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