Germ line polysomy in Atractomorpha similis

GERM LINE POLYSOMY IN

ATRACTOMORPHA

SIMILIS

Gregory Bernard Peters B.Sc.(Hons), A.N.U.

Thesis submitted for the degree of Doctor of Philosophy at the Australian National University

November, 1977

To My Parents

This thesis contains no material which has been accepted for the award of any other degree or diploma in any university and, to the best of my knowledge and belief, it contains no material previously published or written or the result of work by another person, except where due

reference is made in the text.

ACKNOWLEDGEMENTS

I am grateful to my supervisory committee, Dr D,M, Paton, Prof. B. John, Dr G,L.G, Miklos, Dr J. Robbins and Dr J,M, Dearn, for

their guidance during ·the latter half of this project. I am especially indebted to Prof. B, John for suggesting the topic and for his

invaluable assistance during the writing of this thesis.

I also thank Dr O,R, Byrne for his supervision during the early stages of this study. Dr R,N, Nankivell also gave much

assistance and encouragement at that time. To Dr G ,C, Webb I am thankful for assistance in the G- and C-band.ing experiments. Mr M. Adena gave much helpful advice during the early stages in the

construction and implementation of the computer programme used in Chapter Four.

For technical preparation of this thesis I am principally indebted to Mrs T. Raath, who provided patient and cheerful assistance throughout the course of the project. Mr M, Commons and Mr K. Herbert are to be thanked for their skilled preparation of the photographic plates. Other staff members and fellow students of the Botany

Department gave help at various times, and to them I am also grateful.

I thank Ms E, Alfred, Ms B. Britton and Ms K. Su!llm:!rs for much painstaking work in the typing of the thesis.

CHAPTER 1: INTRODUCTION

TABLE OF CONTENTS

Acknowledgements Summary

1.1. The Taxonomy and Distribution of the Genus

Atractomorpha

in Australia

1.2. Biological Features of

Atractomorpha

s

imiLi

s

1.3. The Field Surveys: Rationale and Results

1.4. Rationale for the Thesis

CHAPTER 2: THE CHROMOSOMES OF

Atractomorpha

simiLis

2.1. The Basic Karyotype

2.2. Chiasma Frequency Variation

2.2.1. Chiasma Scores

iv ix

1

3

6

12

14

14

20

20 2.2.2. The Relationship of Chiasma Frequency Variation to Other

Karyotypic Variables 23

2.3. Karyotype Variation

2.3.1. Structural Variation

2.3.1.1. Supernumerary Segment Polymorphism 2.3.1.2. Centric Fusion

27

27

27 31

2.3.2. Numerical Variation ₃₃

2.3.2.1. The Polysomic Polymorphism ₃₃

2.3.2.1.1. Inter- and Intra-Individual Variation 33 2.3.2.1.2. Condensation Cycle and Chiasma

Formation in Normal and Polysomic Copies of Autosome 9 36 2.3.2.1.3. Bivalent and Multivalent Formation

Involving Polysomic Chromosomes 40 2.3.2,1.4. Spermatid Micronuclei 41 2.3.2.2. Aneuploidy in the Embryo

2.3.2.3. Numerical Variation caused by a True B Chromosome in

A

.s

imilis

CHAPTER 3: THE GENETIC BASIS OF GERM LINE POLYSOMY

3.1. Introduction

3. 2. The Selective Breeding of Polysomic Individu·als: Technical

45

49

56

56

Considerations 59

3.2.1. Scoring of the Polysomic Character

3.2.2. The Effect of Inbreeding on Hatchability 3.3.1. Materials and Methods

3.3.2. Transmission of Polysomy: The Mechanism as Revealed by Mating of Selected Individuals and Families

59

63 66

68

3. 3. 3. Maternal versus Paternal Transmission of Germ Line Polysomy 79

3.4. The Long-Term Response to Selection for and against Germ Line Polysomy

3.4.1. The First Laboratory Generation and Its Parents 3, 4. 2. The Inter - Follicular Distribution of Extra A9 's

in Go and G1.

83

84

3.4.3.

The Second Laboratory Generation ₈₈

3.4.4.

_{The Inter-Follicular Distribution of Extra A9's in G2} 92

3.4.5.

The Third Laboratory Generation 93

3.4.6.

The Inter-Follicular Distribution of Extra A9's in G3 95

3.4.7.

The Heritability of Germ Line Polysomy 96

3.4.8.

Further Aspects of the Response to Selection For and

Against Germ Line Polysomy 98

CHAPTER 4: THE GENERATION OF INTRA-INDIVIDUAL VARIATION IN CHROMOSOME

NUMBER 103

4.1. The Structure and Components of the Orthopteran Testis 103

4.2.

The Embryogenesis of the Grasshopper Testis 105

4.3.

Cellular Mechanisms Resulting in Variable Chromosome Number 110

4.4. The Computer Model 113

4.4.1. Aims and Assumptions

4.4.2.

The Mechanics of the Model

113

117

4.5.

Comparison of Simulated and Real Chromosome Number Distributions 124

4.5.1. Salient Features of the Observed Chromosome Distribution and Their Relevance to the Computer Model 124

4.:i.2.

Execution and Results of Computer Simulation

4.6. Conclusions

128

CHAPTER 5: DISCUSSION AND CONCLUSIONS

5.1. The Tranmission of Extra Chromosomes

5.2. The Effect of Various Kinds of Chromatin on the Fitness of the Individual

5.3. The Evolutionary Significance of Extra Chromosomes

APPENDICES

BIBLIOGRAPHY

viii

138

138

150

164

171

ix

SUMMARY

Atractomorpha similis

(Orthoptera, Acridoidea, Pyrgomorphidae)

is distributed along the east coast and adjacent tablelands of Australia

from Sydney, N.S.W., to Cape York Peninsula, Queensland. Populations were

sampled from northern N.S.W. to north Queensland and examined cytologically.

