Chromosomal inversion polymorphism in 2 marginal populations of the endemic Hawaiian species, Drosophila silvestris

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Chromosomal inversion polymorphism in 2 marginal

populations of the endemic Hawaiian species, Drosophila

silvestris

Hl Carson, Em Craddock

To cite this version:

Original

article

Chromosomal inversion

_polymorphism

in

2

_marginal

_populations

of the

endemic Hawaiian

_species,

Drosophila

silvestris

HL

Carson

EM

Craddock

1

University of Hawaii, Department of

Genetics and Molecular

Biology,

John A Burns School

_{of Medicine, Honolulu,}

HI _96822;

2

State

University of

New

_York,

Division

_of

Natural

Sciences,

Purchase,

NY 10577-1400, USA

(Received

9 March

_{1995; accepted}

30

August

1995)

Summary - Chromosomal

_{polymorphisms}

for 11 inversions from 2 demes of

Drosophila

silvestris from the volcano Mauna

_Kea,

Island of Hawaii are distributed over 4 of the

5

_major

chromosome arms. These occur at

frequencies

from 2 to 70%. Seven of these

inversions have been found

widely

distributed in other

populations

of this

species.

The 5 inversions in chromosome 4 were

_{analyzed by}

a new method that

_permits

the determination

of

_{haplotype frequencies}

of linked inversions and the

_scoring

of _genotypes formed

_by

them. This

_variability

appears to

_persist

in a state of balanced

_polymorphism

in these small isolated insular

_populations

and is as _great as or _greater than that found in

_comparable

demes of continental

_species.

chromosomal _variability

_/

small _population

_/

island _biology

_/

inversion

_/

_Drosophila silvestris

Résumé - _{Polymorphisme} d’inversion _{chromosomique} dans 2 _{populations marginales} de _{l’espèce endémique} hawaïenne _Drosophila silvestris. Des

_{polymorphismes}

chromo-somiques concernant 11 inversions, observées dans 2 dèmes de

_Drosophila

silvestris du

Mauna Kea

_(île

de

_Hawaï),

se _{répartissent sur 4 des} 5 bras

_{chromosomiques}

princi-paux. Leur

fréquence

varie de 2 à 70%.

Sept

de ces inversions sont

_{largement répandues}

dans d’autres

populations

de cette

espèce.

Les 5 inversions du chromosome

4

ont été

analysées

selon une méthode nouvelle qui permet de déterminer les

_fréquences

haplotyp-iques d’inversions liées et le

_repérage

des

_génotypes

_qui en résultent. Cette

variabilité,

qui semble _persister sous

forme

d’un

_{polymorphisme équilibré}

dans ces _petites

populations

insulaires

_isolées,

est au moins aussi importante que celle

qu’on

observe dans des dèmes

comparables d’espèces

continentales.

variabilité _{chromosomique}

_/

_{petite population}

_/

_biologie insulaire

_/

inversion

_/

INTRODUCTION

The island of Hawaii is less than half a million _years old

_(Moore

and

_Clague,

1992).

Drosophila

silvestris

_(Perkins)

is endemic to the island and thus has been inferred to be a _young autochthonous

_species

(Carson,

_1982).

_Using

chromosomal

sequences, the

origin

of this

_species

can be traced

_{phylogenetically through}

forms

on

_slightly

older Hawaiian islands back to ancestors on the island of

_Kauai,

which

is about five million years old. When

population genetic

data on D silvestris from

many

populations

around the island are

_{analyzed quantitatively}

_by

hierarchical F

statistics, high

levels of local

_genetic

differentiation are revealed

(Craddock

and

Carson,

1989).

Local demes are

_small,

distinct and restricted

_{demographically,}

ecologically

and

_{altitudinally.}

Periodic destruction of older forested areas

_by

lavas on the

_{currently growing}

shield volcanoes

_requires

such a

_species

to be

_continually

_{recolonizing.}

This

_imposes

a

_{metapopulation}

structure

_(Carson

et

al,

1990).

