Download Te gades

Welcome to Molecular Systema0cs Lab! Russell Meister Molecular Systema0cs 2014 What will we do? •  Common molecular systema0cs lab tools –  Extrac0on, amplifica0on, sequencing, cloning… •  DNA and RNA, sorry, no protein work •  We will focus on animal (insect) mitochondrial sequences •  Have lots of bench top fun! What is Required •  Keep a lab notebook •  Be present in the labs and recep0ve •  One minor homework exercise •  Be a liTle flexible with lab 0me •  Ask ques0ons if you need clarifica0on! • Stop and ask ques0ons! Te#gades: Mul0copy Mitochondrial Madness! (gene)
Photo by Chris Simon Photo by Juan Emilio Background Phylogeny of Family Cicadidae •  Te#gades is a genus of cicadas in the subfamily TeZgadinae Photo by Jose Gerstle Figure by Kathy Hill Background Current Phylogeny of TeZgades Tettigades.auropilosa.12.CL.Alvaro
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Tettigades.chilensis.12.CL.Alvaro.2
Garli GTR + I + G 718bp of COI No par00ons 12 November 2013 Tettigades.ulnaria.06.CL.BI.LGL.06
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Tettigades.ulnaria.Carlos.nr.Pichilemu
Tettigades.procera.06.CL.BI.LGL.02
Tettigades.undata.06.CL.Bi.CAB.01
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Tettigades.???.06.CL.Bi.LGL.01
Tettigades.unipuncta.06.CL.AR.CRB.24.2
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Tettigades.unipuncta.06.CL.AR.TPB.25.3
Tettigades.???.06.CL.BI.LGL.04
Chilecicada.occidentis.13.CL.Campana
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Background •  Te#gades is a genus of cicadas in the subfamily TeZgadinae •  Nothing par0cularly interes0ng (although they are quite fuzzy) in the phenotype… Photo by someone Zammara smaragdina Photo by Andreas Kay Lembeja paradoxa Photo by Kathy Hill Acu8cephala alipuncta Photo by Timothy Emery Background •  Te#gades is a genus of cicadas in the subfamily TeZgadinae •  Nothing par0cularly interes0ng (although they are quite fuzzy) in the phenotype… •  …but the mitochondria are quite interes0ng Why Te#gades? •  Cicadas posses two obligate, bacterial endosymbionts, Hodgkinia cicadicola and Sulcia muelleri A
Microscopy by James Van Leuven •  These endosymbionts are housed in a host made structure called the bacteriome Purple = host. Green = Sulcia Red = Hodgkinia Why Te#gades? A
•  Te#gades, however, has two dis0nct, independently evolving lineages of Hodgkinia* B
Microscopy by James Van Leuven Purple = host Green = Sulcia Blue = Hodgkinia 1 Yellow = Hodgkina 2 Why Te#gades? •  *Not all Te#gades species studied has this double lineage of Hodgkina •  To look at this phenomena phylogene0cally, we decided to sequence the cicadas harboring these bacteria •  Thus we return to the Te#gades mitochonria Mitochondria in General High copy No recombina0on Maternally inherited Faster rates of evolu0on Commonly used in systema0cs/iden0fica0on (barcode region) •  Easily extracted •  “Universal” primers • 
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Te#gades COI Barcode Region •  Direct sequencing (no cloning) was giving some unclear results Te#gades COI Barcode Region •  Direct sequencing (no cloning) was giving some unclear results •  Cloning and sequencing seemed to show promise… Clone sequences •  Cloning and sequencing seemed to show promise… Te#gades ulnaria cloned COI sequences Te#gades Sequencing •  Direct sequencing (no cloning) was giving some unclear results •  Cloning and sequencing seemed to show promise… •  Collaborators did some WGS on the bacteria which produced host mitochondrial genomes, problems ensued WGS sequences vs clones •  Collaborators did some WGS on the bacteria which produced host mitochondrial genomes, problems ensued Te#gades ulnaria cloned COI sequences Conclusion •  Te#gades species appear to have a high number of nuclear copies of COI •  Unfortunately, some of the copies look func0onal, but many are pseudogenes •  This presents a very interes0ng project Ques0ons? •  If not, off to the LAB! Clone sequences Te#gades ulnaria Te#gades undata WGS sequences vs clones Te#gades ulnaria Te#gades undata