Males of the species carry a widespread polymorphism for polysomy of the

megameric Autosome Nine. This polymorphism occurs in most populations

of the species at frequencies as high as 33%. Polysomic males carry

extra chromosomes only in their germ cells, and, within these, the degree

of polysomy always varies between but not within different follicles of

the testis. Up to ten extra copies of Autosome Nine were observed in

a single cell, although the modal frequency, among polysomic individuals

in the field, is one extra copy. Selective laboratory mating of polysomic

males with females from polysomic families resulted in an increase in the

frequency of polysomic males from 24% to 71% after three generations of

selection. Selection against polysomy resulted in the reducing of this

frequency to only 5% after a similar period of selection.

The irregular manner in which this germ line polysomy was

inherited, coupled with cytological observations on the meiotic behaviour

of the extra chromosomes, suggested that factors promoting germ line polysomy,

rather than the extra chromosomes themselves, were transmitted between

generations. Furthermore, computer generation of simulated populations

of cells mosaic for various degrees of polysomy showed that such mosaicism

population initially devoid of any extra chromosomes.

The nature and behaviour of these extra chromosomes is

discussed in relation to the other more commonly occurring types of

dispensable chromatin, namely supernumerary chromosomes and segments,

both of which also occur in

Atractomorpha similis.

Also, the cytogenetic

peculiarities of

A.similis

are contrasted with those of the related

species

Atractomorpha

australis,

with which it was found to be sympatric

in one population. It is proposed that the various chromosomal

abnormali-ties found in these species, of which germ line polysomy is the most

common, are most probably maintained in natural populations in order to

fulfil various functional roles for which each has become especially

adapted. These various manifestations of genomic specialisation should

be considered as part of a spectrum of genie control mechanisms, which

ranges in scale from the interaction which one single gene may have

with another to the adaptive effects resulting from multiplication of

the entire genome as occurs during polyploidisation.

1

CHAPTER 1

INTRODUCTION

1.1. The ·Taxonomy and Distribution of the genus

Atractomorpha

in Australia

Until 1960, only two Australian species of

Atractomorpha

were recognised. These were

A.australis

(Rehn, 1907) and

A

.

crenaticeps

(Blanchard, 1853).

A.similis

(Bolivar, 1884) was regarded as a southern variety of

A.crenaticeps

(Rehn, 1953), the type specimen of which was

collected in New Guinea. In 1960, Bannerjee and Kevan recognised

A.similis

as a separate species on morphological grounds. Subsequently, the range

of

A.crenaticeps

was reduced to include only New Guinea and some nearby islands (Kevan and Chen, 1969).

A.australis

occurs in relatively humid parts of eastern

Victoria, eastern N.S.W. and southeast Queensland. In the northerly part of its range it commonly lives in the cooler, higher altitudes of the

Great Dividing Range.

A.similis

,

on the other hand, is predominantly tropical and sub-tropical in distribution and ranges from Cape York down

the eastern coasts of Queensland and N.S.W. as far as Sydney. Additionally, some isolated populations are found inland in the Murray-Darling drainage

system of N.S.W., northwest Victoria and south-eastern South Australia. There is also one report of the species in southwest Queensland (Fig.1.1).

Elsewhere,

A.similis

is found in Arnhem Land (NT) and the Kimberley region of Western Australia.

Fig. 1.1 The distribution of

A. similis

and

A. aUBtralis

on the eastern Australian mainland. The shaded areas on this map include all those recordings of the two species in the region given by Kevan (1971). Note that the distribu-tions of the two species overlap near Brisbane and down the NSW coast and adjacent ranges. Populations sampled during this project are designated Sl to S25 although

Sites 14 to 25 are shown on Figs. 1.5 and 1.6. Popula-tions of

A. similis

are represented by large round dots whereas those of

A. australis

are represented by triangles. The symbol~ at Site 10 indicates that both species

were present. The collection of

A. aUBtr

a

lis

at this site extends the known range of this species by some

[image:12.673.15.612.20.806.2]

146° E

QUEENSLAND

Tropic of Capricorn

I

I

I

..

..

.

.

:

-'

I

I

I

I

f

:\:

J

A.similis

[ \\] A. a us

t

r a Ii s

12°S _ __.

See Figs.1.5 and 1.6

I

0

3 6°S

I

200 Kilometres

I

2

occurs only in a very confined habitat along the foot of the scarp

in Arnhem Land. The genus

Atractomorpha

is thus represented in Australia by three species. All three are distinct not only on morphological but

also on cytological grounds (Nankivell, 1976).

A.australis

from southern

and central N.S.W. is easily distinguished from

A.similis,

the latter species, especially the females, being more slender (Nankivell, 1976).

This distinction is not equally clear throughout the range of the two

species. In some areas where both species are found within a few

kilometres of each other, or together as at Site 10, it is impossible to

differentiate the two on gross external morphology.

A.similis

from Sites

5, 10 and 11 could not be distinguished in the field from

A.australis

captured at Sites 6 and 10· (Appendix I, and Fig.1.2). It is quite probable,

however, that an examination of genitalic structure would have made

possible the distinction of the two species.

In the present study, the two species were differentiated by

cytological analysis. At meiosis, the great reduction in chiasma frequency

and increase in number of terminal heterochromatic segments in

A.similis

makes species identification a simple task (see Nankivell, 1976 for a

sunnnary of karyotype differences in the Australasian species of

Atractomorpha)

.

Although

A.australis

and

A.similis

overlap in their distribution, no

sympatric populations had been found prior to the present study. The

population at Site 10 was found to contain both species. Of nine males

examined, six were identified cytologically as

A.australis

and two as

A.similis.

The other one contained no scoreable cells and so could not be identified. This site (Fig.l.3a) is unique in that not only is it

the only place where both species have been recorded living together, but it is also the most northern site of

A.australis.

Kevan (1971) reports

the northern limit of the species as 20 km northwest of Brisbane, some

Fig. 1.2:(a) Specimens of

Atractomorpha

from southern Queensland. These individuals were collected

from Sites 5, 6, 10 and 11. Of the two males

collected from Site 10, ml is

A. australis

and m2 is

A. similis,

as identified on

cytological grounds. The precise identification

of the female is not possible because no

suitable cytological preparations were obtained

from this specimen. The individuals from Sites

11 and 5 are

A.

similis,

while that from Site G

is

A. australis.

Note the morphological similarity

of the two species in these individuals. This

contrasts with their differential appearance in the

southern part of their distributions.

