Discipline: Biological Sciences
Subcategory: Ecology
Session: 1
Room: Exhibit Hall A
Merai Dandouch - California State University Channel Islands
Co-Author(s): JA. Ballesteros, UW - Madison, Wisconsin, Madison; EV Setton, UW - Madison, Wisconsin, Madison; G Gainett, UW - Madison, Wisconsin, Madison; C Arango, UW - Madison, Wisconsin, Madison; G Brenneis, UW - Madison, Wisconsin, Madison; G Dilly, CSU Channel Islands, California, Camarillo; S McAtee, CSU Channel Islands, California, Camarillo; C Williamson, CSU Channel Islands, California, Camarillo; L McIntyre, UW - Madison, Wisconsin, Madison; R Moran, UW - Madison, Wisconsin, Madison; A Soler-Membrives,UW - Madison, Wisconsin, Madison; E Cano-Sánchez, UW - Madison, Wisconsin, Madison; P López-González, UW - Madison, Wisconsin, Madison; WC Wheeler, UW - Madison, Wisconsin, Madison; PP Sharma, UW - Madison, Wisconsin, Madison;
Pycnogonida (sea spiders) are an ancient class of marine arthropods that appeared early in the fossil record, likely dating to the Late Cambria/Paleozoic era; they envelop the sister group to the remaining subphylum Chelicerata of Arthropods (Regier 2010). However, due to the inaccessibility of some minute lineages, no phylogenetic works have yet sampled all families. Previous efforts based on a handful of genes have repeatedly yielded unstable tree topologies. In this collaboration, we sequenced transcriptomes from multiple Pycnogonida species, and we present a novel resolved molecular phylogenetic tree of the class. A set of targeted sequencing probes from Pycnogonum and Anoplodactylus were used as references for orthologous loci to 56 exons, 101 ultra-conserved elements (UCE), 15 mitochondrial genes, and ITS regions to define the relationship of 90 de novo Pycnogonida transcriptomes. The 56 exon loci were aligned using a stand-alone tBLASTn package from BLAST+ (ver 2.9) to published sequences (Camacho 2013). Mitochondrial, UCE, and ITS regions were also matched to relevant existing sequences from NCBI. A major challenge lay in the fact that for some loci, the same taxon exon represented by multiple sequences, leading to the difficulty in resolving incomplete exon transcripts. To address this issue, I worked at CSUCI to design criteria for nucleotide similarity cut-offs between unknown taxa and reference sequences, increasing specificity and resolving duplicate transcripts. We combined the 90 unknown Pycnogonid TRINITY transcriptome into a single reference library and looped each exon reference sequence against this. We retrieved optimal alignments at each locus by using a minimum e-value score 1e-10 and chose an overlapping reference region with ≥ 30 bp reads and ≥ 6 taxa per loci using in-house PERL scripts, excel-value sorting, and filtering. The FASTA output files we generated were used to build Multiple Sequence Alignment input files for phylogenetics with convergence achieved using more than 200 loci. Resolution of the new converged phylogenetic tree showed the family Austrodecidae was reconstructed as the sister group to the remaining sea spiders. Additionally, the obscure family Rhynchothoracidae was recovered as the sister group of Pycnogonidae. Beyond sea spider relationships, comparative assessments of gene trees were used to identify the most informative loci, further supporting research into design of targeted sequence capture for Paleozoic taxa studies. Regier, J. C., Shultz, J. W., Zwick, A., Hussey, A., Ball, B., Wetzer, R., … Cunningham, C. W. (2010, February 10). Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature. Camacho C. BLAST+ Release Notes. 2013 Mar 12 [Updated 2019 Apr 1]. In: BLAST® Help [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2008-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK131777/.
Funder Acknowledgement(s): NSF Arthropod Taxonomy Grant
Faculty Advisor: Geoff Dilly, geoffrey.dilly@csuci.edu
Role: I was part of a research team at CSUCI that combined the 90 unknown Pycnogonid TRINITY transcriptome into a single reference library. To retrieve optimal alignments at each locus from the TRINITY reference library, I used a minimum e-value score 1e-10 and chose an overlapping region to reference sequences with ? 30 bp reads and ? 6 taxa per loci using in-house PERL scripts and excel-value sorting and filtering to find best hits.