Plant Speciation: Genetics - UW Courses Web Servercourses.washington.edu › bot113 › summer › LectNotes › ... · Plant Speciation: Genetics (Biol 317, but you knew that already)

Plant Speciation: Genetics (Biol 317, but you knew that already)

2013-08-12

Review: The Biological Species Concept (BSC)

What is a species?

“Groups of actually or potentially interbreeding populations reproductively isolated from all other such groups.” – Ernst Mayr

How do species arise?

“When we understand the origin of reproductive isolation, we understand the origin of species.” – Jerry Coyne

Review of speciation: overview • The process by which new

species arise • Hard to study! How do you

watch “speciation in action”?

• Mostly retrospective study • Much of the literature

focuses on animals (Drosophila especially)

• Plant species are, well, messy!

• Modern Synthesis, speciation-style: combine evolution and genetics to understand speciation

Review of speciation: geography

Ilmari Karonen/Dana Krempels

Reproductive isolation: it’s all about barriers

Pre-mating barriers to gene flow • Geographic: different place • Ecological: different habitats • Phenological: different blooming time • Behavioral: different pollinators • Mechanical: different pollen deposition or different stigma/anther spots

Reproductive isolation: it’s all about barriers

Pre-mating barriers to gene flow • Geographic: different place • Ecological: different habitats • Phenological: different blooming time • Behavioral: different pollinators • Mechanical: different pollen deposition or different stigma/anther spots

Post-mating barriers to gene flow • Gamete incompatibility: pollen tubes are for other pollen • Sperm competition: my sperm is faster than yours • Hybrid inviability: no offspring for you! • Hybrid sterility: DEAD END • Hybrid breakdown: running out of gas

Reproductive isolation: genetic origins?

Pre-mating barriers to gene flow • Geographic: ? • Ecological: adaptation to different environments? • Phenological: flowering time genetics? Emergence from dormancy? • Behavioral: pollinator attraction? • Mechanical: stigma-anther position (herkogamy), pollinator pollen

position?

Reproductive isolation: genetic origins?

Pre-mating barriers to gene flow • Geographic: ? • Ecological: adaptation to different environments? • Phenological: flowering time genetics? Emergence from dormancy? • Behavioral: pollinator attraction? • Mechanical: stigma-anther position (herkogamy), pollinator pollen

position?

Post-mating barriers to gene flow • Gamete incompatibility: pollen tube formation? • Sperm competition: pollen tube speed issues? • Hybrid inviability: synthetic lethal loci? • Hybrid sterility: chromosomal issues? • Hybrid breakdown: incompatible gene interactions?

Quantifying reproductive isolation

• Idea: understand barriers - what, when, how? How much?



• Q1: What would you expect to be stronger overall: prezygotic or postzygotic isolation?




• Q2: Why is quantifying reproductive isolation useful?




• Q2: Why is quantifying reproductive isolation useful?

• Q3: Would you expect it to be different in different systems?

Reproductive Isolation: Mimulus

In other words: ecogeographic isolation does 59% of the job. In the remaining 41% (sympatry), pollinator isolation does 97.6%. ‘nuff said. Good places to start looking, right?

Choosing a plant system for studying the genetics of speciation

When choosing a system, consider: • Ecology – is it known? Can you find it in the

field? • Ease of use – is it easy to grow? Does it make lots

of seeds? • Genetics – do tools exist (genomes, markers,

transcriptomes, etc)? Is it diploid? • Horticultural varieties – do they clarify or

confuse the picture? • Are there related species that would help?

Phylogenetic context!

Some of the many plant systems for studying the genetics of speciation

Props to Hans Hillewaert; Robin Hopkins; Enrico Blasutto; Jörg Hempel; Wikipedia/Takwish, Geographer, Bouba, Roepers; Fir0002/Flagstaffotos

























“Speciation Genes” [a controversial topic]

• Effect size: large vs small • Gene type: structural (enzyme) versus

regulatory (transcription factor) • Mutation type: coding (exon) versus

regulatory (transcription factor binding site) • What about non-genic things like

chromosomal rearrangement?

