Deep-sea shrimp belonging to the family Sergestidae provide a unique system for studying the evolution of bioluminescence.  Most species within the family possess autogenic bioluminescent photophores in one of three distinct forms: lensed photophores, non-lensed photophores, or internal organs of Pesta.  This morphological diversity across Sergestidae has resulted in recent major taxonomic revisions, dividing the two major genera (Sergia and Sergestes) into 15.  Here, we capitalize on molecular data to construct an updated genus-level phylogeny of sergestid shrimp.  DNA was successfully extracted from approximately 87 individuals belonging to 13 of the 15 newly proposed genera.  We implemented a “genome skimming” approach, allowing us to capture mitochondrial genomic data across 19 Sergestidae species.  Additional individuals have been incorporated into the phylogeny through Sanger sequencing of both nuclear (H3, NAK) and mitochondrial (16S and COI) genes.  The resulting molecular phylogeny is compared with previous morphological trees with specific attention to genus-level relationships.  The -sergestes group was rendered non-monophyletic, and the -sergia group was recovered as monophyletic.  Ancestral state reconstructions of light organ type indicate the organs of Pesta is the ancestral state for the family. Non-lensed photophores evolved once across the -sergia group, but were later lost in the deepest living genus, Sergia.  Lensed photophores also evolved once within the genera Prehensilosergia, Lucensosersgia, and Challengerosergia.  Our findings identify preliminary patterns across light organ type and species’ depth distributions, however future research that incorporates finer-scale depth data and more species is needed to confirm our findings. 

PHYLOGENETIC TREE CONSTRUCTION

• Specimens were collected via mid-water trawls in the Gulf of Mexico and Florida Straits. 
• Abdominal muscle tissue was removed from the specimens for genomic DNA extraction using either Qiagen DNEasy Blood and Tissue Kits or a Phenol Chloroform protocol.  
• PCR protocols were employed to target H3, NaK, COI, and 16S for Sanger sequencing. 
  • PCR products were sequenced through the company Genewiz in Boston, MA, USA.
• Genome skimming was employed to extract entire mitochondrial genomes. 
  • Extracted genomic DNA was first sonicated to achieve chromosomal fragmentation of ~200 bps.  
  • Libraries were prepared using NEB Next Ultra II Library Prep kits.
  • Libraries were sequenced through the company Genewiz in Boston, MA, USA on an Illumina HiSeq 3000/4000.
  • Raw NGS sequence data was assembled into complete mitochondrial genomes with NOVOplasty assembling tool, using 16s / COI seed sequences of closely related species acquired through Sanger sequencing, or published in NCBI's online database, Genbank.  
  • Assembled mtGenomes were annotated using MITOS online web tool.
• Genetic sequence data handling, cleaning, and organization was conducted using the bioinformatics software, Geneious Prime. 
  • Raw Sanger sequence data from Genewiz was assembled, cleaned, and trimmed.  NGS sequence data from MITOS annotation was cleaned and trimmed
  • Single-gene alignments of Sanger sequencing and NGS genes were created using MAFFT alignment tool in Geneious.
• Single-gene trees were constructed from Genious alignments using IQTREE phylogenetic software to screen for contaminated sequences and genes lacking phylogenetic signal.
• Final single-gene alignments were concatenated into single multi-gene alignment using Geneious Prime.
• Final multi-gene trees were constructed from multi-gene alignment using IQTREE and MrBayes phylogenetic software.

ANCESTRAL STATE RECONSTRUCTION

• Multi-gene tree was imported to Mesquite software and pruned to include only one individual per species.
• Coding matrix created to assign a number to each species depending on light organ type.
  • 0 = Light organ absent
  • 1 = Organ of Pesta
  • 2 = Non-lensed Photophore
  • 3 = Lensed Photopore
• Maximum likelihood and maximum parsimony ancestral state reconstruction created in Mesquite.