Microbiome evolution along divergent branches of the vertebrate tree of life: what is known and unknown

Vertebrates harbour microbes both internally and externally, and collectively, these microorganisms (the ‘microbiome’) contain genes that outnumber the host's genetic information 10‐fold. The majority of the microorganisms associated with vertebrates are found within the gut, where they influence host physiology, immunity and development. The development of next‐generation sequencing has led to a surge in effort to characterize the microbiomes of various vertebrate hosts, a necessary first step to determine the functional role these communities play in host evolution or ecology. This shift away from a culture‐based microbiological approach, limited in taxonomic breadth, has resulted in the emergence of patterns suggesting a core vertebrate microbiome dominated by members of the bacterial phyla Bacteroidetes, Proteobacteria and Firmicutes. Still, there is a substantial variation in the methodology used to characterize the microbiome, from differences in sample type to issues of sampling captive or wild hosts, and the majority (>90%) of studies have characterized the microbiome of mammals, which represent just 8% of described vertebrate species. Here, we review the state of microbiome studies of nonmammalian vertebrates and provide a synthesis of emerging patterns in the microbiome of those organisms. We highlight the importance of collection methods, and the need for greater taxonomic sampling of natural rather than captive hosts, a shift in approach that is needed to draw ecologically and evolutionarily relevant inferences. Finally, we recommend future directions for vertebrate microbiome research, so that attempts can be made to determine the role that microbial communities play in vertebrate biology and evolution.     

The seasonal structure of microbial communities in the Western English Channel

Very few marine microbial communities are well characterized even with the weight of research effort presently devoted to it. Only a small proportion of this effort has been aimed at investigating temporal community structure. Here we present the first report of the application of high‐throughput pyrosequencing to investigate intra‐annual bacterial community structure. Microbial diversity was determined for 12 time points at the surface of the L4 sampling site in the Western English Channel. This was performed over 11 months during 2007. A total of 182 560 sequences from the V6 hyper‐variable region of the small‐subunit ribosomal RNA gene (16S rRNA) were obtained; there were between 11 327 and 17 339 reads per sample. Approximately 7000 genera were identified, with one in every 25 reads being attributed to a new genus; yet this level of sampling far from exhausted the total diversity present at any one time point. The total data set contained 17 673 unique sequences. Only 93 (0.5%) were found at all time points, yet these few lineages comprised 50% of the total reads sequenced. The most abundant phylum was Proteobacteria (50% of all sequenced reads), while the SAR11 clade comprised 21% of the ubiquitous reads and ～12% of the total sequenced reads. In contrast, 78% of all operational taxonomic units were only found at one time point and 67% were only found once, evidence of a large and transient rare assemblage. This time series shows evidence of seasonally structured community diversity. There is also evidence for seasonal succession, primarily reflecting changes among dominant taxa. These changes in structure were significantly correlated to a combination of temperature, phosphate and silicate concentrations.     

Reintroduction of locally extinct vertebrates impacts arid soil fungal communities

Introduced species have contributed to extinction of native vertebrates in many parts of the world. Changes to vertebrate assemblages are also likely to alter microbial communities through coextinction of some taxa and the introduction of others. Many attempts to restore degraded habitats involve removal of exotic vertebrates (livestock and feral animals) and reintroduction of locally extinct species, but the impact of such reintroductions on microbial communities is largely unknown. We used high‐throughput DNA sequencing of the fungal internal transcribed spacer I (ITS1) region to examine whether replacing exotic vertebrates with reintroduced native vertebrates led to changes in soil fungal communities at a reserve in arid central Australia. Soil fungal diversity was significantly different between dune and swale (interdune) habitats. Fungal communities also differed significantly between sites with exotic or reintroduced native vertebrates after controlling for the effect of habitat. Several fungal operational taxonomic units (OTUs) found exclusively inside the reserve were present in scats from reintroduced native vertebrates, providing a direct link between the vertebrate assemblage and soil microbial communities. Our results show that changes to vertebrate assemblages through local extinctions and the invasion of exotic species can alter soil fungal communities. If local extinction of one or several species results in the coextinction of microbial taxa, the full complement of ecological interactions may never be restored.     

Scaling up: A guide to high‐throughput genomic approaches for biodiversity analysis

The purpose of this review is to present the most common and emerging DNA‐based methods used to generate data for biodiversity and biomonitoring studies. As environmental assessment and monitoring programmes may require biodiversity information at multiple levels, we pay particular attention to the DNA metabarcoding method and discuss a number of bioinformatic tools and considerations for producing DNA‐based indicators using operational taxonomic units (OTUs), taxa at a variety of ranks and community composition. By developing the capacity to harness the advantages provided by the newest technologies, investigators can “scale up” by increasing the number of samples and replicates processed, the frequency of sampling over time and space, and even the depth of sampling such as by sequencing more reads per sample or more markers per sample. The ability to scale up is made possible by the reduced hands‐on time and cost per sample provided by the newest kits, platforms and software tools. Results gleaned from broad‐scale monitoring will provide opportunities to address key scientific questions linked to biodiversity and its dynamics across time and space as well as being more relevant for policymakers, enabling science‐based decision‐making, and provide a greater socio‐economic impact. As genomic approaches are continually evolving, we provide this guide to methods used in biodiversity genomics.     

