Transcriptomes of three species of Tipuloidea (Diptera, Tipulomorpha) and implications for phylogeny of Tipulomorpha

Abstract

Tipulomorpha has long been a problematic taxon in terms of familial composition, phylogenetic relationships among families and position relative to other 'lower' Diptera. Whole-transcriptome shotgun sequencing provides a powerful basis for phylogenetic studies. We performed de novo transcriptome sequencing to produce the first transcriptome datasets representing the families Pediciidae, Limoniidae and Cylindrotomidae using high-throughput sequencing technologies. We assembled cDNA libraries for Pedicia vetusta (Alexander) (Pediciidae), Rhipidia sejuga Zhang, Li and Yang (Limoniidae) and Liogma simplicicornis Alexander (Cylindrotomidae). Using the Illumina RNA-Seq method, we obtained 28,252, 44,152 and 44,281 unigenes, from the three respective species. Based on sequence similarity searches, 12,475 (44.16%), 20,334 (46.05%) and 17,478 (39.47%) genes were identified. Analysis of genes highly conserved at the amino acid sequence level revealed there were 1,709 single-copy orthologs genes across the analyzed species. Phylogenetic trees constructed using maximum likelihood (ML) based on the 1,709 single-copy orthologs genes indicated that the relationship between the four major infraorders of lower Diptera was: Culicomorpha + (Tipulomorpha + (Psychodomorpha + (Bibionomorpha + Brachycera))). Trichoceridae belongs within Tipulomorpha as the sister-group of Tipuloidea. Highly supported relationships within the Tipuloidea are Pediciidae + (Limoniidae + (Cylindrotomidae + Tipulidae)). Four-cluster likelihood mapping was used to study potential incongruent signals supporting other topologies, however, results were congruent with the ML tree.

Fig 1. Phylogenetic hypotheses of lower Diptera relationships from previous analyses.

(A) Hennig [4]. Phylogenetic hypothesis of lower Diptera relationships based primarily on imaginal characters. (B) Wood & Borkent [14]. Cladogram showing relationships between the families of the Nematocera. (C) Oosterbroek & Courtney [9]. Cladogram of the families of nematocerous Diptera. (D) Bertone et al. [10]. 1) Parsimony analysis of combined nuclear ribosomal (28S) and protein-coding (CAD, PGD and TPI) genes (bootstrap values (BV) shown above branches). 2) Majority rule consensus of Bayesian Markov chain Monte Carlo (posterior probabilities (PP) shown above branches and bootstrap values shown below branches). (E) Wiegmann et al. [16]. Combined molecular phylogenetic tree for Diptera (BV shown above branches shown above or below branches). (F) Beckenbach [17]. 1) Mitochondrial phylogenetic tree of major groups of Diptera derived from a Bayesian analysis of all major mitochondrial protein coding genes (PP shown above branches). 2) Bayesian mitochondrial tree using codon positions 1 and 2 for cox1–3, cytb, and atp6 genes, and all alignable sites for the ribosomal genes (PP shown above branches). (G) Lambkin et al. [18]. The Bayes combined majority rule consensus tree (PP shown above branches).

Fig 2. Sequence-length distribution of unigenes.

The X-axis represents the length range bins; the Y-axis is the amount of transcripts present.

Fig 3. Results summary for sequence-homology search against NCBI NR database.

Fig 4. Gene ontology (GO) assignments for the three species.

Results are summarized under three main GO categories: biological process, cellular component and molecular function. The left Y-axis represents the percentage of a specific category of genes in each main category. The right Y-axis represents the number of genes in the same category.

Fig 5. COG functional classification for the three species.

Unigenes of each species with significant homologies in the COG database were classified into 25 COG categories.

Fig 6. KOG functional classification for the three species.

Unigenes of each species with significant homologies in the KOG database were classified into 25 KOG categories.

Fig 7. Phylogenetic tree.

The tree was inferred through a maximum-likelihood analysis of amino acid sequence data of 1,709 single-cope orthologs genes. Branch lengths correspond to the number of changes on that branch. Numbers adjacent to each node are BV.

Fig 8. Phylogenetic tree.

The tree was inferred through a maximum-likelihood analysis of amino acid sequence data of (A) 73 genes involved in ATP binding. (B) 13 genes involved in receptor activity. (C) 43 genes involved in the biological process associated with wing. (D) 7 genes involved in the biological process associated with olfactory. Branch lengths correspond to the number of changes on that branch. Numbers adjacent to each node are BV.

Fig 9. Results of Four-cluster Likelihood Mapping as 2D simplex graphs.

(A) Question 1. (B) Question 2.