Phylogenetics of Tachinidae (Diptera) emphasizing sub-family structure

1. Catharosia sp. (Ph) 55 Hyphantrophagavirilis (Ex) 46 39 Strongygaster sp. (Ta) 63 57 Lespesia aletiae (Ex) 76 Phasia sp. (Ph) 54 76 Blondeliahyphantriae (Ex) Lespesiaaletiae (Ex) Trichopoda sp. (Ph) 40 35 Gymnosoma (Ph) 89 Catharosia sp. (Ph) Hyphantrophaga virilis (Ex) Thelariaamericana (De) Ptilodexia conjuncta (De) Cylindromyiabiontata (Ph) 46 50 3 32 Tachinomyianigricans (Ex) 46 Voria ruralis (De) Thelaria americana (De) Epalpussignifer (Ta) 94 99 Ptilodexia conjuncta (De) 54 13 Panzeria ampellus (Ta) 48 Hyphantrophaga virilis (Ex) Uramya sp. (De) 86 Cylindromyiabiontata (Ph) 26 16 Campylochaetasemiothisae (De) 92 10 Siphonaplusiae (Ta) Siphona plusiae (Ta) 33 100 Panzeriaampellus (Ta) 14 100 Ceracia denata (Ta) Cuterbra sp. (Oe) Lespesiaaletiae (Ex) Epalpussignifer (Ta) 100 2 Uramya sp. (De) 90 11 Winthemiinisinuata (Ex) Ceraciadenata (Ta) 100 92 Blondelia hyphantriae (Ex) Winthemiinisinuata (Ex) 23 Ptilodexia conjuncta (De) Tachinomyianigricans (Ex) 80 99 Trichopoda sp. (Ph) Lespesiaaletiae (Ex) 71 98 6 100 8 Hyphantrophagavirilis (Ex) Gymnosomanitens (Ph) 100 Blondeliahyphantriae (Ex) 14 17 Strongygaster sp (Ta) 88 12 Pollenia sp (Ca) Winthemiini sinuata (Ex) Helicobia sp. (Sa) 20 Cuterbra sp. (Oe) 10 Ceracia denata (Ta) SiphonaPlusiae (Ta) 3 Cylindromyia binotata (Ph) LuciliasericataOu (Ca) Lucilia sericata (Ca) 27 Helicobia sp. (Sa) Pollenia sp. (Ca) 23 Macroychia sp. (Sa) 100 Macroychia sp. (Sa) Musca.domestica (Mu) Campylochaetasemiothisae (De) Siphona plusiae (Ta) Uramya sp. (De) 44 Voria ruralis (De) 63 Strongygaster sp. (Ta) Cylindromyia binotata (Ph) 99 0 Thelaria americana (De) Voria ruralis (De) 18 5 Musca domestica (Ou) 25 Campylochaeta semiothisae (De) 13 Blondeliahyphantriae (Ex) Ceraciadentata (Ta) Ptilodexia conjuncta (De) 9 Thelaira americana (De) 2 10 Uramya sp. (De) 17 Camplochaeta semiothisae (De) Hyphantrophagavirilis (Ex) Campylochaeta semiothisae (De) 13 12 Thelairaamericana (De) 2 3 Ptilodexia conjuncta (De) Winthemiinisinuata (Ex) Epalpus signifer (Ta) Catharosia sp. (Ph) 39 5 0 Panzeria ampellus (Ta) 24 48 Trichopoda sp. (Ph) Gymnosoma fuliginosa (Ph) 7 33 Trichopoda sp. (Ph) 13 11 Voria ruralis (De) Gymnosomanitens (Ph) Catharosia sp. (Ph) 90 28 Strongygaster sp. (Ta) Cylindromyiabiontata (Ph) 10 58 Trichopoda sp. (Ph) 37 Voria ruralis (De) Campylochaetasemiothi (De) Siphona Plusiae (Ta) 25 Pollenia sp. (Ca) 22 Ceracia dentata (Ta) Blondeliahyphantriae (Ex) 14 1 Panzeriaampellus (Ta) Macronychia sp. (Sa) 4 25 9 Helicobia sp. (Sa) Cylindromyia binotata (Ph) Panzeria ampellus (Ta) 8 62 40 Epalpus signifer (Ta) 41 17 99 Cochliomyiamacellaria (Ca) 9 Cylindromyiaeuchenor (Ph) Epalpus signifer (Ta) 99 Ceracia Denata (Ta) Lucilia sericata (Ca) 24 39 Siphona plusiae (Ta) Uramya sp. (De) 26 Musca domestica (Mu) 69 Winthemiini sinuata (Ex) Thelaira americana (De) Phasia sp. (Ph) 26 8 Pollenia sp. (Ca) 48 47 Panzeriaampellus (Ta) Strongygaster sp. (Ta) 14 Cuterebra sp. (Oe) Epalpus signifer (Ta) Trichopoda sp. (Ph) 61 Tachinomyia nigricans (Ex) Epalpus Signifer (Ta) 99 Pollenia sp. (Ca) 7 28 Lespesia aletiae (Ex) 89 Tachinomyianigricans (Ex) Lucilia sericata (Ca) Blondelia hyphantriae (Ex) 51 51 Lespesiaaletiae (Ex) Calliphoranigribarbis (Ca) 38 22 Hyphantrophaga virilis (Ex) 44 60 Hyphantrophaga virilis (Ex) 36 23 Cuterebra sp. (Oe) Helicobia sp. (Sa) 16 Winthemiini sinuata (Ex) Tachinomyianigricans (Ex) 98 Macronychia sp. (Sa) Pollenia sp. (Ca) Ptilodexiaconjuncta (De) 21 Musca domestica (Mu) Cochliomyia macellaria (Ca) Helicobia sp. (Sa) 35 Helicobia sp. (Sa) 94 Macroychia sp. (Sa) 32 Macronychia sp. (Sa) MuscaDomestica (Mu) Musca domestica (Mu) Phylogenetics of Tachinidae (Diptera) emphasizing sub-family structure Figure 2 – Consensus tree from all five genes combined with posterior probabilities Parasitoid flies in the family Tachinidae (Diptera) play important ecological roles in natural and managed environments and hold great promise for biological control. However, study of tachinids and its applications is complicated by a lack of basic knowledge of their biology and taxonomy. The almost10,000 described species of Tachinidae have been difficult to classify with traditional morphological means due to their rapid evolution and a high degree of homoplasy [2,4]. Using molecular DNA techniques we hope to gain a better understanding of the relationships and evolution of this important family of parasitoids. Phassiinae Tachininae Dexiinae Figure 1 – ML trees showing phylogenetic relationships based on each of the five nuclear genes. Conclusions Thanks to Bridget Severt for her work with the TPI gene. Thanks to Jim O’Hara for providing the bot fly. Funding was provided by the Wright State University Biology Awards for Research Excellence (BARE) grant. Table 1 – Genes included for each taxon. Blue boxes indicate sequences that were obtained for each taxon. Exoristinae Tachinids are typically classified into four subfamilies that are generally loosely defined on the basis of several characters. The Tachininae possess a uterus and most have larvae that ambush their hosts. The Exoristinae generally have a setoseprosternum, and the Phasiinae typically parasitize Hemiptera. The Dexiinae possess the only unique morphological feature, a hinged aedeagus [2,4]. Relationships between subfamilies are unclear and their monophyly is questionable. Given the high degree of homoplasy in most morphological characters, DNA sequence data may be helpful in clarifying the phylogeny of Tachinidae. This study uses five nuclear genes (including 18S,28S, CAD, EF1a, TPI for a total of 5100bp) to construct a skeletal phylogeny of tachinids and to provide a framework for future systematic work on Tachinidae. Daniel J Davis and John O. Stireman III Acknowledgements Preliminary results: Monophyly of Tachinidae is strongly supported. This finding confirms the results of previous analysis [1,3]. Pollenia, a Calliphorid genus of earthworm parasitiods, was found to be the closest sister group to Tachinidae. This suggests that tachinids may have arisen from a parasitoid calliphorid ancestor. However, another likely relative Rhinophoridae (isopod parasitoids) is missing from this analysis. There exists a basal split between Exoristinae + Tachininae and Dexiinae+Phasiinae. The Exoristinae and Tachininae are generally monophletic (see 5,6), but Dexiinae (mostly monophyletic, see 6) may arise from Phasiinae (which are paraphyletic). Strongygaster appears within the Phasiinae. This taxon was originally considered a Phasiinae, but was moved to Tachininae since it parasitizes Coleoptera and possesses a uterus. The placement of Strongygaster may have to be reevaluated. Ceraciadentatais placed within the Exoristinae instead of Tachininae. Ceraciashares the reproductive strategy of laying unincubated