“Speciation Genes” [a controversial topic]

• Rieseberg and Blackman 2010: – 41 candidate “speciation genes” – 7 for pre-pollination isolation – 1 to post-pollination, prezygotic isolation – 8 to hybrid inviability – 25 to hybrid sterility – Frequent pathways: anthocyanin (purple color), S RNAse-SI genes

(control self-compatibility), disease resistance genes, chimeric mitochondrial genes (male sterility), pentatricopeptide repeat genes (male sterility)

– Prezygotic mostly regulatory changes – Postzygotic is a mix of both – Often copy number variation or loss of function mutations – Genes often under balancing selection

• How can we draw broad conclusions from only 41 genes, and only 8 prezygotic genes? HERE BE DRAGONS

Example 1: Chromosomal issues • Fishman 2013, Mimulus

lewisii/cardinalis: – Translocations and

inversions between the two species

– Adaptation and flower trait loci clustered in these rearrangements

– Male sterility loci also map to these regions

– Chromosomal rearrangements generate and reinforce gene flow barriers!

Example 2: Soil adaptation

• Mimulus guttatus/cupriphilus – Copperopolis, CA and surrounding copper mines

• Tol1 and Nec1: copper tolerance and hybrid lethality • Tight linkage! So hybrid lethality ‘hitchhikes’ along with

soil adaptation differences • Speciation caused by local adaptation with unrelated

isolation factor

versus

Presgraves 2013

Example 3: Haldane’s Rule in plants!

• Silene latifolia and S. diclinus

• Asymmetric male rarity in crosses – “Haldane’s Rule”

• “Faster-male theory” – male reproductive genes evolve faster

• Not a single gene, but a composite picture

“When in the F1 offspring of two different animal races one sex is absent, rare, or sterile, that sex is the heterozygous sex.” – JBS Haldane

Wikipedia: Sannse, Etxrge; Brothers 2010

Example 4: Multiple barriers, mostly postzygotic isolation

• Solanum pennellii and S. corneliomulleri – exist in sympatry • Premating barriers?

– Flowering time the same – Pollinators seem to be shared

• Postmating barriers? – Pollen-pistil interactions if female is self-incompatible – Multiple factors on both female and pollen sides, but none seem

responsible… – Fruit formation is fine – Embryo development FAIL: embroygenesis checkpoint block, so

hybrid embryos inviable

Sandra Knapp

Example 5: Dobzhansky-Muller

• Mimulus guttatus and M. nasutus

• Dobzhansky-Muller Hybrid Incompatibility Genes – negative interactions among loci

• Many pollen-sterile and female-sterile F2 plants – so expect these interactions

• Often a 2-gene model, but here may have multiple genes involved

Example plant speciation talk titles from Evolution 2012/2013!

Pollination-focused talks: easily half of these • “Selection and speciation: from genotype to phenotype to reproductive isolation”: Phlox (Polemoniaceae) • “Combining quantitative expression of CYCLOIDEA-like genes in Dipsacaceae (Dipsacales) and geometric morphometrics of corolla shape: Further insights into the evolution of bilateral symmetry and radial

symmetry in flowers”: (Dipsacaceae) • “Population genomics of an obligate pollination mutualism: Using RAD sequencing to study coevolution of yuccas and yucca moths”: Yucca (Asparagaceae) • “The functional roles of disassortative pollination and sexual interference in heterostyly: A case study from Darwin's primroses”: Primula(Primulaceae) • “Coevolution, diversification, and biogeography of a specialized insect-plant pollination mutualism on oceanic islands”: Glochidion (Phyllanthaceae) • “The genetics of floral mechanical isolation in the neotropical spiral gingers”: Costus (Costaceae) • “Sex, food and sleep - multiple behaviours in a pollinating fly drive floral divergence in an African daisy”: Gorteria (Asteraceae) • “The role of three floral volatiles in pollinator-mediated reproductive isolation in monkeyflowers (Mimulus)”: Mimulus (Phrymaceae) • “The influence of host shifts on reproductive isolation in a rapid radiation of yucca moth pollinators and cheaters”: Yucca (Asparagaceae) • “Parallel adaptation to hummingbird pollination in Penstemon involves repeated nonfunctionalization of the same flower color enzyme”: Penstemon (Plantaginaceae) • “Selection by hawkmoth and hummingbird pollinators on Polemonium brandegeei (Polemoniaceae): compromise phenotypes or floral mosaics?” Polemonium (Polemoniaceae) • “Selection for speciation: a genetic dissection of reinforcement in Phlox”: Phlox (Polemoniaceae) • “Pollinator-driven adaptation and reproductive isolation in orchids”: Gymnadenia (Orchidaceae) • “Single novel compounds underpin strong reproductive isolation in Australian sexually deceptive orchids: insights into the initiation of pollinator mediated speciation”: Chiloglottis (Orchidaceae) • “Evolution of floral scent in Narcissus and their correlation with pollinators”: Narcissus (Amaryllidaceae) • “The genic basis of pollinator-mediated reproductive isolation”: Ophyrs (Orchidaceae)

