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 MeetingOct 01, 2016
Taxonomy of the Exophthalmus genus complex
June, 2013 Cuba
Dr. Robert (Bob) Anderson (Canadian Museum of Nature)
Franklyn Cala Riquelme,Albert Deler Hernandez(Cuban arachnologist/coleopterist)
Entiminae (Broad-nosed weevils) “Eustylini” (sensu Franz, 2012) Tetrabothynus spectabilis (Klug, 1829) Tetrabothynus
regalisExophthalmus genus complex (Franz 2012)
Exophthalmus genus complex (EGC)
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]
Franz, 2012
Franz, 2012
Franz, 2012
Exophthalmus genus complex (EGC)
Molecular phylogeny of EGC
65 EGC species6 genes, 4747 bpBayesian phylogeny
65 EGC species6 genes, 4747 bpBayesian phylogeny
See poster at back of room
Exophthalmus is polyphyletic
65 EGC species6 genes, 4747 bpBayesian phylogeny
Molecular phylogeny of EGC
Molecular phylogeny of EGC
65 EGC species6 genes, 4747 bpBayesian phylogeny
Exophthalmus is polyphyletic
Tropirhinus Schoenherr, 1823
PachnaeusSchoenherr, 1826
DiaprepesSchoenherr, 1823
ExophthalmusSchoenherr, 1823
Reclassification of EGC
65 EGC species6 genes, 4747 bpBayesian phylogeny
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
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.)
Statistical historical biogeography of Exophthalmus genus complex
What is the history of the geographic range evolution that has shaped the current distributions of Caribbean weevils?
Historical biogeography of EGC
Historical biogeography of EGC
Cuba
JamaicaJump dispersal
BioGeoBEARS
Dated molecular phylogeny Current distribution ranges Statistical method of
inference of range evolution
Hispaniola
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%
CaribbeanCentral Am
erica
Molecular profiling of host plant associations in Neotropical Entiminae
Are host plant associations of Neotropical weevils phylogenetically conserved?
Host plant associations
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
Phylogeny of weevils
Plant familiesHost plant associations
Burseraceae (R
osids: Sapindales)
Calophyllaceae (R
osids: Malpighiales)
Euphorbiaceae (R
osid: Malpighiales)
Fabaceae (all) (Rosids: Fabales)
Fagaceae (Rosids: Fagales)
Melastom
ataceae (Rosids: M
yrtales)
Meliaceae (R
osids: Spindales)
Moraceae (R
osids: Rosales)
Phyllanthaceae (R
osids: Malpighiales)
Picram
niaceae (Rosids: P
icramniales)
Rham
naceae (Rosids: R
osales)
Adoxaceae (A
sterids)
Aquifoliaceae (A
sterids)
Asteraceae (A
sterids)
Prim
ulaceae (Asterids)
Rosaceae (R
osids)
Rubiaceae (A
sterids)
Sapotaceae (A
sterids)
Solanaceae (A
sterids)
Dryopteridaceae (Fern)
Lauraceae (Magnoliids: Laurales)
Nyctaginaceae (C
aryophyllales)
Polygonaceae (C
aryophyllales)
Pinaceae (P
inophyta)
Piperaceae (M
agnoliids)
Ranunculaceae (R
anunculales)
Trebouxiophyceae (Algae)
Musaceae (M
onocot)
Poaceae (M
onocot)
Expected pattern of phylogenetic conservation of host associations
Phylogeny of weevils
Plant familiesHost plant associations
Burseraceae (R
osids: Sapindales)
Calophyllaceae (R
osids: Malpighiales)
Euphorbiaceae (R
osid: Malpighiales)
Fabaceae (all) (Rosids: Fabales)
Fagaceae (Rosids: Fagales)
Melastom
ataceae (Rosids: M
yrtales)
Meliaceae (R
osids: Spindales)
Moraceae (R
osids: Rosales)
Phyllanthaceae (R
osids: Malpighiales)
Picram
niaceae (Rosids: P
icramniales)
Rham
naceae (Rosids: R
osales)
Adoxaceae (A
sterids)
Aquifoliaceae (A
sterids)
Asteraceae (A
sterids)
Prim
ulaceae (Asterids)
Rosaceae (R
osids)
Rubiaceae (A
sterids)
Sapotaceae (A
sterids)
Solanaceae (A
sterids)
Dryopteridaceae (Fern)
Lauraceae (Magnoliids: Laurales)
Nyctaginaceae (C
aryophyllales)
Polygonaceae (C
aryophyllales)
Pinaceae (P
inophyta)
