Franz Zhang et al Weevil Workshop 2016 Neotropical Entiminae Systematics evolution and beyond
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The document discusses research on the Neotropical Entiminae weevil subfamily, including a molecular phylogeny that showed the Exophthalmus genus complex is polyphyletic. Historical biogeography analysis found Caribbean island ancestry with jump dispersal between islands accounting for 25% of range evolution. Molecular profiling of gut contents identified host plant associations and multiple bacterial symbionts across weevil specimens using next-generation sequencing.
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Franz Zhang et al Weevil Workshop 2016 Neotropical Entiminae Systematics evolution and beyond
- 1. Neotropical Entiminae – Systematics, evolution and beyond Guanyang Zhang, Nico Franz, et. al. Arizona State University Biodiversity Knowledge Integration Center Hasbrouck Insect Collection School of Life Sciences 2016 International Weevil Meeting Oct 01, 2016
- 2. Taxonomy of the Exophthalmus genus complex
- 3. June, 2013 Cuba Dr. Robert (Bob) Anderson (Canadian Museum of Nature) Franklyn Cala Riquelme, Albert Deler Hernandez (Cuban arachnologist/coleopterist)
- 4. Entiminae (Broad-nosed weevils) “Eustylini” (sensu Franz, 2012) Tetrabothynus spectabilis (Klug, 1829) Tetrabothynus regalis Exophthalmus genus complex (Franz 2012)
- 6. Exophthalmus genus complex (EGC) 9 genera, 131 species; Caribbean and Neotropical mainland Exophthalmus Schoenherr, 1823 85 described species; Caribbean [~45 species] and Neotropical mainland [~40] Genera of EGC s.s. (Franz, 2012) [No. species] Chauliopleurus Champion, 1911 [4] Compsoricus Franz, 2012 [3] Diaprepes Schoenherr, 1823 [19] Exophthalmus Pachnaeus Schoenherr, 1826 [7] Rhinospathe Chevrolat, 1878 [2] Tropirhinus Schoenherr, 1823 [4] Tetrabothynus Labram & Imhoff, 1852 [2] Not sampled by Franz (2012) Naupactopsis Champion, 1911 [5]
- 7. Franz, 2012
- 8. Franz, 2012
- 10. Molecular phylogeny of EGC 65 EGC species 6 genes, 4747 bp Bayesian phylogeny 65 EGC species 6 genes, 4747 bp Bayesian phylogeny See poster at back of room
- 11. Exophthalmus is polyphyletic 65 EGC species 6 genes, 4747 bp Bayesian phylogeny Molecular phylogeny of EGC
- 12. Molecular phylogeny of EGC 65 EGC species 6 genes, 4747 bp Bayesian phylogeny Exophthalmus is polyphyletic
- 13. Tropirhinus Schoenherr, 1823 Pachnaeus Schoenherr, 1826 Diaprepes Schoenherr, 1823 Exophthalmus Schoenherr, 1823 Reclassification of EGC 65 EGC species 6 genes, 4747 bp Bayesian phylogeny
- 14. 190 terminals, 6-gene, Maximum likelihood Eustylini (13 genera) Geonemini (7 genera) Naupactini (>4 genera) Tanymecini (Pandeleteius) Eudiagogini (Promecops) Polydrusini (Apodrosus) Anypotactini (Polydacrys) Tribal classification follows Alonso- Zarazaga & Lyal (1999) and integrates subsequent modifications Neotropical Entiminae
- 15. Polydrusini (Apodrosus) Tanymecini (Pandeleteius) Anypotactini (Polydacrys) “Eustylini” (Eustylus) + “Naupactini” | (EustylusLitostylus)Eudiagogini (Promecops) “Eustylini” (Scelianoma Franz & Girón, 2009) “Geonemini” (Apotomoderes, Ischionoplus, Lachnopus) + “Naupactini” (Artipus) | (ArtipusApotomoderes) “Eustylini” (Brachyomus, Compsus, Exorides, Xestogaster) “Eustylini” (Exophthalmus genus complex s.s.)