All other reproductive isolation discussions • “Chromosomal rearrangements and the genetics of speciation in Mimulus lewisii and M. cardinalis”: Mimulus (Phrymaceae) • “Genetics of hybrid lethality between sympatric species of Mimulus”: Mimulus (Phrymaceae) • “Divergent selection and clinal variation between incipient species of Mimulus”: Mimulus (Phrymaceae) • “Intraspecific variation for hybrid sterility QTL in Solanum”: Solanum (Solanaceae) • “Genetic architecture of isolation between two species of Silene with sex chromosomes and Haldane's rule”: Silene (Caryophyllaceae) • “Endless forms most beautiful: population processes and the origin of diversity in Begonia”: Begonia (Begoniaceae) • “Speciation and introgression in a Mimulus species pair”: Mimulus (Phrymaceae) • “Does a jack-of-all-temperatures have a large geographic range?”: Mimulus (Phrymaceae) • “Parallel Genomic Evolution [But Not Parallel Speciation] in Annual Sunflowers”: Helianthus (Asteraceae) • “Pre- and postzygotic reproductive barriers between sympatric wild tomato species”: Solanum (Solanaceae) • “Mating patterns at an artificial zone of secondary contact between two allopatric and highly divergent populations of Arabidopsis lyrata spp. Petrea”: Arabidopsis (Brassicaceae) • “Hybridization between two rewardless lady's slipper orchids (Cypripedium): conservation threat or life as usual?”: Cypripedium (Orchidaceae) • “The genetic basis of flowering time evolution in the annual plant Brassica rapa”: Brassica (Brassicaceae) • “Parent-offspring conflict and the evolution of reproductive isolation in Mimulus ”: Mimulus (Phrymaceae)

Why study plant-pollinator interactions? A historical perspective

“The rapid development as far as we can judge of all the higher plants within recent geological times is an abominable mystery... Saporta believes that there was an astonishingly rapid development of the high plants, as soon [as] flower-frequenting insects were developed and favoured intercrossing. I shd [sic] like to see this whole problem solved.” (1879)

George Richmond (1809-1896)

Why study plant-pollinator interactions? A modern perspective

“Our major conclusion is that two sets of key factors - traits increasing sexual selection in animals, and traits promoting animal pollination in plants - appear to increase the rate of speciation.” (Coyne and Orr, 2004)

Many factors influence attraction…

Pattern

Texture

Color

Size

Shape

Scent

Phenotype to genotype to significance

Pink Salmon-yellow

Phenotype: Color


Genotype: YUP

Pink Salmon-yellow

Phenotype: Color


Pink Salmon-yellow

Phenotype: Color

Genotype: YUP

Visit

s/hr

(x0.

001)

0

2

4

6

8

10

12

14

16

18

YUP (pink) yup (salmon)

Bumblebees

Hummingbirds

Significance: Visitation

Case study 1: Petunia color

Species: Petunia integrifolia and P. axillaris Pollinators: bumblebees, hawkmoths Trait: anthocyanin (purple) color Genetic underpinnings: ANTHOCYANIN2 (AN2) activates biosynthesis of anthocyanins Genetic details: regulatory gene, changes in coding sequence Phenotypic significance: transgenic flowers - bees and hawkmoths switched preference – so reproductive isolation!

Case study 2: Petunia scent

Species: Petunia axillaris and P. exserta Pollinators: hawkmoths, hummingbirds Trait: methyl benzoate (scent) Genetic underpinnings: ODORANT1 (ODO1) activates biosynthesis of methyl benzoate Genetic details: regulatory gene, changes in its expression Phenotypic significance: near isogenic lines – hawkmoths preferred scented flowers, even if red – so reproductive isolation!