The

_species

shows

_high

_variability

for inversions and

_isozymes

_(Craddock

and

_Johnson,

₁₉₇₉₎

as well as

_geographical

variation for

_{morphological characters, including}

some that are related to

_courtship

behavior

_(Carson,

_1982).

Variation due to chromosomal inversions in this

_species

is

_spread

over 4 of the 5

major

chromosomes of the _genome.

_Using

a new method that

_{permits recognition}

of chromosomal

_haplotypes,

we

_present

a detailed

_description

of the

_segregating

chromosomal

_variability

in this

_{species, using population samples}

from 2

_localized,

high-altitude

demes.

MATERIALS AND METHODS

Samples

were collected at sites about 9 km

_apart

on Mauna Kea. The Maulua site is in the North Hilo District at

_Spring

Water

_Camp

_(altitude

1539

_m)

in dense

Metrosideros-Cibotium forest. The Hakalau site

_(Hakalau

Wildlife

_Refuge

of the United States Fish and Wildlife

_Service)

is near Pua

_Akala,

South Hilo District.

At 1890 _{m, it} is not far below the tree line on the mountain and

_represents

the

highest

altitude from which a

_sample

of this

_species

has been obtained. At this

site,

there is an

_overstory

of

_large

Acacia koa and Metrosideros collina trees. Both

samples

were collected

_by

_baiting

with fermented mushroom and banana within

quite

small areas; the most distant

_baiting

_positions

at each

_locality

were no more

than 50 m

_apart.

Maulua was

_sampled

in

_January

1979

_(Maulua-1)

and

_again

in

October 1980

_(Maulua-2)

and Hakalau in March and November

_1990,

and

_April

1991. The last 3

_samples

are

_homogeneous

(P

>

_0.25)

and have been

pooled.

The

gametic

frequencies

of the inversions at Maulua were

reported

by

Craddock and

Carson

_(1989);

the

_present

_paper

_presents

an extension of the

_analysis

to include

haplotype

data,

as

explained

below.

In both

_{populations, segregating}

inversions are

_present

in chromosomes

_X,

_2,

3 and

_4;

most of these are

_{geographically widespread}

entities in the

_species.

This _paper

concentrates

_particularly

on variation in chromosome 4. In both

populations,

5 inversions

_segregating

in substantial

_frequencies

are

_present.

A feature of this

_study

is the

_development

of methods to determine the

_{haplotype frequencies}

_(linkage

inversions,

most of which are

separable by crossing

over in the

visibly

_large

sections

of chromosome between the inversions

_{(fig 1).}

Each wild

_specimen

was

_{separated by}

sex at

_capture

and was

_brought

to

the

_laboratory

and

_{analyzed separately.}

Wild males were crossed to females of

laboratory

stock

_T94X,

which is

_{homokaryotypic}

for _gene

_arrangement

_(X,

_{2, 3m,}

4k

2

,

4t,

5: this formula should be read

_{’homozygous}

standard

_X,

standard

_2,

standard

_5,

_homozygous

for inversion m in chromosome 3 and

_homozygous

for

inversions

k

2

and t in chromosome

_4’).

Examination of routine aceto-orcein smears

of the

salivary gland

chromosomes of at least 7

_F

₁

larvae was then used to infer the chromosomal

_composition

of each wild male. The lack of

_crossing

over in the male

(see

Carson and

_Wisotzkey,

₁₉₈₉₎

_permits

a direct

reading

of the _sequence of each

chromosome as it occurred in the wild

population.

The

_analysis

of females was

_{accomplished by obtaining}

_progeny from each wild

specimen

(isofemale method)

and

_inferring

2

_{parental zygotic}

_karyotypes

from

the wild from the

_composition

of 12 or more

_F

₁

_{progeny. In}

_{prior studies,}

_many

sequential

combinations have remained

ambiguous

because of

_tight

_synapsis

of the

homologues

and thus could not be recorded. In this

_study,

2 methods were used that

permitted

accurate

_diagnosis

of all

_{sequential haplotypes}

present

in the wild

parents.