(b) Two laboratory bred individuals of

A. similis

[image:15.662.12.611.17.774.2]

S11:

f

S6:m

1

I

m

1cm

L...J

'

(

\

S10:f m1 m2

S5:

f

m

Fig. 1.3:

Fig. 1.4:(a)

Site 10; Splinter Creek, looking south along western bank of the creek. Both

A. austraZis

and

A. simiZis

were collected at this site, where they were found randomly dispersed on

the vegetation.

Abnormal spermiogenesis in an individual of

Atractomorpha

from Site 10. Note the wide

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TABLE 1.1 RESULTS OF INTER-SITE SINGLE-PAIR MATINGS

*

A: Matings between different populations of

A.simiZis

Copulation

Female Parent Male Parent Observed Hatchlings

Site 5 Site 9

9 5

9 1

11 8

8 11

8 5

B: Between-species matings

Site 6

6

2

5

Laboratory-bred

A. simi

Zis

Site 9 5 11 2

A. austraZis,

Sullivan's Creek, A.C.T.

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

(4)

+

.

(2**)

*

More than one single-pair mating was set up in most cases. Normally, three pairs were so mated.

** These two hatchlings were the total progeny of ten single-pair matings.

Adults

+

+

+

+

[image:19.666.20.650.15.793.2]

3

hybridise at Site 10, as they do copulate in the laboratory (Table 1.1). ·The one undetermined male from Site 10 might possibly·have been hybrid as

it appeared to be sterile. Its testes were undeveloped, no meiotic

divisions were found, and the few sperm present were abnormal in appearance (compare Figs. 1.4a and b). However, considering the extremely low

viability of hybrids seen in the laboratory, it seems unlikely that hybrid individuals, sterile or otherwise, would be detected in such a small

population sample as that taken from Site 10. Previous attempts to hybridise the species in the laboratory have all proved unsuccessful

(Nankivell, 1976). Inter-specific matings reported in this study also failed to produce any viable progeny, except in one mating, where a single sterile adult female resulted (Table 1.1).

1.2. Biological Features of

Atractomorpha

simiZis

In his 1971 paper, Kevan listed 114 recordings of

A.simiZis

or its earlier synonyms. This species is particularly well known between Townsville and Mosman on the north Queensland coast and 47 of the 114 reports relate to this area. Like other species of the genus,

A.simiZis

lives in moist situations and feeds on dicotyledonous herbs (Key, 1969, p.341).

4

of low vagility and occurs, over the less humid parts of its range, in

small discrete populations several kilometres apart. Only in the relatively

humid "refuge area" in New Guinea, around Cairns and near the N.S.W./

Queensland border are continuous populations found. "Refuge areas" are

those which, as a result of their particularly favourable climates, were

the only areas inhabitable by many species during periods when the climate

of the entire region was much drier than at present. These areas and the

times at which they were acting as refuges are discussed in detail by

Keast (1961). In these areas,

A.simiZis

is present along roadsides, in

overgrown areas in and around cultivated fields and in domestic gardens.

In less humid areas, the species is restricted to the banks of creeks and

rivers.

A.simiZis

has a preference for fleshy dicotyledonous food plants

and hence is much more common in weed-infested or cultivated areas than in

native vegetation. The advent of European settlement has probably led to

a wider distribution of this endemic species. However, despite its

widespread abundance, it is difficult to catch in large numbers because it

often lives amongst dense ground cover. Its tendency to crawl rather than

fly from plant to plant makes it difficult to find in thick vegetation.

A.simiZis

has no winter diapause, unlike

A.austraZis

which is

univoltine except possibly in the "refuge area" near the N.S.W./Queensland

border, near the northern limit of its range (Site 6, Fig.1.1). Judging

by the generation times observed in the laboratory,

A.simiZis

could go

through 2.5 generations per year in its north Queensland habitat. A great

deal of generation overlap was observed in all north Queensland populations.

Field records made during collection note the presence of most instars

occurring contemporaneously. Collections were made during April and

September and at both times individuals at all stages of maturity were

5

year. Populations in north Queensland are perennial. Specimens of

A.similis

raised in the laboratory have survived up to the age of 14 months

and females may still lay eggs up to the age of 12 months.

The north Queensland climate varies most with respect to rainfall,

and the year may be divided into two seasons: dry, May to October, and wet,

November to April (Atlas of Australian Resources, 1959). This division

becomes less pronounced inland, as total rainfall drops dramatically.

This trend is shown in the map of the region (Fig.1.5). The average annual

rainfall in the area ranges from approximately 2500 mm in some coastal

areas to 1000 metres southwest of Mareeba, around Site 21. It should be

noted that above average rainfall has been recorded in this region for

every year since 1970. The isohyets shown in Fig.1.5 are those supplied

by the Bureau of Meteorology for 1975. In that year, mean rainfall in

the Barron Meteorological District, which includes the area of this study,

was 15% above average. The altitude of sampled population sites ranges

from six metres (Site 17, Fig.1.6) to 900 metres (Site 20). Variation in

the vegetation is largely correlated with rainfall, and ranges from

tropical rain forest on the eastern edge of the Tableland and the coastal

plain, from which it has been almost totally removed by man, to dry

sclerophyll woodland around Site 21 and further west.

On the humid coastal plains, grasshoppers are relatively

plentiful, and are dispersed evenly rather than concentrated in small

pockets, as they are further inland. The nature of these two types of

site is shown in Fig.1.7. The inland populations are often confined to

disturbed areas in the sclerophyll forest. Such sites are usually beside

roads or under bridges at river or creek crossings. The disturbed areas

around the bridge earthworks are often colonised by noxious and other weedy

species such as Noogoora burr

(X

anth

i

wn chinen

s

e

),

blackberries

(

Rubu

s

Fig. 1.5: The frequency of germ-line polysomic males in populations of

A. similis

taken from the

Cairns/Atherton Tableland region of North

Queensland (see inset of Fig. 1.1 for

location and scale). Isohyets, drawn from

the regional annual rainfall map for 1975

(Australian Bureau of Meteorology) are

labelled at the bottom of the map with the

annual rainfall in millimetres. Grasshopper

collection sites are denoted as S14 to S25

and the relative frequencies of polysomic

individuals at each site are represented

as unshaded sectors in the pie diagrams.