Case study 3a: Mimulus color

Species: Mimulus lewisii and M. cardinalis Pollinators: bumblebees, hummingbirds Trait: carotenoid deposition (yellow color) Genetic underpinnings: YELLOW UPPER (YUP) controls carotenoid deposition Genetic details: WE.HAVE.NO.IDEA! (very frustrating) Phenotypic significance: near isogenic lines – shifts in bumblebee and hummingbird preference – so reproductive isolation!

Case study 3b: Mimulus color

Species: Mimulus lewisii and M. cardinalis Pollinators: bumblebees, hummingbirds Trait: anthocyanin deposition (purple color) Genetic underpinnings: ROSE INTENSITY (ROI1) represses anthocyanin deposition Genetic details: regulatory gene, regulatory changes Phenotypic significance: F2 experiments show anthocyanin is important, so probably reproductive isolation!

Case study 4: Mimulus nectar guides

Species: Mimulus lewisii and M. cardinalis Pollinators: bumblebees, hummingbirds Trait: nectar guides (yellow stripes) Genetic underpinnings: guideless mutant has no guides Genetic details: regulatory gene, unclear what species differences are Phenotypic significance: mutants have less bumblebee visitation, so could lead to reproductive isolation

Case study 5: Antirrhinum veination

Species: Antirrhinum majus and others Pollinators: bumblebees Trait: petal veination pattern Genetic underpinnings: Venosa controls veining pattern production Genetic details: regulatory gene, changes in its expression Phenotypic significance: pale flowers (B) are equally attractive to bumblebees as dark ones (A) – can compensate for color loss!

Case study 6: Mimulus scent

(this is what keeps me up at night. honest!)

Mimulus as a model system: pollination syndrome evolution

~150 species (western US and Australia)

Huge variation in floral form (color, shape, perhaps scent?)

Diverse pollinator assemblage

Section Erythranthe (7 species): 2 independent bee to hummingbird transitions Beardsley 2003

Mimulus as a model system: ease

Short generation time (3 months)

Large seed set (1000-2000 seeds/fruit)

Grows at high density Ecology well studied

(80+ years), established field sites

Genetics: M. guttatus sequenced and assembled; M. lewisii and M. cardinalis sequenced; 500 Mb genome, 2n = 16

Toby Bradshaw

Mimulus section Erythranthe: an ideal system for pollination study

Bee-pollinated Wide pink nonreflexed corolla Small nectar volume Inserted sexual organs Higher elevation (4000-10,000 ft) Scent?

Bird-pollinated Narrow red reflexed corolla Large nectar volume Exserted sexual organs Lower elevation (0-7000 ft) Scent?

Bumblebee neurons respond to three key scents in Mimulus lewisii…

myrcene

limon

ene

ocimene

… which are found at different abundances in M. cardinalis

Mimulus lewisii

Mimulus cardinalis

Three critical scents, one precursor

Geranyl pyrophosphate (not volatile)

Limonene Ocimene Myrcene

Limonene synthase Myrcene synthase

Ocimene synthase

Three critical scents, one precursor, two enzymes

Geranyl pyrophosphate (not volatile)

Limonene Ocimene Myrcene

Limonene-myrcene synthase

Ocimene synthase

Enzyme assay for myrcene/limonene: M. lewisii

Mimulus lewisii myrcene/limonene synthase

myrcene limonene

(in E. coli)

Mimulus lewisii

ocimene

Myrcene:limonene ratio of 0.08

Myrcene:limonene ratio of 0.1

Myrcene/limonene synthase

Ubiquitin C (control)

M. cardinalis

M. lewisii

Myrcene/limonene synthase is not expressed in M. cardinalis

Enzyme assay for ocimene

myrcene limonene ocimene limonene

ocimene absent

(in E. coli) (in E. coli)

Ocimene synthase

Ubiquitin C (control)

M. cardinalis

M. lewisii

Ocimene synthase is expressed late in the flowering process

Two different mechanisms for lack of scent in Mimulus cardinalis

• Limonene/myrcene synthase (LMS): – Expressed in M. lewisii but not in M.

cardinalis

– M. lewisii allele functional, M. cardinalis allele pseudogenized

• Ocimene synthase (OS): – Expressed in both species – M. lewisii allele functional, M.

cardinalis allele not (16 AA differences) – Causative SNP(s) unknown

X X

pFGC5941

kanR basta (herbicide) resistance

35S promotor

In planta testing: transgenics!