- 16. Statistical historical biogeography of Exophthalmus genus complex
- 17. What is the history of the geographic range evolution that has shaped the current distributions of Caribbean weevils? Historical biogeography of EGC
- 18. Historical biogeography of EGC Cuba Jamaica Jump dispersal BioGeoBEARS Dated molecular phylogeny Current distribution ranges Statistical method of inference of range evolution Hispaniola
- 19. Historical biogeography of EGC Caribbean island ancestor dispersed to Central America Jump dispersal between islands accounted for 25% ancestral range evolution events; within- island (area) diversification 75% Caribbean Central America
- 20. Molecular profiling of host plant associations in Neotropical Entiminae
- 21. Are host plant associations of Neotropical weevils phylogenetically conserved? Host plant associations
- 22. Phylogeny of weevils Host plants Host plant associations ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Extracted ingested plant DNA from weevil digestive tracts Amplified and sequenced two plant markers: rbcL and matK Molecular cloning Queried sequences against Genbank database to assign plant taxonomy (family) 42/49 (86%) weevils successful 16/42 (38%) associated with 2 or more plant families 28 families as host plants
- 23. Phylogeny of weevils Plant families Host plant associations Rosids Asterids Monocots Others Burseraceae(Rosids:Sapindales) Calophyllaceae(Rosids:Malpighiales) Euphorbiaceae(Rosid:Malpighiales) Fabaceae(all)(Rosids:Fabales) Fagaceae(Rosids:Fagales) Melastomataceae(Rosids:Myrtales) Meliaceae(Rosids:Spindales) Moraceae(Rosids:Rosales) Phyllanthaceae(Rosids:Malpighiales) Picramniaceae(Rosids:Picramniales) Rhamnaceae(Rosids:Rosales) Adoxaceae(Asterids) Aquifoliaceae(Asterids) Asteraceae(Asterids) Primulaceae(Asterids) Rosaceae(Rosids) Rubiaceae(Asterids) Sapotaceae(Asterids) Solanaceae(Asterids) Dryopteridaceae(Fern) Lauraceae(Magnoliids:Laurales) Nyctaginaceae(Caryophyllales) Polygonaceae(Caryophyllales) Pinaceae(Pinophyta) Piperaceae(Magnoliids) Ranunculaceae(Ranunculales) Trebouxiophyceae(Algae) Musaceae(Monocot) Poaceae(Monocot) Expected pattern of phylogenetic conservation of host associations
- 24. Phylogeny of weevils Plant families Host plant associations Rosids Asterids Monocots Others Burseraceae(Rosids:Sapindales) Calophyllaceae(Rosids:Malpighiales) Euphorbiaceae(Rosid:Malpighiales) Fabaceae(all)(Rosids:Fabales) Fagaceae(Rosids:Fagales) Melastomataceae(Rosids:Myrtales) Meliaceae(Rosids:Spindales) Moraceae(Rosids:Rosales) Phyllanthaceae(Rosids:Malpighiales) Picramniaceae(Rosids:Picramniales) Rhamnaceae(Rosids:Rosales) Adoxaceae(Asterids) Aquifoliaceae(Asterids) Asteraceae(Asterids) Primulaceae(Asterids) Rosaceae(Rosids) Rubiaceae(Asterids) Sapotaceae(Asterids) Solanaceae(Asterids) Dryopteridaceae(Fern) Lauraceae(Magnoliids:Laurales) Nyctaginaceae(Caryophyllales) Polygonaceae(Caryophyllales) Pinaceae(Pinophyta) Piperaceae(Magnoliids) Ranunculaceae(Ranunculales) Trebouxiophyceae(Algae) Musaceae(Monocot) Poaceae(Monocot)