The number of individuals sampled at each

[image:23.666.6.612.20.776.2]

S 2 2 ~

S 2 1 ~

1 O O 0

160 0

2 400

3200

I

I

146°E

170

s

J.+.-,-Fig. 1.6: The topography of the Cairns/Atherton Tableland region, showing the positions of

[image:25.660.6.607.17.772.2]

D

<

200m

~

.-:::::·

:-:

2 0 0 - 1 0 0 0

m

-:··

_{. ::::-·}

·:·:-146

°

E

Fig. 1. 7: The two major kinds of site at which populations of

A. similis

are found in no_rth Queensland. (a) A site (S18) on the humid coastal plain, 18 km north of Cairns.

A.

simiLis

was found in the

grassy region stretching from the foreground to the sugar cane. (b) A typical riparian site

[image:27.665.7.613.18.780.2]

6

Polygonaceae and Compositae.

Some of these species provide ideal food souTces for

A.simiZis,

which rarely eats grasses or xerophytic shrubs. The abovementioned species are hydrophilic and, in drier areas of the region, their growth is primarily limited by lack of rainfall. During the winter drought, the plants, and hence the grasshoppers, live in a much smaller area and in reduced numbers. The grasshoppers need moist· bare earth or sand in which to lay their eggs and the sides of river banks, ploughed fields or roadsides provide ideal laying sites.

1.3. The Field Surveys: Rationale and Results

This study began with the specific aim of surveying the chromosome variation present in widespread populations of the grasshopper,

A

.s

imiZis.

To this end, the initial field collection surveyed populations of this species from northern N.S.W. to north Queensland, a range of some 2000 kilometres (Fig.1.1). Only relatively small numbers of individuals from each site were sampled at this stage since the intention was to maximise the number of populations examined.

TABLE 1.2 INITIAL SURVEY: FREQUENCIES OF GERM LINE POLYSOMIC MALES

IN

A.SIMILIS

POPULATIONS

Polysomic Males Sample Size

Site: II Name Number Frequency

5 Kedron Brook 0 0 3

7 North Yandina 0 0 5

8 Catfish Creek 2 0.33 6

9 Raglan 1 0.17 6

10 Splinter Creek 0 0 2

11 Barambah Creek 1 0.25 4

12 Black River 0 0 15

13 Cordelia 0 0 13

14 Innisfail 0 0 14

15 Atherton 1 0.10 10

16 Mt.Molloy 2 0.20 10

17 Rocky Point 2 0.20 10

18 Reed Road* 2 0.13 15

x

=0.10 ~ =113

TABLE 1.3 OCCURRENCE OF POLYSOMIC AND B CHROMOSOMES

IN

A.AUSTRALIS

POPULATIONS

Name

S*2 Musk Valley Creek

S 3 Crystal Creek

S 4 Nixon's Creek

S 6 Mt Glorious

S10 Splinter Creek

*

Site

Individual

II

1 2 3 4 5 1 2 3 1 2 3 4 5 6 1 2 3 1 2 3 4 5 6

Number of Extra.Chromosomes Per Follicle

Polysomics B's Follicle

II

1 2 3 4

2 1 2 0 0 0 0

1 2 2

2 0 6 3

0 2 0 1 2 2 0 3 0 0 2** 0 0 0 0 0 1

Follicle ti

1 2 3 4

0

0 0 0 1 0

0 0

0

0 0 0 0

1 1 1 2 0 0 0 2 0 0 0 0 1 0 0 0 0

supernumerary chromosomes are not homologous with any member of the

standard set (White, 1973, p.312; Rees, 1974) whereas,. as shown in Fig. 7

1.8, the extra chromosome in

A.similis

occasionally establishes a clear

chiasmate connection with the megameric chromosome of the standard set, A9.

Individuals carrying such extra chromosomes are thus unmistakably polysomic.

The occurrence of polysomy on this scale is rare in any organism

and is especially so among animals (Khush, 1973, Ch.12). It was, therefore, of considerable interest to attempt an analysis of the nature, the extent and the consequences of such polysomy. Since the first collection had shown that polysomy in

Atractomorpha similis

was most frequent in the Cairns/Atherton Tableland region of north Queensland, this area was chosen

as the site for a detailed examination of the phenomenon.

In the Atherton Tableland/Cairns region, the maximal climatic

change over the shortest distance occurs along a line running WSW from the coastline near Cairns. The Cairns/Mareeba/Dimbulah road provides a

reasonable approximation to this line. Reports of

A

.

similis

from several sites along this road were recorded in Kevan's 1971 review. The species extends southwest from Dimbulah, on the road to Chillagoe, for at least 32 kilometres, as far as Eureka Creek (Site 21), where the annual rainfall

is approximately half that found in Cairns. Unfortunately, this transect does not afford maximal variation in altitude or, therefore, in mean

monthly temperature. The Tableland varies between 350 and 450 metres above sea level along this road. Further south around Ravenshoe the altitude

reaches 900 metres and more. One population was sampled in this region (Site 20, Fig.1.6) which is as high as any known habitat of

A

.

similis

in Australia. An additional site from the humid coastal plains near Innisfail

was chosen, as this area has a mean rainfall of approximately 2500 mm per

annum, the highest in the region, and indeed, the equal of the highest rainfall

Fig. 1.8:

Fig. 1.9:

Pachytene of male meiosis in

A. sirrrilis.

This cell is trisomic for the megameric Autosome 9, and

the 3 copies of A9 are associated in what appears

to be a trivalent, designated 9:iii. One

interstitial and one terminal chiasmata are

present in this trivalent. The centromeres

of the 3 AG's are marked with small bars.

Late diplotene of male meiosis, showing the

presence of a univalent B chromosome. Also

shown are 2 heteromorphic bivalents, including

a very large homozygous segment on Autosome 7.

The characteristic megameric bivalent, designated

9m, is also identified, as is the X. Note the

close proximity of the X, the megameric 9m and

the heterochromatic half of the B. Such

associations are often observed, but do not

usually persist beyond diakinesis. This cell

(Site 19), 90% of them died during or soon after transportation to the

laboratory. Among samples from the other seven sites visited during this

same expedition, survival to the time of fixation varied between 36 and

64% (Table 1.4 ) .

