Population genetics Population genetics ofof

Liothyrella Liothyrella neozelanicaneozelanica

in Breaksea Soundin Breaksea SoundErik SuringUniversity of Otago,

Dunedin, New Zealand

Marine Science 480Research Project

Marine Gene FlowMarine Gene FlowGene flow occurs by larval dispersalLarval life history characteristics

– Larval type – passive or swimming– Duration of planktonic phase– Settlement characteristics– Spawning time

Geographic factors– Past or present physical barriers to genetic

exchange– Fiord hydrography may limit gene flow

Fiord DynamicsFiord DynamicsSpecial type of estuarine flow (Stanton and Pickard 1981)

Liothyrella neozelanicaLiothyrella neozelanica

Brachiopoda:Articulata:Terebratulidae– Reported in Fiordland as far north as

Doubtful Sound– Found attached on overhangs– Aggregated distribution (Richardson

1981)– Lecithotrophic, brooded larvae

(Chuang 1994)– Found at depths below 20 meters

Terebratella sanguineaTerebratella sanguinea

Articulate brachiopod– The most common and widely

ranged brachiopod in Fiordland– Less gregarious (Richardson

1981)– Attached or free-lying– Found between 5 and 35 meters

Sample LocationsSample Locations

Inner and Outer locations from Breaksea Sound– 32 animals from inner

location– 27 animals from outer

location– Collected during

summer, 1999

Breaksea Sound

DoubtfulSound

Dusky Sound

Breaksea SoundBreaksea Sound

Genetic DifferentiationGenetic DifferentiationNo genetic exchange – populations

equilibrate at different allele frequencies– Longer and more complete isolation causes

increased fixation of differencesGenetic markers quantify the amount of

genetic variation– Different markers record different processes

and show different scales of variation

Population QuestionsPopulation QuestionsHow much genetic variation is there in L.

neozelanica?Is the population structure of an inner fiord

site different from an outer fiord site in Breaksea Sound?

Is the genetic differentiation (Fst) between inner and outer populations different between species?

How differently do separate genetic markers resolve genetic population structure?

Allozyme ElectrophoresisAllozyme ElectrophoresisIndirect measure of sequence variation

– Based on electrophoretic changes in proteins that represent allelic changes - phenotypes

– Some loci can be affected by selection, does not register all variation

Technique is well documented– Relatively cheap and easy, though polymorphic

enzyme systems can be hard to find (Parker et al. 1998)

Allozyme Electrophoresis Allozyme Electrophoresis MethodsMethods

Cellulose-acetate gels– Homogenize tissues– Add to gel– Run gel– Stain gel

• Hexokinase (Hk)• Glucose-Phosphate

Isomerase (Pgi)• Peptidase (Pep)

Amplified Fragment Length Amplified Fragment Length Polymorphism (AFLP)Polymorphism (AFLP)

Also an indirect measure of sequence variation– Cut entire genome with restriction enzymes– Amplify cut fragments– Compare size and number of fragments

AFLP resolves finer differences than allozyme electrophoresis

Allozymes – Hardy-Weinburg Allozymes – Hardy-Weinburg EquilibriumEquilibrium

BreakseaInner

BreakseaOuter

Hk Het Obs 14.3 10.9Het Exp 17.0 10.0P value 0.43 1

Pep Het Obs 24.2 20.7Het Exp 21.0 23.0P value <0.0001 <0.0001

Pgi Het Obs 8.2 0.0Het Exp 5.0 2.0P value 0.002 0.019

Overall P value <0.0001 <0.0001

Enz

yme

Allozymes – Peptidase Allele Allozymes – Peptidase Allele FrequenciesFrequencies

Breaksea Inner Breaksea Outer

Allele frequencies in each group for HK and PGI were very similar

Allozymes – FAllozymes – Fstst

Liothyrella neozelanica Fst

Breaksea Inner

Breaksea Outer 0.031*

Terebratella sanguinea Fst

George Inner

Dusky Inner

George Outer -0.0067

Dusky Outer 0.0227**

* p = 0.05, ** p = 0.01

FFstst Comparisons ComparisonsPopulations Mean Fst

L. neozelanica Breaksea Sound 0.031Paracyanthus sternsiiCalifornia coast – Planktonic larvae(Hellberg 1996)

0.004

Evechinus chloroticusAll New Zealand – Planktonic larvae(Mladenov et al. 1997)

0.019

Balanophyllia elegansCalifornia coast – Crawling larvae(Hellberg 1996)

0.195

AFLPAFLPBands were visible for some animals but

overall the gel was underdevelopedThis could be remedied by

– Using increased radioactivity– Using a different DNA extraction method

We have some of the problems have been worked out and may have useful data soon…

HeterozygosityHeterozygosityTwo loci, especially peptidase, were not in

Hardy-Weinburg equilibrium– Banding patterns may not represent allelic

variation– Banding may have been improperly scored– Natural selection may favor certain alleles– Inbreeding– Small populations– Small sample size

No conclusions can be made about disequilibrium without further tests

Genetic DifferentiationGenetic Differentiation

There is significant population structure within Breaksea Sound in L. neozelanica.

Not much allozyme allelic variation (except in peptidase)

There are also indications that L. neozelanica may have greater within fiord population differentiation than T. sanguinea– Perhaps because of short planktonic phase and

aggregated distribution limits gene flow

Technique ComparisonTechnique ComparisonAllozymes may evolve too slowly to be

informative in this situation– Fiords have been inundated for less than 10,000

years (Pickrill et al. 1992)AFLP may show finer genetic

differentiation In Lingula anatina DNA sequences have

revealed genetic structure not seen with allozymes (Endo et al. 2001)

Further WorkFurther WorkFind a technique with greater resolution

– L. neozelanica seems to have low variabilityAnalyze a greater number of sites

– More inner and outer fiord replicates– More sites within the fiord

Make comparisons with different species– Species with planktotrophic larvae– Underway by Cecile Perrin and Gigi Ostrow

ConclusionsConclusionsThere is significant population structure

within Breaksea FiordL. neozelanica has low allozyme variabilityL. neozelanica may have greater within

fiord differentiation than T. sanguineaFurther study of the interactions between

the fiord environment and larval life histories is promising

Thank you!Thank you!

Gigi OstrowDr. Stephen WingDr. Michael Roy

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