- 25. Phylogeny of weevils Plant families Host plant associations Plant major groups Rosids Asterids Monocots Others Burseraceae(Rosids:Sapindales) Calophyllaceae(Rosids:Malpighiales) Euphorbiaceae(Rosid:Malpighiales) Fabaceae(all)(Rosids:Fabales) Fagaceae(Rosids:Fagales) Melastomataceae(Rosids:Myrtales) Meliaceae(Rosids:Spindales) Moraceae(Rosids:Rosales) Phyllanthaceae(Rosids:Malpighiales) Picramniaceae(Rosids:Picramniales) Rhamnaceae(Rosids:Rosales) Adoxaceae(Asterids) Aquifoliaceae(Asterids) Asteraceae(Asterids) Primulaceae(Asterids) Rosaceae(Rosids) Rubiaceae(Asterids) Sapotaceae(Asterids) Solanaceae(Asterids) Dryopteridaceae(Fern) Lauraceae(Magnoliids:Laurales) Nyctaginaceae(Caryophyllales) Polygonaceae(Caryophyllales) Pinaceae(Pinophyta) Piperaceae(Magnoliids) Ranunculaceae(Ranunculales) Trebouxiophyceae(Algae) Musaceae(Monocot) Poaceae(Monocot)
- 26. Bacterial symbionts in weevils https://www.focusforhealth.org/human-microbiome-chronic-illness/
- 27. “Nowhere else aside from the cicadas do so many symbiotic sites exist as in this insect family [Curculionoidea] - P. Buchner (1965, p. 160) Why study bacterial symbionts in weevils?
- 28. Known bacterial symbionts in weevils based on DNA sequences Symbiont Weevil- specific Functions in weevils Nardonella Yes Growth & development [?] Curculioniphilus Yes ? Klebsiella No Nitrogen fixation Rickettsia No ? Serratia No ? Sodalis No Nutrient provision, cuticular synthesis (?) Spiroplasma No ? Wolbachia No Oocyte production [?]
- 29. Limited taxonomic sampling in previous studies Bacteria sequenced? Curculionoidea - Six non-curculionid weevil families not sampled - Diversity within Curculionidae (true weevils) poor represented - 7/25 subfamilies of Curculionidae sampled
- 30. Objective: Survey and identify bacterial symbionts across weevils using next generation sequencing (NGS)
- 31. Specimen & taxonomic sampling 246 weevil and other beetle specimens dissected and total gut content used for DNA extraction 124 with usable PCR amplicons (115 weevils, 9 other beetles) 4 families and 17 subfamilies (1 and 7 previously) Curculionoidea
- 32. - Gut content subjected to bead-beating & DNA extracted with Qiagen DNeasy Blood & Tissue kit - 16S V3-V5 region amplified with primer pair F515- R909 (394 bp) - Primers barcoded to allow for multiplexing/pooling - PCR products purified, normalized and pooled - Library prepared and sequenced with NGS platform Illumina MiSeq (paired-end) Molecular experiments
- 33. Bacterial OTUs across samples Each column represents a sample Each color represents a genus-level OTU Size of bar indicates relative abundance (% of sequences in a sample) 11,396,976 seqs 947 OTUs 4,619-459,088 reads/sample Median = 65,615
- 34. OTU/distance method failed to identify 44.5% sequences Each column represents a sample Each color represents a genus-level OTU Size of bar indicates relative abundance (% of sequences in a sample) assigned only to Enterobacteriaceae not assigned to any taxonomic groups Wolbachia Sodalis Rickettsia Symbionts 44.5% sequences not assigned to a genus
- 35. Why did OTU/distance method fail? Genetic distance (%) Frequency Nardonella sequences show >3% genetic distances
- 36. Sequence filtering Initial data set 11,396,976 Remove singleton sequences & merge redundant reads 282,587 Select sequences with >100 reads 4,217 Filtering step No. sequences Phylogeny-based taxonomic assignments Phylogeny-based taxonomic assignments
- 37. Phylo-method: (1) build reference phylogeny • Create reference 16S sequence database of 1,209 sequences • Align 4217 post-filtered sequences with reference database sequences • Reconstruct phylogeny of 5,426 sequences on supercomputing cluster Phylogeny-based taxonomic assignments
- 38. Phylo-method: (2) screen for target symbionts Phylogeny-based taxonomic assignments Nardonella !!