First,

in almost _every chromosome smear it was observed that in a small number

of

_cells,

_usually

_passed

over in routine

_analysis,

the 2

_homologous

members of a

polytene

chromosome had

_fortuitously

separated

from one

_another,

at least in

_part.

Accordingly,

the _sequence of at least one of these

asynapsed

strands could be read

by

direct visual

_sequencing

of the

_banding

order.

_Reading

of one such

_homologue

permits

the inference of the sequence of the other.

Second,

it was

_possible

to remate

wild females to T94X males without

introducing

ambiguity.

These wild

populations

and 4 that the

_paternal

contribution of each chromosome of the

_laboratory

stock is

effectively

marked.

Accordingly,

these methods

_permit

the

inference,

from a

_single

isofemale,

of 2

complete

’wild’

_{zygotic karyotypes.}

Where the

_laboratory

_{sperm is}

_{accepted by}

the wild female after progeny has

already

been

produced

from a wild

male,

it is

possible

to infer which

_zygotic

combination

_represents

that of the wild female.

_Analysis

is

facilitated

_by

the fact that _sperm

_precedence

in D silvestris

_by

the second male

_(P2)

is above

80%

_(Wisotzkey

and Carson

_1986).

Two cases of

_multiple

insemination

_by

a second wild male were

_recognized

in this

_study.

RESULTS

Table I

_{gives gametic frequencies}

of the inversions

_present

without

_regard

to their association with one another. The

_frequency

of inversion

41

2

differs between the Maulua-1 and Maulua-2

_samples

_{(P $ 0.001).}

Tables II and III

_give

the

_frequencies

of the

_haplotypes

observed in chromosomes 3 and

_4,

_{respectively.}

In the

_latter,

the

5 inversions form 3

_regional

_groups over the

_length

of the chromosome:

k

2

is distal:

t and

_p

₃

act

as alleles in

_segregation

and are

central;

1

2

and

m

2

are allelic and

proximal

(fig

1;

see also

_photograph

in Carson

1987).

_Crossing

over within

_regions

1 and 2 in

_{triple heterozygotes}

is rare

(9/1586

and

7/1521, respectively)

when

the

_zygote

is

_t/+

in the center. In 2

_{instances, however,}

data were obtained on

crossing

over in females from informative

_karyotypes

_8/11

and

_{7/12 (see}

table

_III).

Both of these are

t/t

in the

_mid-region

of the

_chromosome,

affording

observation

of crossovers in an

_enlarged

central

_{homokaryotypic}

section

_(regions

1 +

2;

fig

1).

The first of these data sets was obtained from a

_virgin

_laboratory

female from stock U34B4

_(Kohala

_Mountains)

crossed to a known

_{homokaryotypic}

male. The _progeny

showed 14 crossovers in 50 observed

_gametes

_(0.28).

A second case

(karyotype 7/12)

was found in a wild female from Hakalau. Of 18

_gametes

_observed,

8 were crossovers

(0.44).

Although

these data are _sparse,

they

demonstrate that intrachromosomal

recombination among the inversions in chromosome 4 occurs and can account for the _presence of some rare

_haplotypes

in the

_population.

The theoretical maximum

number of

_haplotypes

in chromosome 4

_{generated by}

such

_crossing

over is

_18;

16 of

these have been observed at Maulua and 14 at Hakalau. In chromosome

_3,

_only

3

haplotypes,

of a

_possible

_4,

have been observed.

Table IV lists the inversion

_genotypes

observed in the 3

samples

and their

frequencies.

The number

_(N)

of different inversion

_genotypes

_{theoretically possible}

from each

_{sample given}

at the bottom of the table is calculated as N =

k(k

₊

1)/2,

where k

represents

the number of chromosome 4

_haplotypes

observed in each

sample.

Even in the

_larger

_sample

from

_{Hakalau, only}

a little more than one-third

(0.38)

of the

_possible

variation has been revealed.