8

This field survey would perhaps have been more successful if

material had been fixed in the field, as it was during the preceding field

collection. However, a major aim of the last collection trip was to provide

enough live animals for establishment of breeding stocks containing polysomic

chromosomes. As the suspected frequency of individuals carrying these

chromosomes was conservatively estimated at 10%, at least 20 live males

from each site would have been required in order to ensure the availability

of a reasonable number of polysomic parents for future breeding.

The second major aim of this field survey was to test whether

the frequency of polysomic individuals in different populations could be

correlated with any environmental variable. The Cairns/Atherton Tableland

region is geographically and climatically heterogenous to a far greater

degree than any region of comparable size throughout the range of

A.similis.

It was therefore expected that if maintenance of extra chromosomes in these

populations was achieved by any environmental selection pressure, then a

correlation between some measurable variable and frequency of polysomy

would be most easily detected within this region. A similar rationale

II

has been used elsewhere. Bosemark (1967) studied the frequency of

accessory chromosomes in the plant

Phlewn phleoides

on a Baltic island

which was chosen specifically for its heterogeneity of soil type. The

istand was 140 x 15 km, a size sufficiently large to provide significant

environmental variation, but small enough to ensure that all plants present

would not be so genetically diversified as to have evolved different

responses to any given value of a relevant environmental parameter. In the

TABLE l.4 Site II 18 19 20 21 22 23* 24 25

THE FREQUENCIES OF EXTRA CHROMOSOMES IN POPULATIONS SAMPLED IN NORTH QUEENSLAND

N 48 8 16 20 18 36 31 48 Site Altitude (Metres) 20 90 900 400 450 500 450 350

Frequency of % Survivial

Polysomic Between Collection Males and Examination

0.15 64

0 9

0.19 40

0.25 36

0.33 25

0.33 51

0.19 52

0.27 64

~ = 225 X = 0.23

these prerequisites than does any other area available.

The wide-ranging but smaller population samples of the first field collection yielded information on chromosomal variation

9

in the species over long distances. On this scale, any local peculiarities could be the product of genetic isolation as well as adaptation to

local selection factors. The results of the earlier collection are shown in Tables 1. 2 and 1. 3. What one can conclude from these is

that polysomy in

A.simiZis

seems to be more connnon in the Cairns/Atherton Tableland populations than elsewhere. Nankivell (1976) also found a relative absence of polysomy in southern populations of the species. He

observed no polysomy in Dee Why (Sydney) and Balranald (southwestern N.S.W.) populations, but did observe trisomy for A9 in one individual from Coff's Harbour (north coast of N.S.W.). This individual also contained a single B chromosome (Nankivell, 1976, his Fig. 3), similar in appearance to the only other B chromosome known in this species

(Fig.1.9), which was found, during this study, in two individuals from Site 23 in north Queensland (Fig.1.5), 1600 km from Coff's Harbour.

B chromosomes occur much more frequently in

A

.

a:ustraZis

.

Nankivell (1976) reports a large and a small B chromosome in the Gilderoy (Melbourne) population. Two similar species of extra chromosomes were found in this study, in the Site 4 population of

A.a:ustraZis.

Extra chromosomes of one kind or another were found

in

A

.

australis

populations from Sites 2, 3, 4, 6 and 10 (Fig.1.10).

These appear similar in morphology to either the large or the small B chromosome seen in the Gilderoy population (c.f. Nankivell, 1976, his Fig.?.), despite the fact that Gilderoy and the populations sampled here are at least 1000 km apart.

Fig. 1.10:

Fig. 1.11:

Extra chromosomes found in populations of

A

.

austraZis.

(a) AB chromosome found at S2 (Musk Valley Creek). The heterochromatic end of the Bis associated with the X chromosome which is itself associated with the megameric bivalent (labelled 9m). (b) A small extra chromosome, designated 9e, which was found at S3 (Crystal Creek). This cell is therefore

tetrasomic for A9. The individual was mosaic with respect to the number of extra copies of 9 present and the inset includes part of a cell from another follicle in the same individual, containing only three extra A9's, here associated as a trivalent (9e:iii). (c) The large B chromosome found at S4 (Nixon's Creek). This individual contains one B, although two others from this same site had

two B's each. (d) The small chromosomes labelled u were observed in an individual from S6 (Mt. Glorious). From their degree of condensation, it is probable that these chromosomes are unpaired megamerics from an essentially polysomic

individual and that the bivalent labelled 9ii represents a pair of additional A9's. (e) The semi-heterochromatic B chromosome found in one

A. austraZis

male from S10 (Splinter Creek).

Diagrammatic representations of the B chromosomes found during this study in both

Atractomorpha

·s.

C

.

,

~ Sp

A-l ~'\ '

.-.

- -

.

.

'

d

Q)~

I

52

t'

:/

u\

S4

9e

,

,

e

•

•u

I

9ii

J

x

I

xtl

S10

S23

'

...

C

4 and 6 (Table 1.3) could possibly be an extra copy of A9 rather

than a true B. The megameric chromosome of

A.australis

is smaller and less distinctive than its counterpart in

A

.

similis,

and so

identification of extra copies of it is more difficult. However, the

small chromosomes from Site 3 do occasionally exhibit a striking

similarity to the megameric pair (Fig.l.lOb).

·This small extra· chromosome and the similar one from Site 4

(Fig.l.lOc) are present in variable numbers in the germ line, unlike the

large B chromosome coI!IlTlon at Site 4 and the smaller Bat Site 2 which

are present in invariant numbers within an individual. When three

of the small unstable chromosomes are present, they can form a

trivalent (Fig.l.lOb). The behaviour of these small chromosomes, as

will be demonstrated in Chapter Two, is remarkably like that of the

polysomic chromosomes common in

A

.s

imilis.

If the small extra chromosomes of

A

.

australis

are extra copies of the megameric, then, in the population sampled at Site 3, such polysomy appears more

frequently than does polysomy in any known

A.similis

population

{Compare Tables 1.2 and 1.3). Up to six extra A9's were seen in the

germ line of

A

.

australis

.