- 39. Phylo-method: (3) verify symbionts Representative sequences (1-3 species/genus) closely related to target symbiont collected from Genbank Aligned with putative symbiont sequeneces Reconstruct phylogeny Repeat steps 2 – 3 (sequence binning and phylogenetic verification) for all clades of target sequences Phylogeny-based taxonomic assignments
- 40. Diversity of symbionts in weevils In Enterobacteriaceae 6 lineages (genera) of symbionts found in weevils 41 (36%) weevil samples host Nardonella symbionts Nardonella Kleidoceria Novel symb.? Curculioniphilus SodalisGibsiella Newly identified symbiont sequences Previous sequences
- 41. Evolution of symbionts in weevils Nardonella symbionts coevolved with hosts at shalow phylogenetic levels Weevil Nardonella
- 42. Specimen & data management, Weevil tissue collection
- 43. http://tinyurl.com/EGcomplex (Specimen) Data management Symbiota Collections of Arthropods Network Reproducibility Exposes issues Data/specimen reuse Enables future work http://tinyurl.com/SCANdatabas e
- 47. Available to all Hands-on Collaboration & annotation at specimen level
- 48. Weevil Tissue Collection tinyurl.com/weeviltissuecollection See poster at back of room
- 49. Acknowledgements Undergraduate student mentees/collaborators Boris Dimov, Julina Jones Natalia Rahman, Mary Walsh, Zhen Geng, Joe Hunter, Pan Lin, Bukola Obayomi, Pavithra Paravastu, Juyan Pourturk, Will Sides, Sara Tanveer, Richard Thompson, Don Tram, Usmaan, Basharat, Daniel Vargas Lin Pan, weevil taxonomy Juyan Pourturk, DNA extraction, PCR, molecular cloning Will Sides, specimen imaging Sara Tanveer, PCR, sequencing
- 50. NSF CAREER # 1155984 (to N. Franz) USDA (US Department of Agriculture) Agreement No. 58-1275-1-335 (to N. Franz) ASU School of Life Sciences (Postdoctoral Collaborative Grant) ESA STEP (Students in Transition, Early-career Professional) award Acknowledgements
- 51. © Melvyn Yeo Thank you!
- 52. Discussion Specimen/DNA vouchering and access Legacy for future generations Best practices What is really meant by “taxon sampling” “Taxon” versus “taxonmic names” (concept labels) Higher-level “taxonomic names” may or may not be monophyletic Whose taxon concept Data publication/sharing – open, collaborative, realtime, quantum-volume
Editor's Notes
- Alright. That wraps up the Zelus monography. By working on assassin bugs, I gained well-rounded training in the theories and methods of taxonomy, systematics, and collection-based research. Reduviids will continue to fascinate and inspire me and they fit really well with curiosity-driven systematics research. However, I was acutely aware that their economic relevance is relatively limited compared to some other insects. When I was just finishing my PhD, I identified and secured a postdoctoral position to work on weevils. In the following part of my current research, I will share with you some of my latest research that expanded not only my taxonomic expertise, but also my conceptual and methodological arena.
- To reconstruct this biogeographic history, I will need three things. a phylogeny, which I already had, but now it is dated using a molecular dating method integrating fossil calibrations. I also need to know the extant distributions, which are readily available, indicated as colored boxes. I also a need a method of inference.. There are many here. I used a so-called statistical method implemented in the program BioGeoBEARS. What it does is to estimate the range evolution scenarios at ancestral nodes.What does that mean. Let’s look at the resuls.
- To reconstruct this biogeographic history, I will need three things. a phylogeny, which I already had, but now it is dated using a molecular dating method integrating fossil calibrations. I also need to know the extant distributions, which are readily available, indicated as colored boxes. I also a need a method of inference.. There are many here. I used a so-called statistical method implemented in the program BioGeoBEARS. What it does is to estimate the range evolution scenarios at ancestral nodes.What does that mean. Let’s look at the resuls.
