“Gut microbial communities of velvet worm Euperipatoides rowelli (Onychophora) across deadwood microhabitats in southeastern Australia”

Abstract This study investigated the gut microbial community of the velvet worm Euperipatoides rowelli, a saproxylic invertebrate residing in deadwood microhabitats within historical refugia in the Tallaganda forest, New South Wales, Australia. The eucalyptus-dominated temperate forest is marked by unique topography, hydrology, and historical influences from Pleistocene glacial cycles. We described patterns in gut microbial community structure and composition across eight sites. The velvet worms were unfed for two weeks prior to dissection to lessen the influence of transient prey-associated microorganisms. Amplicon sequencing of the V4-V5 region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform, with positive and negative controls integrated to assess contamination and ensure data quality. Alpha and beta diversity metrics demonstrated minimal overall variation among sites, suggesting broadly similar community structure. However, a closer look at individual taxa revealed contrasting patterns. Proteobacteria dominated the gut microbiome, with genus A37b (Rickettsia) forming part of the core microbiome in 50% of individuals; its presence varied among sites. In contrast, Spiroplasma displayed substantial site-specific variation in relative abundance. These findings suggest that, while overall diversity remained relatively stable, particular taxa displayed heterogenous distributions across historical refugia. Collectively, our findings suggest that gut microbiomes of velvet worms are shaped more by environmental filtering within deadwood microhabitats than by host geographic differences, reflecting a combination of stochastic and niche-driven assembly processes. Impact Statement We present the first characterization of the gut microbiome in the saproxylic velvet worm Euperipatoides rowelli from temperate Australian forests. Despite marked host genetic divergence, microbial communities showed minimal spatial structuring, suggesting substantial environmental filtering within deadwood microhabitats. The limited variation and transient taxa may indicate microbial associations are more likely environmentally acquired rather than host-specific. These findings broaden our understanding of the microbiome assembly in underexplored invertebrates and underscore the influence of ecological context over site-based spatial differentiation in shaping gut microbial communities. Competing Interest Statement The authors have declared no competing interest. Footnotes Data statement: Raw 16S rRNA sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number (PRJNA1337573). Metadata, QIIME2 pre-processing scripts, and R scripts for diversity and taxonomic analyses are available via GitHub at https://github.com/ILFExodus1414. Processed data files, including amplicon sequence variants (ASVs), taxonomic assignments, and diversity metrics, are provided as supplementary files. Abbreviations - DESeq2 - Differential Expression Sequencing 2; - EOD - Extra-Oral Digestion; - LMM - Linear Mixed-Effects Model; - rRNA - Ribosomal Ribonucleic Acid; - VW - Velvet Worm; - CLR - Center-Log Ratio; - PERMANOVA - Permutational Multivariate Analysis; - PERMADISP - Permutational Multivariate Analysis of Dispersion; - NMDS - Non-Metric Multidimensional Scaling; - PCoA - Principal Coordinate Analysis