Temporal and Taxonomic Variability in Response of Fauna to Riparian Restoration

Evaluating the success of restoration projects requires well‐designed studies. Among the decisions that need to be made are what taxonomic groups to study and when to conduct the monitoring. To explore how these decisions can influence assessments of restoration success, we examined species richness and composition data collected over several years on different terrestrial fauna (landbirds, rodents, bees, and beetles) at Sacramento River restoration and remnant riparian sites. Our selection of study organisms enabled us to ask whether variability in species richness among restoration sites is less for vagile taxa than for sedentary taxa, and if invertebrates display greater variability among sites than vertebrates. Our results demonstrate that responses to restoration can vary depending upon the season when it is assessed, and the taxa that are studied. For all taxa except bees, there was considerable variability in the relative performance of taxa at restoration sites from one sampling date to the next, such that the relative ranking of the sites often changed dramatically. Comparisons of β ‐diversity (variability in species richness across sites) revealed that certain taxonomic groups were more spatially variable in their response to restoration than others. Among vertebrates, sedentary taxa (rodents) had significantly higher variability in species richness across sites than highly vagile taxa (birds); however, no such pattern was observed for invertebrates. Overall, vertebrates had lower variability than invertebrates, suggesting that evaluations of restoration success based on a few better‐known taxonomic groups (e.g., birds, rodents) may be inadequate to represent the biodiversity response of other groups (e.g., insects).     

Phylogenetic analysis of RAD‐seq data: examining the influence of gene genealogy conflict on analysis of concatenated data

One of the major issues in phylogenetic analysis is that gene genealogies from different gene regions may not reflect the true species tree or history of speciation. This has led to considerable debate about whether concatenation of loci is the best approach for phylogenetic analysis. The application of Next‐generation sequencing techniques such as RAD‐seq generates thousands of relatively short sequence reads from across the genomes of the sampled taxa. These data sets are typically concatenated for phylogenetic analysis leading to data sets that contain millions of base pairs per taxon. The influence of gene region conflict among so many loci in determining the phylogenetic relationships among taxa is unclear. We simulated RAD‐seq data by sampling 100 and 500 base pairs from alignments of over 6000 coding regions that each produce one of three highly supported alternative phylogenies of seven species of Drosophila. We conducted phylogenetic analyses on different sets of these regions to vary the sampling of loci with alternative gene trees to examine the effect on detecting the species tree. Irrespective of sequence length sampled per region and which subset of regions was used, phylogenetic analyses of the concatenated data always recovered the species tree. The results suggest that concatenated alignments of Next‐generation data that consist of many short sequences are robust to gene tree/species tree conflict when the goal is to determine the phylogenetic relationships among taxa.     

Assessing the conservation status of soil‐dwelling vertebrates: Insights from the rediscovery of Typhlops uluguruensis (Reptilia: Serpentes: Typhlopidae)

Soil‐dwelling amphibians and reptiles are relatively poorly studied and understood. Difficulties in sampling these taxa in their subterranean habitats might impede assessments of their conservation status. We explore this issue with a case study of the burrowing scolecophidian snake Typhlops uluguruensis, endemic to the Uluguru Mountains in the Eastern Arc of Tanzania. Despite recent standard faunistic surveys, there have been no reported sightings or collections of T. uluguruensis since the type series of four specimens was collected in 1926. Intensive replacement of forest by agriculture in the vicinity of the type locality had led to concern about the conservation status of this and other species. We report the rediscovery of T. uluguruensis in low intensity agriculture adjacent to human habitation, and close to the type locality. We compare the new material with the type series, and discuss the implications of this rediscovery for conservation assessments of small, soil‐dwelling lower vertebrates. We advise caution in determining conservation status when, as is usually the case, no special sampling of the soil has been carried out. Additionally, relatively neglected disturbed habitats should also be given more attention. Standard sampling methods for soil‐dwelling vertebrates need to be further developed and established.     

Lateral bending of the lumbar spine during quadrupedalism in strepsirhines

Much research has been devoted to spinal kinematics of nonmammalian vertebrates, while comparatively little is known about the locomotor role of spinal movements in mammals, especially primates. This study, conducted at the Duke University Primate Center, examines the function of lateral spinal bending during quadrupedal walking among a diverse sample of strepsirhines. The taxa studied include Loris tardigradus (1), Nycticebus coucang (1), N. pygmaeus (1), Cheirogaleus medius (2), Varecia variegata (2), Eulemur fulvus (2), and a total sample size of 261 strides. Lateral bending varies among the taxa with respect to both magnitude and effects of velocity, and does not appear to be correlated with body size. In addition, the timing of lateral bending during a stride appears to differ from that reported for other (nonmammalian) tetrapods. On average, maximum lateral flexion occurs just after ipsilateral foot touchdown, which may be functionally associated with touchdown of the contralateral forelimb during diagonal sequence gait. For some of the taxa, lateral flexion coincides more closely with foot touchdown as velocity increases, suggesting a functional role in increasing hindlimb stride length. Both of these timing patterns contrast with those reported for lizards. Finally, although lorids as a group have been described as having a "sinuous" gait, this study shows more pronounced lateral flexion in Nycticebus than in Loris.