- Alright. That wraps up the Zelus monography. By working on assassin bugs, I gained well-rounded training in the theories and methods of taxonomy, systematics, and collection-based research. Reduviids will continue to fascinate and inspire me and they fit really well with curiosity-driven systematics research. However, I was acutely aware that their economic relevance is relatively limited compared to some other insects. When I was just finishing my PhD, I identified and secured a postdoctoral position to work on weevils. In the following part of my current research, I will share with you some of my latest research that expanded not only my taxonomic expertise, but also my conceptual and methodological arena.
- Now I’m turning to the last chapter of my adventures. I will talk about I wrangled with 11 million sequences generated using the Illumina MiSeq next generation sequencing platform. I will also share my story of initially following but soon revolting against the current doctrines of bacterial sequence diversity and taxonomy analyses.
- Why study symbionts of weevils besides the obvious reason that I work on weevils? A good reason is that weevils are known to harbor diverse symbionts. Paul Buchner, a pioneer of endosymbiosis research, said this, “Nowhere else aside from the cicadas do so many symbiotic sites exist as in this insect family”. He was referring to what we now understand as Curculionoidea or weevils.
- During the recent year, molecular studies have revealed eight groups of symbionts in weevils. Notably, two of those, Nardonella and Curculioniphilus are found only from weevils. Both symbionts were discovered quite recently, Nardonella in 2004 and Curculioniphilus in 2010. For most of these symbionts, we do not yet know what they do. Klebsiella fixes nitrogen and Sodalis provision nutriends and may also participate in cuticular synthesis.
- And again, previous studies were limited in their taxonomic scopes. The six non-curculionid familes were never sampled. And within the Curculionidae, only 7 subfamilies were previously sampled.
- So I would like to survey and identify bacterial symbionts across a large taxonomic sample of weevils by sequencing the 16S gene using next generation sequencing technologies.
- We dissected 246 weevils and other beetles. Of those, 124 produced usable PCR products. This sample represents 4 families of weevils and 17 subfamilies of the Curculionidae. Those are significant increases compared to previous studies.
- For the molecular experiments, we extracted DNA from gut content and amplified a 400 bp fragment of the 16s gene. In order to uniquely identify each sample so that they can be combined for multiplexing, the PCR primers were barcoded. Every sample had a unique combination of forward and reverse barcodes. The sequencing was done on a Illumina Miseq system, specifically using paired sequencing.
- This graph is produced by QIIME. The bar chart is a visualization of the diversity, distribution and abundance of OTUs across samples. Each column is a sample. Each color in the chart represents an OTU at the genus level (if the OTU could be assigned to a genus, but that is not always the case). And the size of a bar indicates the relative abundance of an OTU. Altogether there are 947 OTUs. We can immediately see that the color orange is present in many samples and also quite abundant. Red, blue and green are widespread as well. So what OTUs do those represent?
- It turned out that orange and blue are OTUs assigned only to the family Enterobacteriaceae. This is a large family and includes the famous E. coli and many symbiotic bacteria such as Buchnera. The red color is an aggregation of OTUs not assigned to any taxonomic groups. Altogether those sequences not classified to the genus-level comprise almost half of my dataset. This method did find some common symbionts, such Wolbachia, Rickettsia and Sodalis. However, we did not find either of the two weevil-specific bacteria, Nardonella and Curculioniphilus.
- Filtering the sequences was straightforward and was done with Shell and Python scripting. First we removed singleton sequences and merged redundant sequences. What does that mean? In an illumina next generation sequencing data set, many sequencing reads are repetitions of the same sequence or are redundant reads. We merged those into unique sequences. But many other sequencing reads only appear once. Those are called singletons sequences and are most likely due to sequencing errors. We removed those singletons The resulting data set contained nearly 300,000 unique sequences. But 300,000 sequences are still too many to run a phylogenetic analysis. The next thing we did was to select sequences that have at least 100 redundant reads. The rational is that symbionts usually occur in large numbers and their sequences should be abundant in the data set. We are then down to 4000 unique sequences. We are going call this the “100 + dataset”.