In order to test for conformance to

_expectation

under the

_{Hardy-Weinberg}

equilibrium,

the observed inversion

_genotypes

of chromosome 4 listed in table IV have been divided into 4

_classes,

those that are

_{homokaryotypic,}

and those that are

heterokaryotypic

for

_1,

2 or 3 inversions

_{(table V).}

For the

chi-square

values

_given

in this

table,

the number of

_degrees

of freedom is

_given

as two

_(2).

_{Conservatively,}

this number

_might

be viewed as less than

_this,

since some of the

_haplotypes

are

deviations from

expectation

by

Maulua-1 and Hakalau would be

_only

of

_marginal

significance

at the

5%

level. We conclude that the observed numbers do not

_depart

significantly

from

_expectation.

Inversion

genotypes

in chromosome 3 were also

_compared

with

_expectation

based

on the

frequencies

of the 3

haplotypes

(table

VI).

There _appears to be no deviation from

_expectation.

DISCUSSION

Chromosomal

_polymorphism

in D silvestris has been shown to be

reasonably high

throughout

the distribution of the

species,

which shows

_striking

altitudinal clines as

about the

_populations

described here is the almost

_genome-wide

distribution of the chromosomal

_{polymorphism.}

Chromosome 5 is

_{monomorphic throughout}

this

species

and also in the other 4 members of its cluster of

_closely

related

_species

on

Hawaii, Maui,

Molokai and Oahu. The

_polymorphism

in these demes is

_highlighted

by

its extensive nature in chromosome

_4,

in which 16

_(Maulua-2)

and 14

_(Hakalau)

haplotypes

have been

_recognized

and their

_zygotic

combinations observed.

Compared

with a well-studied case of a continental

_species

_{(table VII),}

it is clear that the current

populations

of this

_newly

formed insular

_species

are far

from

_depauperate

in

_genetic

variation.

_Indeed,

this

_tendency

is even

_expressed

in

altitudinally marginal populations

such as the one described from Hakalau. These

findings

confirm earlier estimates for other Hawaiian

_species

_(Ayala

_1975;

Craddock and Johnson

_1979).

What forces within these small

_populations

are

responsible

for the retention

of this

_variability?

The data from the

_analysis

of the

_haplotypes

of chromosome 4

suggest

that

_they

combine at random at _syngamy

_according

to the

_{Hardy-Weinberg}

equilibrium

and that no

_major

viability

differences are

_apparent

between the various

Respectively,

for Maulua

_(—1

and

_-2)

and Hakalau: total number of genotypes observed:

16,

38,

68; number of different genotypes observed: 12,

27, 40;

number of different

Four of these chromosome 4

_haplotypes

from a different

_geographical

_area,

however,

have been studied in

_laboratory

_experiments

_(Carson

_1987;

Carson and

Homokaryotypes, however,

were

greatly under-represented

relative to

heterokaryo-types

among those flies that bred

successfully

in the

laboratory. Accordingly,

we

_suggest

that a

comparable

heterotic

reproductive

success _may occur in these more

complex

natural

populations, making

this

_yet

another case of differential

reproduction favoring

heterokaryotypic

individuals. As each inversion covers a

substantial section of

_chromosome,

the amount of

_{genic heterozygosity}

retained

is also

_expected

to be

_large.

Although

these insular

_populations

of D silvestris have been

_apparently

naturally

subjected

to

_repeated

recolonizations and bottlenecks on these

_growing

shield

volcanoes, they

nevertheless appear to retain a

_large

amount of

_{genetic variability}

that is manifested even in

_marginal,

_relatively

isolated demes.

ACKNOWLEDGMENTS

We thank K Kaneshiro, W Perreira and C Simon for

_assisting

in the

_collecting

and L Freed

and R Cann for

logistical

support in the field. R Wass, of the Fish and Wildlife Service

kindly

issued a

_Special

Use permit for the collections in the Hakalau Forest Reserve. T

Lyttle

and R

Wisotzkey

gave valuable advice on statistical matters. We are also

grateful

for the work of L Teramoto

_Doescher,

who reared the larvae and made the chromosome

smears. Work

supported by

NSF _grant BSR 84-15633 to the

_University

of Hawaii.

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FJ

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