Phylogenetic analysis of the fecal microbial community in herbivorous land and marine iguanas of the Galápagos Islands using 16S rRNA-based pyrosequencing

Abstract

Herbivorous reptiles depend on complex gut microbial communities to effectively degrade dietary polysaccharides. The composition of these fermentative communities may vary based on dietary differences. To explore the role of diet in shaping gut microbial communities, we evaluated the fecal samples from two related host species--the algae-consuming marine iguana (Amblyrhynchus cristatus) and land iguanas (LI) (genus Conolophus) that consume terrestrial vegetation. Marine and LI fecal samples were collected from different islands in the Galápagos archipelago. High-throughput 16S rRNA-based pyrosequencing was used to provide a comparative analysis of fecal microbial diversity. At the phylum level, the fecal microbial community in iguanas was predominated by Firmicutes (69.5±7.9%) and Bacteroidetes (6.2±2.8%), as well as unclassified Bacteria (20.6±8.6%), suggesting that a large portion of iguana fecal microbiota is novel and could be involved in currently unknown functions. Host species differed in the abundance of specific bacterial groups. Bacteroides spp., Lachnospiraceae and Clostridiaceae were significantly more abundant in the marine iguanas (MI) (P-value>1E-9). In contrast, Ruminococcaceae were present at >5-fold higher abundance in the LI than MI (P-value>6E-14). Archaea were only detected in the LI. The number of operational taxonomic units (OTUs) in the LI (356-896 OTUs) was >2-fold higher than in the MI (112-567 OTUs), and this increase in OTU diversity could be related to the complexity of the resident bacterial population and their gene repertoire required to breakdown the recalcitrant polysaccharides prevalent in terrestrial plants. Our findings suggest that dietary differences contribute to gut microbial community differentiation in herbivorous lizards. Most importantly, this study provides a better understanding of the microbial diversity in the iguana gut; therefore facilitating future efforts to discover novel bacterial-associated enzymes that can effectively breakdown a wide variety of complex polysaccharides.

Figure 1

Sampling sites and collection details. Fecal samples from LI and MI were collected in Fernandina, Plaza Sur, Santa Fe and San–Cristobal of Galápagos Islands. NA denotes that LI are no longer extant on San–Cristobal.

Figure 2

Bacterial distribution evaluated at the phylum taxonomical level. (a) The gut microbiota of herbivorous reptiles was predominated by Firmicutes, unclassified Bacteria and Bacteroidetes. (b) The low-abundance phyla were grouped together as ‘others', and their specific abundances were listed. ^*denotes that Archaea were detected in all herbivorous reptiles, at abundance ranging from 0.1 to 0.2% of total microbial community, except in MI. LI, land iguanas; MI, marine iguanas; GI, green iguanas; GT, giant tortoises.

Figure 3

Host-specific clustering differences in bacterial lineages. The abundance and occurrence of bacterial lineages present in feces of each individual reptile were compared and plotted on multidimensional scaling plot (MDS). Apparent clustering was observed in relation to host species (circled in dashed lines), regardless of the sampling location. Abbreviations denote the sampling locations, that is, F, Fernandina; P, Plaza Sur; SF, Santa Fe; EL, El-Salvador; SC-L, San Cristobal-Loberia; SC-C, San Cristobal-Corola ; SC-G, San Cristobal-–Galapaguera.

Figure 4

Bacterial distribution evaluated at a finer taxonomical level. The classes Bacteroidia and Clostridia were consistently present at high abundances in all herbivorous reptiles. A further evaluation within Bacteroidia and Clostridia showed differences in the profiles and abundance of bacterial groups among the herbivorous reptiles.

Figure 5

Archaeal population detected in fecal microbiota. No Archaea were detected in MI. The genus Methanobrevibacter, Methanosarcina and Methanocorpusculum were detected in the herbivorous reptiles that consumed terrestrial plant material. Triangles denote that only one LI at each location, respectively, was shown to harbor Methanocorpusculum. F-LI, land iguanas in Fernandina; P-LI, land iguanas in Plaza Sur; SF-LI land iguanas in Santa Fe.

Figure 6

Comparison of microbial richness among the herbivorous reptiles. Microbial richness was defined based on number of OTUs (defined at 97% gene similarity). ^*denotes fairly significant difference evaluated at 90% confidence interval (CI), ^**significant at 95% CI, ^***highly significant at 99% CI.