A.similis

and

A

.

australis

may both commonly exhibit apparent germ line polysomy, but they do not show such similarity in their

possession of true B chromosomes. In

A.australis,

a

large-sized stable B chromosome was found to be very common at Site 4

and intermediate sized stable B's were found at Sites 2 and 10 (Table

1.3). These occurred in four out of six individuals at Site 4, one

out of five at Site 2 and one out of six at Site 10. All three B

chromosomes were semi-heterochromatic, and are shown in Fig.1.11.

10

this B chromosome was also heterochromatic for half its length and thus

similar in appearance to the B reported in an individual from Coff's

Harbour, N.S.W. (Nankivell, 1976, his Fig.3).

11

A striking feature of the findings of the last field collection

is that there is no sign{ficant difference between the frequencies of

polysomy at any of the seven sites visited. These frequencies ranged from

15 to 33% with a mean of 24% (N = 217) (Table 1.4) if Site 19 from

which only nine males were sampled is excluded. By contrast, out of a

total of 68

A.similis

collected earlier from Sites 5 to 14, only four were polysomic. These sites all lie south of the Cairns/Atherton Tableland

district. Two of the polysomic individuals were found at Site 8, the

others at Sites 9 and 11. AG-test of the difference between this sample

and the Cairns/Atherton Tableland sample gives a highly significant result.

G = 11.852 as calculated by means of the 2x2 test of independence (Sokal

and Rohlf, 1969, p.591). Such a G value indicates that the probability

of both samples coming from one homogeneous population is less than

0.005. The null hypothesis, that there is no difference in frequency of

polysomy over the range examined, is thus rejected.

Although only four polysomic males were found in the southern

and central range of

A.similis,

it should be noted that polysomy was also found in this species at Coff's Harbour, N.S.W. (Nankivell, 1976). It

therefore seems that germ line polysomy, although it is more connnon in

the north, occurs throughout most of the species range in eastern Australia.

The same is probably true of

A

.

australis

,

although true B chromosomes are an equally frequent phenomenon in the latter species.

The small numbers of

A

.si

milis

sampled from populations in southern Queensland make it difficult to state whether these sites differ

significantly from those further north in respect of the frequency of

Tableland region were from southern Queensland. What does seem certain, therefore, is that the populations between Townsville and Innisfail

12

(Sites 12, 13, 14 and 19) are relatively devoid of polysomy, compared with

the Cairns/Atherton Tableland populations. There is no recognisable

environmental factor common to these sites which could be responsible for

this. The annual rainfall at Innisfail is approximately 1000 mm compared

with 300 mm p.a. at Townsville. Most other environmental variables change as a result of this rainfall differential.

From the field data presented here, it is not possible to

identify any environmental factor which correlates with the frequencies of polysomy found in natural populations of

A

.similis.

These frequencies do vary, but only over distances of hundreds of kilometres, which are far in excess of the distance over which populations of this invagile species could have been in recent genetic contact.

1.4. Rationale for the Thesis

In summary, the survey of natural populations of

Atractomo

r

pha

s

imilis

revealed the occurrence of male germ line autosomal polysomy in 14 of the 20 populations sampled. As some of these 14 populations were small and isolated, containing, for example, an estimated one to

13

in natural populations are these: (1) Any adaptive advantage or disadvantage

suffered by the individual as a result of germ line polysomy; (2) The

efficiency with which a basis for polysorny is transmitted from one

generation to the next. In essence, this thesis attempts an investigation

of these two factors, with emphasis on the second, as this is a cytological

rather than an ecological study. The data presented in this chapter shed

some light on the first factor: the adaptive significance of germ line

polysorny. It is clear that if there is any such adaptive significance,

it is only marginal, as there is no drastic net reduction or increase in

fitness associated with germ line polysomy. The equivalent viability of

polysomic and non-polysomic laboratory stocks, discussed in later chapters,

reinforces this conclusion.

The remainder of this thesis falls into four major sections, each

of which is concerned with a different aspect of the polysomic system in

A.s

imilis.

The aims of these sections can be briefly summarized as follows: (1) To define the precise nature of the polysomic variation encountered

within and between individuals and to consider it against the more general

background of the other forms of cytological variation encountered within

this species. (2) To investigate the basis of transmission of the polysomic

state and to consider the results of laboratory matings, including selection

both for and against polysomic parents; (3) To attempt a computer simulation

of the patterns of variation observed within this polysomic system in order

to define more clearly the causal factors involved in the maintenance of

this system and (4) To compare the observed behaviour of the polysomic

chromosomes with that of supernumerary chromosomes and other forms of

CHAPTER 2

THE CHROMOSOMES OF

ATRACTOMORPHA SIMILIS

2.1. The Basic Karyotype

The karyotype of

Atractomorpha

similis

is that of a typical

pyrgomorphid grasshopper. The male has 2n

=

18

+

XO, the female

18

+

XX. All the chromosomes are rod-shaped (Fig.2.1). There is a

current controversy as to whether such chromosomes have terminal or

sub-terminal centromeres. Proponents of the latter claim that a small,

usually invisible, arm is present in all rod chromosomes which are hence

acrocentric (Prokofieva, 1935; Muller, 1938). In some organisms, it

is known that experimentally produced telocentrics, formed by fission 14

of a metacentric at the centromere, show impaired centromeric activity,

manifest in lagging at anaphase (White, 1973, p.204). Clearly such

unstable chromosomes would be expected to be rapidly lost from natural

populations. The arguments in favour of telocentric chromosomes are

based on the observations that (a) some spontaneous novel telocentrics,

which also arise by fission of a metacentric, are certainly not unstable

at either mitosis or meiosis (Southern, 1969; John and Hewitt, 1968)

and that (b) in many species, and particularly Orthopterans, the

centromeres appear to be genuinely terminal, as they seem to be in

A.similis

(cf. Fig.2.2a and b).

For the purpose of this thesis all the chromosomes of

A.similis

will be referred to as telocentric. Autosome Nine is distinct

Fig. 2.1: The mitotic karyotype of

Atractomorpha

simiZis

(9). This cell is from a female embryo with the

conventional chromosome complement of 18

+

2X.

