of the total number of sequences per clone library. Observed numbers of OTUs at 99% sequence similarity are provided below the figure, as well as the estimated diversity for each library using the Chao1 estimator.

Figure 3. Phylogeny of Firmicutes-associated 16 S rRNA gene sequences derived from kakapo.

16 S rRNA gene-based phylogenetic analysis of Firmicutes recovered from kakapo samples. Solid junctions represent >90% bootstrap support, and hollow junctions >75%. Kakapo-derived sequences are in bold. Dashed lines indicate sequence length <1200 bp. Branch lengths were calculated using the maximum-likelihood method RAxML, using sequences >1200 bp in length, and short sequences were added subsequently using the Parsimony Interactive tool in ARB. Bootstrap values were calculated using maximum parsimony with 5000 resamplings. Scale bar, 10% sequence divergence.

Figure 4. Phylogeny of Gammaproteobacteria-associated 16 S rRNA gene sequences derived from kakapo.

16 S rRNA gene-based phylogenetic analysis of Gammaproteobacteria found within kakapo samples. Solid junctions represent >90% bootstrap support, and hollow junctions >75%. Kakapo-derived sequences are bolded. Dashed lines indicate sequence length <1200 bp. Branch lengths were calculated using the maximum-likelihood method RAxML, using sequences >1200 bp in length, and short sequences were added subsequently using the Parsimony Interactive tool in ARB. Bootstrap values were calculated using maximum parsimony with 5000 resamplings. Scale bar, 10% sequence divergence.