Piperaceae (M
agnoliids)
Ranunculaceae (R
anunculales)
Trebouxiophyceae (Algae)
Musaceae (M
onocot)
Poaceae (M
onocot)
Phylogeny of weevils
Plant familiesHost plant associations
Plant major groups
Rosids
Asterids
Monocots
Others
Burseraceae (R
osids: Sapindales)
Calophyllaceae (R
osids: Malpighiales)
Euphorbiaceae (R
osid: Malpighiales)
Fabaceae (all) (Rosids: Fabales)
Fagaceae (Rosids: Fagales)
Melastom
ataceae (Rosids: M
yrtales)
Meliaceae (R
osids: Spindales)
Moraceae (R
osids: Rosales)
Phyllanthaceae (R
osids: Malpighiales)
Picram
niaceae (Rosids: P
icramniales)
Rham
naceae (Rosids: R
osales)
Adoxaceae (A
sterids)
Aquifoliaceae (A
sterids)
Asteraceae (A
sterids)
Prim
ulaceae (Asterids)
Rosaceae (R
osids)
Rubiaceae (A
sterids)
Sapotaceae (A
sterids)
Solanaceae (A
sterids)
Dryopteridaceae (Fern)
Lauraceae (Magnoliids: Laurales)
Nyctaginaceae (C
aryophyllales)
Polygonaceae (C
aryophyllales)
Pinaceae (P
inophyta)
Piperaceae (M
agnoliids)
Ranunculaceae (R
anunculales)
Trebouxiophyceae (Algae)
Musaceae (M
onocot)
Poaceae (M
onocot)
Bacterial symbionts in weevils
https://www.focusforhealth.org/human-microbiome-chronic-illness/
“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?
Known bacterial symbionts in weevils based on DNA sequences
SymbiontWeevil-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 [?]
Limited taxonomic sampling in previous studies
Bacteria sequenced?
Curc
ulio
noid
ea - Six non-curculionid
weevil families not sampled
- Diversity within Curculionidae (true weevils) poor represented
- 7/25 subfamilies of Curculionidae sampled
Objective: Survey and identify bacterial symbionts across weevils using next generation sequencing (NGS)
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)
Curc
ulio
noid
ea
- 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
Bacterial OTUs across samples
Each column represents a sampleEach color represents a genus-level OTUSize of bar indicates relative abundance (% of sequences in a sample)
11,396,976 seqs947 OTUs
4,619-459,088 reads/sampleMedian = 65,615
OTU/distance method failed to identify 44.5% sequences
Each column represents a sampleEach color represents a genus-level OTUSize of bar indicates relative abundance (% of sequences in a sample)
assigned only to Enterobacteriaceaenot assigned to any
taxonomic groups
Wolbachia
Sodalis
Rickettsia
Symbionts
44.5% sequences not assigned to a genus
Why did OTU/distance method fail?
Genetic distance (%)
Freq
uenc
y Nardonella sequences show >3% genetic
distances
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-basedtaxonomic assignments
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
Phylo-method: (2) screen for target symbionts
Phylogeny-based taxonomic assignments
Nardonella !!
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
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 sequencesPrevious sequences
Evolution of symbionts in weevils
Nardonella symbionts coevolved with hosts at shalow phylogenetic levels
Weevil Nardonella
Specimen & data management,Weevil tissue collection
http://tinyurl.com/EGcomplex
(Specimen) Data management
Symbiota Collections of Arthropods Network
ReproducibilityExposes issuesData/specimen reuseEnables future work
http://tinyurl.com/SCANdatabase
Available to allHands-onCollaboration & annotation at specimen level
Weevil Tissue Collection
tinyurl.com/weeviltissuecollectionSee poster at back of room
Acknowledgements
Undergraduate student mentees/collaboratorsBoris 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
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
© Melvyn Yeo
Thank you!
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