- Filtering the sequences was straightforward and was done with Shell and Python scripting. First we removed singleton sequences and merged redundant sequences. What does that mean? In an illumina next generation sequencing data set, many sequencing reads are repetitions of the same sequence or are redundant reads. We merged those into unique sequences. But many other sequencing reads only appear once. Those are called singletons sequences and are most likely due to sequencing errors. We removed those singletons The resulting data set contained nearly 300,000 unique sequences. But 300,000 sequences are still too many to run a phylogenetic analysis. The next thing we did was to select sequences that have at least 100 redundant reads. The rational is that symbionts usually occur in large numbers and their sequences should be abundant in the data set. We are then down to 4000 unique sequences. We are going call this the “100 + dataset”.
- Doing phylogeny-based taxonomic assignments involved several steps. The first part is we need to build a phylogeny. To do that we created a reference 16S sequence database by filtering and modifying the greengenes database and we made sure that symbionts are well represented in this database. Those sequences were aligned with our own sequences of the 100+ dataset. Then we ran a phylogenetic analysis. Surprisingly, it took only a day to run the analysis on a supercomputing cluster.
- The second step was to put sequences into bins according to their phylogenetic position on the phylogeny. In the picture here, the names starting with BEP are my sequences and they cluster with the weevil symbiont Nardonella. I picked all the sequences in the neighborhood of Nardonella. What this does is find clusters of sequences that have a possible close phylogenetic relationship to a symbiont. However, this does not confirm the identity of the sequences, primarily because the reference database is not complete. I’m going to call those putative symbiont sequences. It is like saying we found an insect that closely resembles a butterfly, but we really don’t know what it is or whether it is new until we compare it with all known butterflies. What we will need to do next is take the putative symbiont sequences and closely related sequences with known identities and construct another phylogeny. And I will call this step ‘phylogenetic verification’.
- In phylogenetic verification, I first collected representative sequences, usually 1-3 species per genus, which are closely related to the target symbiont. And then I aligned those sequences with my own putative symbiont sequences.
- This is a phylogeny of Nardonella and its relatives, which are in the family Enterobacteriaceae. The shaded branches are my putative symbiont sequences and the ones not shaded are published sequences with known identities. First impression is that they come out at different places on the phylogeny. There is a large clade of sequences, which we can identify as Nardonella. Two sequences cluster with a bacterium called Kleidoceria, previously found from Lygaeid hemipteran bugs. There is cluster that is sister to a number of genera, which might represent a novel symbiont. I found a single sequence of Curculioniphilus and several that clustered with Sodalis.
- This is a phylogeny of Nardonella and its relatives, which are in the family Enterobacteriaceae. The shaded branches are my putative symbiont sequences and the ones not shaded are published sequences with known identities. First impression is that they come out at different places on the phylogeny. There is a large clade of sequences, which we can identify as Nardonella. Two sequences cluster with a bacterium called Kleidoceria, previously found from Lygaeid hemipteran bugs. There is cluster that is sister to a number of genera, which might represent a novel symbiont. I found a single sequence of Curculioniphilus and several that clustered with Sodalis.
- Alright. That wraps up the Zelus monography. By working on assassin bugs, I gained well-rounded training in the theories and methods of taxonomy, systematics, and collection-based research. Reduviids will continue to fascinate and inspire me and they fit really well with curiosity-driven systematics research. However, I was acutely aware that their economic relevance is relatively limited compared to some other insects. When I was just finishing my PhD, I identified and secured a postdoctoral position to work on weevils. In the following part of my current research, I will share with you some of my latest research that expanded not only my taxonomic expertise, but also my conceptual and methodological arena.
- We dissected 246 weevils and other beetles. Of those, 124 produced usable PCR products. This sample represents 4 families of weevils and 17 subfamilies of the Curculionidae. Those are significant increases compared to previous studies.