• Stable RNAi lines: – Myrcene/limonene

knockdown – Ocimene knockdown – Double knockdowns

• Allows: – Confirmation of

function – Assays of pollinator

response

Site of synthase fragment introduction

Myrcene/limonene knockdown

myrcene limonene ocimene

Mimulus lewisii flowers

Myrcene/ limonene synthase knockdown flowers ~ M. cardinalis plus ocimene

Changes in scent only; no visual changes!

6.7% wt

1.6% wt

Ocimene knockdown

myrcene limonene ocimene

Mimulus lewisii flowers

Ocimene synthase knockdown flowers ~ M. cardinalis plus myrcene, more limonene

Changes in scent only; no visual changes!

0.7% wt

Okay, so what?

(1) We’ve got phenotypes that differ between species that seem important: myrcene/limonene and ocimene (2) We’ve got their genetic underpinnings: myrcene/limonene synthase, ocimene synthase, transgenics (3) Does scent really matter from an evolutionary standpoint? Does it change pollinator behavior? Could this be involved in reproductive isolation?

• Greenhouse experiments: preference, constancy: captive, free-flying bees

• Knockdowns: LMS, OS (no lim/myr; no oci)

X X

wild type no oci: DONE!

no myr/lim: DONE!

X

Assessing phenotypic significance

Greenhouse experiment setup • Greenhouse: 15’ x 20’ space, two benches • 48 plants (24 each wild-type, transgenic knockdowns) • Hexagonal (equidistant) random array • Bombus impatiens (Eastern US species, commercial hive) • Training: 8-10 days; testing: 3 days • Observations: dawn to noon

Wild-type and transgenics, equal flowers per type each day

Training (no observation other than occasional

bee foraging verification)

Continuous observation

(voice recorder)

Continuous observation

(voice recorder)

Switch out plants

Wild-type only

40%

42%

44%

46%

48%

50%

52%

54%

56%

Days 1-3 Day 1 Day 2 Day 3

M. lewisii (normal) Limonene/myrcene RNAi

Bees are indifferent to the loss of limonene and myrcene

* p = 0.34 p = 0.12 p = 0.03 p = 0.55

% o

f visi

ts (t

otal

n =

135

5)

Bees respond strongly to the loss of ocimene

% o

f visi

ts (t

otal

n =

220

2)

40%

42%

44%

46%

48%

50%

52%

54%

56%

Days 1-3 Day 1 Day 2 Day 3

M. lewisii (normal) Ocimene RNAi

* * p = 0.006 p = 0.15 p = 0.94 p = 0.0008

Is there constancy?

• Myrcene/limonene: 32 bee foraging bouts; permutation test, p = 0.96648 – no constancy

• Ocimene: 46 bee foraging bouts; p = 0.95431 – no constancy

• Shuffled plant locations in simulation and tested ocimene bee tracks for 100,000 iterations to get p-values (“permutation test”)

• Even if bees don’t prefer one type over the other, constancy could keep them reproductively isolated!

Phenotype to genotype to significance Phenotype difference: Ocimene

X X

Phenotype to genotype to significance Phenotype difference: Ocimene Genotype

difference: OS

X X results from

Phenotype to genotype to significance Phenotype difference: Ocimene

X X results from

40%

45%

50%

55%

60%

Wild-type Low ocimene

* Visitation difference

p = 0.006

Genotype difference: OS

Acknowledgments

Bradshaw Lab Toby Bradshaw Mary Sargent Yuan Yaowu Brian Watson Riane Young James Vela Janelle Sagawa David Haak Christina Owen Marina Kovic Funding NSF GRF*, DDIG, and FIBR NIH UW Plant Biology Fellowship GenOM Project (UW/NHGRI) ARCS

Riffell Lab Jeff Riffell Elischa Sanders Marie Clifford Billie Medina Biology Department Doug Ewing Jeanette Milne Paul Beeman Erin Forbush Janneke Hille Ris Lambers Dave Hurley

* This material is based on work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-0718124

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Plant Speciation: Genetics - UW Courses Web Servercourses.washington.edu › bot113 › summer › LectNotes › ... · Plant Speciation: Genetics (Biol 317, but you knew that already)