Only three of the chromosomes pairs can be reliably

recognised at mitotic metaphase. namely the X,

which is the largest, the AS, which has a conspicuous

procentric segment, and the A9, the smallest element

in the complement. The other chromosomes have

been graded simply on size. This is not always

a reliable guide because of the high frequency

[image:45.690.5.636.21.802.2]

8

2

4

9

'

7

4

₅

3

8

5

2

X

9

₃

s

X

A1

A2

A3

A4

Fig. 2.2:(a)

Fig. 2.3:(a)

Anaphase II of meiosis in

A. similis

to indicate the terminal position of the centromeres

(denoted by small bars). Note that the

megameric half-bivalent (9m)has already separated.

A large heteromorphic segment (r--1) is present on

one of the autosomes. (b) Anaphase I of an

extra univalent A9 in

A.

sunilis

showing that its centromere (indicated by bars) is also terminal.

Late mitotic prophase in a standard male of

A

.

similis

showing the uniformly condensed nature of all the chromosomes. Centromeric

positions are indicated by bars. (b) Diplotene

of male meiosis in

A. similis

showing the various heterochromatic segments occurring in this species.

Seven of these are polymorphic (r---,) in this

individual. Because of the internal differentiation

a

b

9in

,

-9m,

.

I

I

-• Sp

I

a

_b

X

, ~ . _

.

' '

~

c,x

·

,

I

tf

~]\

I I

~

,

~r

·

7

f''I.,

.~~.

-,_

2

,1!

...,

:

,

, v

i~

•

,

13;

1 ;

~

₊

• l ~

~~

••j\

\ ~# I · ~ ·

~I

_,.,,.

\

,, l''

I

-'

.

, s,,. •

behaviour, which is common in grasshoppers, identifies the A9 as the

megameric chromosome, as first described by Corey (1938).

The chromosomes of

A.similis

are distinguished from those

of other species of the genus by the presence of distinct terminal

or sub-terminal heterochromatic blocks in all chromosomes other than

Al. Smaller blocks are present at the centric ends of all the

autosomes. None of these heterochromatic segments are apparent at

mitotic prophase (Fig.2.3a), but most are well defined at prophase

of meiosis (Fig.2.3b). There is considerable variation in the number

and the size of the heterochromatic blocks present in

A.similis.

Fig.2.4 shows a C-banded meiotic cell from a male heteromorphic for

four terminal and one sub-terminal segments, as well as two probable

procentric segments. If the commonest arrangement is taken as a

standard then the chromosomes can be classified as shown' in Table 2.1.

This table also indicates the extent of the variation seen in respect

of heterochrornatic segments. This variation is discussed in more

detail in Section 2.3.1. By reference to the features described in

Table 2.1, it is possible to classify individual members of the

complement by the symbols Al to A9 as illustrated in Fig.2.5.

The X chromosome in the male is negatively heteropycnotic

from metaphase I to telophase I (Fig.2.5), but not differentially

pycnotic in mitosis (Fig.2.3a). It is also negatively heteropycnotic

15

in most spermatogonial mitotic metaphases. The X chromosome commonly

behaves in this manner in the short-horned grasshoppers (White, 1973).

Other notable features of the X in

A.similis

are its large size, it being

in fact the largest chromosome, and its characteristic sub-terminal

Fig. 2.4: Diakinesis in a C-banded cell heteromorphic for 3 terminal, 2 sub-terminal and 3 centric

segments. The segments in Al, A3 and A7 involve variation in the amounts of both C-band positive and C-band negative chromatin. Note that the X chromosome is C-band positive only at its

centric end and at the site of the sub-terminal band which is visible after conventional staining. In this cell, the A9 bivalent appears to have undergone 2 crossover events. This coupled with

9

5

X

2

_I 514 _I

X

A1

A2

A3

. I

AS

Al

Al

A9

I I

\;

l

.

.

TABLE 2.1

Chromosomes

X

A 1

2

3

DIAGNOSTIC FEATURES OF THE KARYOTYPE OF

A.SIMILIS

Conventional Meiotic Appearance

positively heterochromatic in prophase. Negatively heterochromatic from metaphase I to telophase I. Non-staining sub-terminal band usually present, and visible up to and sometimes beyond telophase I.

very small amount of heterochromatin around centromere, and sometimes visible at

sub-terminal and terminal distal end.

small centric heterochromatic block, and

small sub-terminal distal block.

small centric heterochromatic block, slightly larger sub-terminal distal block, and smaller terminal block.

Conventional C-banding

(all have

centric C-bands)

small terminal and large

·sub-terminal C-bands

two large sub-terminal and occasionally a small terminal band

broad sub-terminal and small terminal bands

narrow sub-terminal and small terminal bands

Supernumerary Segments

Major super-numerary segments

Minor super-numerary segments

as revealed by C-banding

non-staining band may be of variable

width

terminal band may be present, interstitial bands may vary in

size

sub-terminal band may be of variable width.

4

5

6

7

8

large distal block small centric block large distal block large centric block, appears bipartite at times

similar to A5, but all heterochromatic blocks are smaller here

often

indistinguishable

small amount of condensation at and near centric end. Distal end free of

heterochromatin.

small amount of centric heterochromatin, distal end euchromatic

9 adjacent blocks of centric terminal and (megameric) sub-terminal heterochromatin, interstitial

euchromatic segment, and small distal terminal euchromatic segment.

large sub-terminal distal heterochromatic block.

all have large terminal band

centric band only when segment absent same as

A7

five evenly spaced and sized bands along length of A9

large distal heterochromatic block on one pair

very large ·

segment, 20%

euchromatic large distal heterochromatic block

small interstitial band sometimes present,

distal and centric segments of variable size

centric band of variable size

16

division and, to a lesser extent, in mitotic division.

It is difficult to consistently distinguish particular

chromosomes at mitosis in this species because all of them are rod-shaped.

In meiosis, the problem of identification is somewhat alleviated by the

presence of large and distinctive heterochromatic blocks in bivalents

such as A9, and their absence in Al. However, the considerable amount of

segment polymorphism mentioned earlier makes identification by

heteropycnotic character somewhat unreliable. Identification of mitotic

chromosomes is not assisted at all by reference to heterochromatin,

because, apart from the negatively staining X band, heterochromatic regions

are not visible in condensed mitotic chromosomes. As the aim of this

project was the analysis of the extensive polysomy found in natural

populations, the identification of polysomic as distinct from supernumerary

elements in both spermatogonial and somatic mitoses was essential.

