Molecular phylogeny of historical micro-invertebrate specimens usingde novosequence assembly

Abstract

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

A broadly resolved molecular phylogeny of New Zealand cheilostome bryozoans as a framework for hypotheses of morphological evolution

“…Trimmed reads were de novo assembled with SPAdes 3.13 (Bankevich et al, 2012) using k-mers of 21, 33, 55, 77, 99 and 127. The mitogenome and rRNA operon of each sample were identified separately with blastn (Altschul et al, 1990) using blast+ against a database constructed from broadly sampled cheilostome sequences already deposited in NCBI (Orr et al, 2020). An E-value of 1.00e-185 and maximum target sequence of 1 were used to filter any blast hits of non-cheilostome origin.…”

“…Eight published (Orr et al, 2019a(Orr et al, , 2020 New Zealand samples (BLEED 48, 104, 127, 196, 344, 694, 1267 and1687) were included in the subsequent workflow to bring the total number of samples to 229. Further, the mitogenomes and rRNA operons of 38 non-New Zealand bryozoans (Orr et al, 2020), were aligned with our samples to compile a broader cheilostome ingroup and ctenostome outgroup taxon sample.…”

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 9, 2020. ; https://doi.org/10.1101/2020.12.08.415943 doi: bioRxiv preprint al., 2012) using k-mers of 21, 33, 55, 77, 99 and 127. The mitogenome and rRNA operon of each sample were identified separately with blastn (Altschul et al, 1990) using blast+ against a database constructed from broadly sampled cheilostome sequences already deposited in NCBI (Orr et al, 2020). An E-value of 1.00e-185 and maximum target sequence of 1 were used to filter any blast hits of non-cheilostome origin.…”