eggs upon its host with Winthemiini, a Exoristinae. The placement of Ceraciamay have to reevaluated. There is weak support for Cylindromyia + Voria, that is likely due to long branch attraction. Further sequence data will likely support a monophyletic Dexiinae. Introduction Construct a phylogenetic framework for the Tachinidae using multiple nuclear genes (18S, 28S, CAD, EF1a, and TPI) . Assess the monophyly of the Tachinidae and its four subfamilies. Identify the sister-group to Tachinidae Clarify positions of problematic taxa including Strongygaster and Ceracia Objectives Results Department of Biological Sciences, Wright State University Tachinds from 5 different tribes of each of the four subfamilies were sampled along with 5 taxa from the closely related taxa from the families of Sarcophagidae, Calliphoridae, and Oestridae. DNA was extracted using QiagenPuregene Core Kit. Each sample was amplified by using PCR for each gene (18S, 28S, CAD(1), CAD(2), EF1a, and TPI). Amplification was verified using gel electrophoresis. The PCR product was cleaned by using EXO SAP and then sent to the Arizona University Genetics Core for sequencing [3]. The sequences were assembled, edited, initially aligned with Codon Code Aligner. Three missing gene sequences for various taxa and all five sequences for MuscaDomesticawere obtainedfromGenBank. CAD, EF1a, and TPI were aligned in MEGA using Muscle. 18S and 28S were aligned in RNAsalsa using structural constraints from RNAfold. Phylogenetic trees were constructed in MEGA using maximum likelihood with the best model for each gene. The genes were then combined into a concatenated set and a tree was constructed using Bayesian inference in Mr. Bayes. Results and Discussion References Using a large number of nuclear genes for a wide range of tachinid taxa, we have started to construct a phylogenetic framework for Tachinidae. The most useful genes for this level analysis were (in order) TPI, CAD, EF1α, 28S and 18S. Each individual gene gave us mixed results but a Bayesian consensus tree is beginning to provide useful insights into relationships within Tachinidae. The slowly evolving 18S gene appears highly homoplasious and may have been detrimental to the analysis. We are adding more nuclear protein coding genes and improving coverage to obtain a robust and reliable phylogenetic framework for Tachinidae. Materials and Methods [1] Kutty, S. A., Pape, T., Wiegmann, B.M., Meier, R. (2010). Molecular phylogeny of the Calyptrate (Diptera: Cyclorrphapha) with an emphasis on the superfamilyOestroidea and the position of Mystachinobiidae and McAlpine’s fly. Systematic Entomology. 35, 614-635. [2] McAlpine, J.F., Peterson, B.V., Shewel, G.E., Teskey, H.J., Vockeroth, J.R., Wood, D.M. (1987). Maual of NearcticDiptera Volume 2. Research Branch, Agriculture Canada. 1147-1158. [3] Stireman, J.O. (2002). Phylogenetic relationships of tachinid flies in subfamily Exoristinae (Tachinidae: Diptera) based on 28S rDNA and elongation factor1-α. Systematic Entomology. 27, 409-435. [4] Stireman, J.O., O’Hara, J.E., Wood, M.D. (2006). Tachinidae: evolution, behavior, and ecology. Annual Review of Entomology. 51, 525-555. 28S CAD 18S Legend Ta - Tachininae Ex - Exoristinae De - Dexiinae Ph – Phassiinae Ca – Calliphoridae Sa – Sarcophagidae Oe – Oestroidae Mu - Mucidae EF1α TPI 3 3 1 6 5 4 2 Bruce Marlin, bugguide.net Texhoma, bugguide.net Muhammad MandiKiram, Wikipeida.com