The identification of specific chromosomes is frequently

facilitated by techniques of chromosome banding. These were introduced

in 1971 (Caspersson et al., 1971; Arrighi and Hsu, 1971; Drets and Shaw,

1971) and include several different types of staining and chemical

pretreatment of the chromosomes. Banding techniques may be broadly

classified into two types. One type, involving only mild pretreatment,

includes the G-, R- and Q-banding processes. The other, known as

C-banding,demands more drastic pretreatment. In the G and R techniques,

chromosomes are stained with Giemsa, whereas the Q technique involves

the use of fluorescent dyes such as Quinacrine mustard. The G, Rand Q

techniques give essentially similar banding patterns. The human karyotype

has been extensively studied by these methods, and roughly corresponding

hands are produced in all three cases, although R-bands have reversed

stain intensities (Paris Conference, 1971: Standardization in Human

technique, as well as all the G-bands. Because only mild pre-treatment is required to give G-bands, many simple techniques have been reported, each giving similar results (references given in Paris Conference, 1971). Common pretreatment techniques involve trypsin digestion or heating in solutions of Na2P04, SSC, distilled H20 or NaOH solution (Comings et al., 1973). In the present study, trypsin pre-treatment was used, following the method of Seabright (1971), with some modifications (Webb, 1976).

There is as yet no satisfactory explanation of the molecular nature of chromosome bands. Evidence (McKay, 1973; Comings et al., 1973; Schneider and Nagl, 1976) suggests that G-bands result from a disruption of chromosome regions containing particular acidic proteins. The

preferential removal of the cations associated with these acidic groups, and their replacement with some component of the Giemsa stain may lead to the formation of a darker staining band. This partial denaturation also removes some of the DNA, mainly from the non-staining regions. Severe pre-treatment, such as that used to produce C-bands, results in much greater DNA loss, again mainly from the non-staining regions.

C-bands generally appear in a small segment of each chromosome. Their distribution corresponds approximately to that of constitutive heterochromatin. Hence in humans, C-bands occur around the centromeres and in the long arm of the Y chromosome. In

A

.s

imilis

,

Fig.2.5 shows that C-bands are present near the centromeres as well as at the distal ends of most chromosomes, corresponding in many but not all cases to the large heterochromatic blocks seen with normal staining.

G-bands, by contrast, are distributed more evenly over the entire genome. In humans, G- (and

Q-

and R-) bands occur over nearly all chromosome arms. Unlike C-bands, they are not limited to the region immediately adjacent to the centromere, and, in humans, are rarely found at chromosome tips (Paris Conference, 1971). In Orthopterans generally,

Fig. 2 .5:

Fig. 2.6:

The diagnostic features of the karyotype of

A. similis

at meiotic prophase. In this figure, dots have been placed adjacent to the distinguishing

features of each of the chromosomes. These features

arc discussed in Table 2.1. Centromeres of all the

chromosomes are represented by the small bars. The

two larger brackets (r---i) indicate heterochromatic

segments in A7 and A8 respectively. Segment

polymorphism is so common in this species that

individuals are rarely, if ever, found without

any such segments.

A spermatogonial mitosis after G-banding. This

cell is from an individual carrying the stable

B chromosome derived from the population at S23.

The cell is also abnormal in that it contains

only one A9. The Band the A9 are the only

members of the complement which do not G-band

throughout their entire length, although there

is a tendency for a number of the autosomes to

stain lightly at or near their centromeric ends

(bars). The centromeres of the Band the A9 are

indicated by arrows. The A9 is not stained

at or adjacent to its centromere. It has two

interstitial G-positive bands. With the

exception of the small band adjacent to the

centromere, the centric half of the Bis

X

A1

....

•

•

•

•

_I

A4

AS

'

I

A7

AS

t

A2

-•

•

A6

/

•

Al

. I

A3

•

•

•

•

18

almost the entire genome is G-band positive, and differential staining

is usually restricted to some of the highly condensed heterochromatic

blocks of the megameric chromosome, and specific segments of supernumerary

chromosomes (Webb, 1976). -Fig.2.6 shows a G-banded spertnatogonial mitosis

from

A.similis

which behaves in a similar fashion to other Orthopterans in that only the B chromosome, which is G-positive for half its length,

and the megameric A9 are not homogeneously stained.

Daniel and Lam-Po-Tang (1973) showed that in the chromosomes of

the vole

Microtus agrestis,

as in humans, most but not all constitutive heterochromatic blocks correspond to C-bands. Similar observations can

be made from the distribution of C-bands in

A

.s

imilis

.

Figs.2.7 and 2.8

show C-banded chromosomes from an adult spermatogonial mitosis and a

male embryo respectively. In both cases, it is evident that the C-bands

generally correspond to the heterochromatic regions visible in a

non-meiotic cell (Fig.2.5), but there are some exceptions to this. Some

of the heterochromatin of the megameric A9 is not C-banded. Conversely,

some bands appear to correspond to no visible heterochromatic blocks

of appropriate size. This is the case with the band on the distal end

of the X, and the two large sub-terminal bands on Al (Fig.2.5).

There is some parallel between this C-banding distribution

and that of the particular heavy DNA satellite isolated from

A.similis

by Miklos and Nankivell (1976).

In situ

hybridisation with an RNA

copy of this satellite showed that it was localised in the distal

heterochromatic blocks of all autosomes other than AS, but was absent from

the centric blocks. This satellite copy also annealed to the negatively

staining sub-terminal band on the X. However, some of the heterochromatin

of A9 behaved in an exceptional manner. Here, the labelled RNA copy

annealed only at or near the distal end despite the fact that both C-bands

Fig. 2.7:

Fig. 2.8:

C-banded spermatogonial mitosis in a cell containing the normal male

chromosome complement. Heteromorphic C-banded segments occur on Al, A3,

AS,

A6, A7 and A8. There also appears to be some differential C-banding of the A9 homologues, but the A9 often C-bands erratically.