Implications to the Evolution of Firefly Signaling Systems...Antennal type Bioluminescence....

A Molecular Phylogeny of

Lampyridae

Implications to the

Evolution of Firefly

Signaling Systems

Gavin J. Martin, Nathan P. Lord, Michael F. Whiting,

Marc A. Branham & Seth M. Bybee

Outline Introduction to Lampyridae

Part 1

Vision/opsin

Part 2

Antennal/bioluminescence

Lampyridae Latrielle, 1817

~2000 species, ~83 genera

Cosmopolitan

Humid to arid

Bruce Marlin Sam Stewart

Lampyridae Defined by:

7 ventrites in male (6 in Luciolinae)



Usually 11 antennal segments




Antennal sockets in front of eyes

Trochanters attached obliquely to

femora




Antennal sockets in front of eyes

Trochanters attached obliquely to

femora

Tarsal claws simple (some

exceptions)


Head when retracted partially

covered by pronotum


Head when retracted partially

covered by pronotum

Free labrum

Sickle-shaped mandibles

Abdominal spiracles usually in

pleural fold

Elytra with epipleura

Basics of insect vision

Apposition (most insects)

Superposition (Fireflies, most beetles &

nocturnal insects)

What is an

Opsin?

Research Objectives

1. Use Next Generation Sequencing to assess

opsin number (copies and classes)

2. Assess ancestral state of bioluminescence

3. Determine potential association between

antennal type and use of bioluminescence

Methods Opsin phylogeny:

Trasncriptomes from 13 species were generated

as follows:

RNA extracted from whole body using

Nucleospin columns

cDNA generated using Illumina TruSeq v2

Sequenced on Illumina HiSeq 2000

101 paired-end reads

Genbank for rest of insects

Transcriptome assembly

Galaxy pipeline

PoPoolation; Trinity

PIA BLAST

Methods

Sequences aligned in Geneious using the

MAFFT L-INS-i strategy, and afterward

checked by eye

ML analysis

RAxML; 200 replicates, 1000 bootstrap pseudo

replicates

GTR +Γ model (Partition Finder)

All analyses were run on the BYU Fulton

Supercomputer

Results

Pyractomena dispersa

opsin generated

using Swiss Model

Ultra-violet opsin

Long-wavelength opsin

Only two opsin classes found (LW & UV) with

no duplicates

If only two copies of opsins are expressed,

what else can explain the ability for fireflies to

differentiate complex flash patterns?

Conclusions

Firefly

Communication Most well known for luminescence

Not all species using luminescence, many use

pheromones, while some use a combination of

both

Research Objectives

1. Use Next Generation Sequencing to assess

opsin number (copies and classes)

2. Assess ancestral state of bioluminescence

3. Determine potential association between

antennal type and use of bioluminescence

Materials 70 Taxa

3 outgroup taxa

16S, 18S & COI sequences used from Genbank

Morphological data from 410 character matrix

Jeng 2008

Male antennal characters were coded according

to the following categories:

Simple, expanded, branching

0: Simple 1: Expanded 2: Branching

Methods

Sequences aligned in MAFFT using E-INS-i

for 16S and 18S, and G-INS-i for COI

Bayesian analysis: Mr. Bayes with posterior

probability for support values (detailed

methods available for those interested)

Characters mapped and ancestral states

reconstructed in Mesquite under parsimony

framework

Posterior Prob.: >90

Black: Non-Bioluminescent

White: Bioluminescent

Gray: Equivocal

Bioluminescence

Oxynopterus spRhagophthalmus ohbai

Plateros spPterotus obscuripennisBrachylampis blaisdelli

Ceylanidrilus spStenocladius sp

Stenocladius shirakiiStenocladius flavipennisStenocladius yoshikawai

Mimophaeopterus spFlabellotreta obscuricollis

Flabellotreta spDrilaster auxillarisDrilaster ohbayashiDrilaster borneensis

Drilaster spCyphonocerus ruficollis

Pollaclasis bifariaPristolycus sangulatus

Luciola ovalisLuciola parvulaLuciola lateralisBourgeoisia sp

Luciola cruciataLuciola filiformis

Luciola italicaLuciola sp

Luciola kuroiwaeCurtos sp

Curtos costipennisCurtos okinawanus

Lamprohiza splendidulaPhausis reticulataVesta saturnalis

Vesta spBicellonycha wickershamorum

Photuris congenerPhoturis pennsylvanicaPhoturis quadrifulgens

Photuris aff lucicrescensPhoturis tremulans

Micronaspis floridanaLucidota atra

Lucidina biplagiataPhosphaenus hemipterus

Pyropyga decipiensPyropyga nigricans

Ellychnia californicaEllychnia corruscaPhotinus australisPhotinus tanytoxis

Photinus pyralisPhotinus floridanus

Photinus punctulatusPyractomena palustrisPyractomena angulataPyractomena borealis

Aspisoma spPleotomodes needhami

Pleotomus pallensLampyris noctiluca

Microphotus angustusParaphausis eximiaDiaphanes formosusPyrocoelia atripennis

Pyrocoelia rufaPyrocoelia amplissima

Pyrocoelia fumosaLychnuris formosana

Pyrocoelia abdominalisPyrocoelia discicollis

Pyrocoelia matsumurai kumejimensis

Simple = White

Expanded = Green

Branching = Black

Present = White

Not Present = Black

Antennal type Bioluminescence































Simple Antennae

Simple = White

Expanded = Green

Branching = Black

Present = White

Not Present = Black
































Branching Antennae

Simple = White

Expanded = Green

Branching = Black

Present = White

Not Present = Black
































Expanded

Antennae

Simple = White

Expanded = Green

Branching = Black

Present = White

Not Present = Black


Ancestral state of sexual communication in

Lampyridae is found to be non-bioluminescent

With one exception (Ototretinae), simple

antennae are associated with use of

bioluminescence

With the exception of two genera (Pterotus and

Vesta), branching antennae are associated with

non-bioluminescence

Conclusions

Future Directions Narrow the vision...

Increase taxon sampling to include all subfamilies

and additional outgroups

Incorporate signaling system data and photic

organ/antennal morphology

Test for statistical correlations (e.g. BayesTraits)

Ototretinae

Cyphonocerinae

Lampyrinae

Luciolinae

Photurinae

Posterior Prob.: >90

Pterotinae

Acknowledgements I especially like to thank the members of my committee

and my co-authors without whom this project would not be possible

I would additionally like to thank the following:

Members of the Whiting and Bybee lab group:

Joseph Mugleston, Haley Wightman, David Morris, Anton Suvorov, Yelena Pacheco, Taylor King, Preston Arnold, Michael Naegle, Michael Swindle

Brigham Young University Graduate Student Fellowship and NSF for funding

Brigham Young University Fulton Supercomputer for computing resources

Questions??

Methods Cont.

Mr. Bayes

39,095,000 generations

2 parallel runs

8 chains each

Data partitioned by gene and morphology

according to Partition Finder

MK + Γ model for morphology

GTR + Γ + I model for all genes

Stationarity checked in Tracer

Implications to the Evolution of Firefly Signaling Systems...Antennal type Bioluminescence....

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