Accurate assessments of biodiversity are crucial to advising ecosystem-monitoring programs and understanding ecosystem function. Nevertheless, a standard operating procedure to assess biodiversity accurately and consistently has not been established. This is especially true for meiofauna, a diverse community (>20 phyla) of small benthic invertebrates that have fundamental ecological roles. Recent studies show that metabarcoding is a cost-effective and time-effective method to estimate meiofauna biodiversity, in contrast to morphological-based taxonomy. Here, we compare biodiversity assessments of a diverse meiofaunal community derived by applying multiple taxonomic methods based on comparative morphology, molecular phylogenetic analysis, DNA barcoding of individual specimens, and metabarcoding of environmental DNA. We show that biodiversity estimates are strongly biased across taxonomic methods and phyla. Such biases affect understanding of community structures and ecological interpretations. This study supports the urgency of improving aspects of environmental high-throughput sequencing and the value of taxonomists in correctly understanding biodiversity estimates.

Summary Stone surfaces are extreme environments that support microbial life. This microbial growth occurs despite unfavourable conditions associated with stone including limited sources of nutrients and water, high pH and exposure to extreme variations in temperature, humidity and irradiation. These stone‐dwelling microbes are often resistant to extreme environments including exposure to desiccation, heavy metals, UV and Gamma irradiation. Here, we report on the effects of climate and stone geochemistry on microbiomes of Roman stone ruins in North Africa. Stone microbiomes were dominated by Actinobacteria, Cyanobacteria and Proteobacteria but were heavily impacted by climate variables that influenced water availability. Stone geochemistry also influenced community diversity, particularly through biologically available P, Mn and Zn. Functions associated with photosynthesis and UV protection were enriched in the metagenomes, indicating the significance of these functions for community survival on stones. Core members of the stone microbial communities were also identified and included Geodermatophilaceae, Rubrobacter, Sphingomonas and others. Our research has helped to expand the understanding of stone microbial community structure and functional capacity within the context of varying climates, geochemical properties and stone conditions.

The phylogenetic relationships among certain groups of gastropods have remained unresolved in recent studies, especially in the diverse subclass Opisthobranchia, where nudibranchs have been poorly represented. Here we present the complete mitochondrial genomes of Melibe leonina and Tritonia diomedea (more recently named T. tetraquetra), two nudibranchs from the unrepresented Cladobranchia group, and report on the resulting phylogenetic analyses. Both genomes coded for the typical thirteen protein-coding genes, twenty-two transfer RNAs, and two ribosomal RNAs seen in other species. The twelve-nucleotide deletion previously reported for the cytochrome oxidase 1 gene in several other Melibe species was further clarified as three separate deletion events. These deletions were not present in any opisthobranchs examined in our study, including the newly sequenced M. leonina or T. diomedea, suggesting that these previously reported deletions may represent more recently divergent taxa. Analysis of the secondary structures for all twenty-two tRNAs of both M. leonina and T. diomedea indicated truncated d arms for the two serine tRNAs, as seen in some other heterobranchs. In addition, the serine 1 tRNA in T. diomedea contained an anticodon not yet reported in any other gastropod. For phylogenetic analysis, we used the thirteen protein-coding genes from the mitochondrial genomes of M. leonina, T. diomedea, and seventy-one other gastropods. Phylogenetic analyses were performed for both the class Gastropoda and the subclass Opisthobranchia. Both Bayesian and maximum likelihood analyses resulted in similar tree topologies. In the Opisthobranchia, the five orders represented in our study were monophyletic (Anaspidea, Cephalaspidea, Notaspidea, Nudibranchia, Sacoglossa). In Gastropoda, two of the three traditional subclasses, Opisthobranchia and Pulmonata, were not monophyletic. In contrast, four of the more recently named gastropod clades (Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia) were all monophyletic, and thus appear to be better classifications for this diverse group.

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