DOI: 10.5061/dryad.n5tb2rbt1
title: Amino acids (AA) all genes for: Beyond Drosophila: resolving the rapid radiation of schizophoran flies with phylotranscriptomics

description: Background: The largest radiation of animal life since the end Cretaceous
 extinction event 66 million years ago is that of schizophoran flies: a
 third of fly diversity including Drosophila lab fruit flies, house flies,
 and many other well and poorly known true flies. Rapid diversification has
 hindered previous attempts to elucidate the phylogenetic relationships
 among major schizophoran clades. A robust phylogenetic hypothesis for the
 major lineages containing these 55,000 described species would be critical
 to understand the processes that contributed to the diversity of these
 agriculturally, medically, and forensically important flies. We use
 protein encoding sequence data from transcriptomes, including 3,145 genes
 from 70 species, representing all superfamilies, to improve the resolution
 of this previously intractable phylogenetic challenge. Results: Our
 results support a paraphyletic acalyptrate grade including a monophyletic
 Calyptratae and the monophyly of half of the acalyptrate superfamilies.
 The primary branching framework of Schizophora is well supported for the
 first time, revealing the primarily parasitic Pipunculidae and
 Sciomyzoidea s.l. as successive sister groups to the remaining
 Schizophora. Ephydroidea, Drosophila’s superfamily, is the sister group of
 Calyptratae. Sphaeroceroidea has modest support as the sister to all
 non-sciomyzoid Schizophora. We define two novel lineages corroborated by
 morphological traits, the Modified Oviscapt Clade containing Tephritoidea,
 Nerioidea, and other families, and the Cleft Pedicel Clade containg
 Calyptratae, Ephydroidea, and other families. Support values remain low
 among a challenging subset of lineages, including Diopsidae. The placement
 of these families remained uncertain in both concatenated maximum
 likelihood and multi-species coalescent approaches Rogue taxon removal was
 effective in increasing support values compared with strategies that
 maximize gene coverage or minimize missing data. Conclusions: Dividing
 most acalyptrate fly groups into four major lineages is supported
 consistently across analyses. Understanding the fundamental branching
 patterns of schizophoran flies provides a foundation for future
 comparative research on the genetics, ecology, and biocontrol.
description: <b><i>Sample collection, preservation, and
 transcriptome sequencing</i></b> Novel transcriptome data for this
 manuscript originated from three sources, 1,000 Insect Transcriptome
 Evolution Project (1KITE), North Carolina State University (NCSU), and the
 National University of Singapore (NUS). The laboratory and data processing
 workflows were similar and compatible for data from all three
 sources. Generally, to preserve tissue for RNA sequencing, specimens were
 collected live into RNAlater and stored at -20 °C, or into 95% ethanol and
 stored at 80 °C. Their cuticle was broken to allow the preservative to
 penetrate the exoskeleton and enter the muscle tissue. Samples were
 examined in an ice bath under dissecting microscopes to verify vouchers
 and perform identifications based on museum comparisons and primary
 literature. Extractions were performed with the RNeasy kit (Qiagen,
 Valencia, CA) on thoracic tissue unless the flies were very small. New
 transcriptome samples underwent library preparation using the NEBNext (New
 England Biosciences, Ipswich, MA, USA) Ultra RNA Library Prep Kit for
 Illumina kit, following the manufacturers guidelines. RNA was bound to
 Agencourt AMPure XP Beads (Beckman Coulter, Inc., Brea, CA, USA) on a
 magnetic plate and the sample underwent a series of washes. A reverse
 transcription reaction was performed, followed by a PCR enrichment,
 yielding a size-selected non-directional cDNA library that was sequenced
 as paired-end reads on an Illumina system (Illumina, San Diego, CA, USA).
 Double indexes were used where possible to reduce sample misidentification
 during demultiplexing. Samples were multiplexed with up to eight taxa per
 lane on Illumina MiSeq and 22 taxa per lane on Illumina HiSeq. Double
 indexes were used where possible to reduce sample misspecification during
 demultiplexing. Read quality was checked with FastQC v. 0.11.5 to assess
 whether further trimming was necessary. Trimmomatic v. 0.32 was used to
 remove adapter contamination and low-quality sequences. Trinity v. 2.2 and
 2.4 were used to assemble the reads into contigs.
 <b><i>Orthology search and
 Alignment</i></b> We used an ortholog reference set
 comprising 3,145 gene single copy protein coding genes, termed
 “Mecopterida”. This set includes annotated genomes and transcriptomes from
 five reference species: <i>Drosophila melanogaster</i>,
 <i>Glossina morsitans</i>, <i>Aedes aegypti</i>,
 <i>Bombyx mori</i> (silkworm moth, an outgroup), and
 <i>Danaus plexippus</i> (monarch butterfly, an outgroup) from
 OrthoDB7. Orthograph was used to assign orthology to all target taxa using
 the relaxed reciprocal blast hit criterion and otherwise default
 settings. Stop
 codons were masked and each gene was aligned individually with the L-INS-i
 algorithm implemented in MAFFT v. 2.273 [42]. Outlier sequences were
 identified and realigned or removed. Ambiguously aligned positions were
 identified with Aliscore v. 2.0 and removed with Alicut v 2.1. Genes with
 no information content were identified with MARE v. 0.1.2 and removed.
 Pal2Nal v. 14 correlated nucleotides to the amino acid-based
 alignment.
description: See BeyondDrosophilaDataDryadReadme.txt file for exact
 explanation of each file. Alignments analyzed for
 this project:  1: Amino acids (AA) all genes
 2: AA Mare reduced 1130 genes 3: AA Mare
 reduced 1130 genes, Alistat reduced to 80% coverage by site
 4: Nucleotides all genes, 3rd codon positions removed (All codon
 positions remain in the file) 5: Nucleotides reduced
 1130 genes, 3rd codon positions removed (All codon positions remain in the
 file)