BackgroundUnderstanding the phylogenetic relationships among species is one of the main goals of systematic biology. Simultaneously, credible phylogenetic hypotheses are often the first requirement for unveiling the evolutionary history of traits and for modelling macroevolutionary processes. However, many non-model taxa have not yet been sequenced to an extent such that statistically well-supported molecular phylogenies can be constructed for these purposes. Here, we use a genome-skimming approach to extract sequence information for 15 mitochondrial and 2 ribosomal operon genes from the cheilostome bryozoan family, Adeonidae, Busk, 1884, whose current systematics is based purely on morphological traits. The members of the Adeonidae are, like all cheilostome bryozoans, benthic, colonial, marine organisms. Adeonids are also geographically widely-distributed, often locally common, and are sometimes important habitat-builders.ResultsWe successfully genome-skimmed 35 adeonid colonies representing 6 genera (Adeona, Adeonellopsis, Bracebridgia, Adeonella, Laminopora and Cucullipora). We also contributed 16 new, circularised mitochondrial genomes to the eight previously published for cheilostome bryozoans. Using the aforementioned mitochondrial and ribosomal genes, we inferred the relationships among these 35 samples. Contrary to some previous suggestions, the Adeonidae is a robustly supported monophyletic clade. However, the genera Adeonella and Laminopora are in need of revision: Adeonella is polyphyletic and Laminopora paraphyletically forms a clade with some Adeonella species. Additionally, we assign a sequence clustering identity using cox1 barcoding region of 99% at the species and 83% at the genus level.ConclusionsWe provide sequence data, obtained via genome-skimming, that greatly increases the resolution of the phylogenetic relationships within the adeonids. We present a highly-supported topology based on 17 genes and substantially increase availability of circularised cheilostome mitochondrial genomes, and highlight how we can extend our pipeline to other bryozoans.

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25Resolution of relationships at lower taxonomic levels is crucial for answering many 26 evolutionary questions, and as such, sufficiently varied species representation is vital. This 27 latter goal is not always achievable with relatively fresh samples. To alleviate the difficulties 28 in procuring rarer taxa, we have seen increasing utilization of historical specimens in building 29 molecular phylogenies using high throughput sequencing. This effort, however, has mainly 30 focused on large-bodied or well-studied groups, with small-bodied and under-studied taxa 31 under-prioritized. Here, we present a pipeline that utilizes both historical and contemporary 32 specimens, to increase the resolution of phylogenetic relationships among understudied and 33 small-bodied metazoans, namely, cheilostome bryozoans. In this study, we pioneer 34 sequencing of air-dried bryozoans, utilizing a recent library preparation method for low DNA 35 input. We use the de novo mitogenome assembly from the target specimen itself as reference 36 for iterative mapping, and the comparison thereof. In doing so, we present mitochondrial and 37 ribosomal RNA sequences of 43 cheilostomes representing 37 species, including 14 from 38 historical samples ranging from 50 to 149 years old. The inferred phylogenetic relationships 39 of these samples, analyzed together with publicly available sequence data, are shown in a 40 statistically well-supported 65 taxa and 17 genes cheilostome tree. Finally, the 41 methodological success is emphasized by circularizing a total of 27 mitogenomes, seven from 42 historical cheilostome samples. Our study highlights the potential of utilizing DNA from 43 micro-invertebrate specimens stored in natural history collections for resolving phylogenetic 44 relationships between species. 45 46