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Studies on natural populations of four Melanoplus species (Orthoptera : Acrididae) from Quebec, Canada
ARTICLE · AUGUST 1984 Source: OAI
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Sumaia Abukashawa University of Khartoum
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'1
STUDIES ON NATURAL POPULATIONS OF FOUR
MELANOPLUS SPECIES (ORTHOPTERA:ACRIDIDAE)
FROM QUEBEC, CANADA
BY
Sumaia Mohamed Abukashawa
A thesis submitted ta the faculty of Graduate Studies and
Research in partial fulfillment of the requirements for
The degree of Master of Science
Department of Entomology Macdonald College of McGill University Montreal, Quebec, Canada @ August 1984
v ( , .. ln the name of God, ..tost Gracious, Most Merciful. 1 Proclaim! (or Re~d) ln the name of thy Lord and Cherisher, Who created -----
Created ~~n, out of A (mere) clot of congealed blood:
Proc1aim! And thy Lord Is ~'ost Bountiful, - ( He who taught (The use of) the Pen. ----
Taught man tha t Which he kne~ not.
~1-Quran: Chapter XCVI. Verses 1 - s.
J j j r
(
ABSTRACT
M. Sc. Sumaia Mohamed Abukashawa Entomology
CYTOGENETIC STUDIES ON NATURAL POPULATIONS OF FOUR MELANOPLUS SPECIE5 FROM QUEBEC, CANADA
Natural populations of four distinct taxa in the
grasshopper genus Melanoplus Stal (2n Ô = 22 + XO) were
studied over a period of three years. A detailed
description of the chromosome complement and the meiottc
system of Melanoplus bivittatus, ~. ~. borealis, M. f.
femurrubrum and ~. ~. sanguinipes ls presented. Intra- and
1 ( inter-specifie chromosome variability, with special reference to chromosome structure, supernumerary
chro~osomes and chiasma frequency and distribution, was
investigated. The species differed trom one another in
respect of the size and the shape of the autosomes.
!!. !. femurrubrum popul"ations were polymorphie for a
meiotically stable B-chromosome which assoeiated with the , X. The supernumeraries of M. bivittatus, ,M. b. borealis
and M. !. sanguinipes were found to have an autosomic
nature. A unique structural variant of the standard B was
( i encountered in ~. ~. sanguinipes. Distinct cases of
spontaneous fra~mentation of A elements were revealed in a
population of ~. bivittatus. The meiotic behaviour and
chromatic expression of these centric fragments provide
evidence on the possible origin and evolution of
supernumeraries.
Chiasmata were found to be localized in aIl four
species. Significant effects of supernumer4ries on chiasma
frequency and distribution were detected in M. s.
sanguinipes where an increase in chiasma frequency ( accompanied the presence of the B-chromosomes. The study revealed that a high percentage of the karyomorphs within
the four Melanoplus species lacked one or ~more of the
chromosomes. The missing chromosomes were not strictly the
smaller autosomes~ large, medium, small and even
X-chromosomes were involved.
( ii ( RESUME
M.Sc. Sumaia Mohamed Abukashawa Entomologie
ETUDES CYTOGENETIQUES SUR LES POPULATIONS SAUVAGES DE QUATRE ESPECES DE MELANOPLUS DU QUEBEC, CANADA
Les populations sauvages de quatre différentes espèces
de l'orthoptère Melanoplus Stal (2n ~ = 22 + XO) furent
étudieés durant une p~riode de trois ans. Une déscription
dêtaille~ du compl~ment chromosomal et du système meiotique
de Melanoplus bivittatus, ~. ~. borealis, M. f. femurrubrum
et ~. ~. sanguinipes est pr~sente~. La variabilit~
chromosomal intra- et inter-spécifique a êté étudieé en
fonction de la structure chromosomale, des chromosomes '
supernuméraires et de la fréquence et la distribution des
chiasmes. Les différentes espèces varient entre elle~ en
ce qui concerne la taille et la forme des autosomes. Les
populations de ~. !. femurrubrum sont polymorphes pour un
chromosome 8, stable A la méiose et qui est associé au
chromosome X. Il a Até d'terminé que les chromosomes
supernu.éraires de M. bivittat~s, M. b. borealis et M. s.
s~nguinipes sont de nature autosomique. Une variante
structurale unique du chromosome B a 't' découverte chez
( iii
( ~. ~. sanguinipes. Des cas de fragmentation spontanée d'éléments A ont êté révèlées dans une population de ~. bivittatus. Le comportement meiotique et l'expression chromatique de ces fragments centriques fournissent la preuve de l'origine et de l'évolution possible des chromosomes supernuméraires.
Il fut détérminé ques Ips chiasmes sont localizés dans les quatre espèces. Des effets significatifs des supernuméraires sur la fréquence et la distribution des chiasma furent detéctés chez ~. ~. sanguinipes où la croissance de la fréquence des chiasmes si lie â la présence des chromosomes B. L'étude révèle aussi qu'un ou plusieurs chromosomes manquaient dans un pourcentage élevé des karyomorphes de ces quatre esp~ces. Les chromosomes manquants nrétaient pas nécessairement les petits autosomes: des longs, moyens, petits, et même des
1 chromosomes X furent impliqueée.
iv Short Ti tle
CYTOGENETICS OF FOUR MELANOPLUS SPECIES
Sumaia Mohamed Abukashawa
vi ( •
ACKNOWLEDGBMBN'l'S
l wish to express my sincere gratitude to Dr. V.R.
Vickery, my research director, for his continuous interest,
advice and encouragement throughout this investigation, and
for unlimited access to his personal reprint collection.
He has always been friendly, helpful and cooperative.
l am grateful to Dr. O.K. MeE. Kevan, Director of the
Lyman Museum and Research Laboratories, for facilities
, enjoyed in this institution.
l am very grateful to the staff of the Entomology
Department for their valuable assistance. Special thanks
are due to Dr. R.K. Stewart, Chairman of the Entomology
Department, for his valuable corrections.
l wish to acknowledge with gratitude the contribution
of Mr. C.C. Bsuing"who helped in collecting the material
and providing accurate information on collecting areas.
Sincere appreciation is extended to Dr. M. J~vahery for his
help on several field trips. Mr. P. Genier was extremely
helpful in printing some of the original photomicrograpba.
vii
( .. ' ,.
•
1 would like to thank Mr. P. Langlois for reproducing
the photographs presented in this the sis and for his help
and vatuable advice.
• The assistance of J. Schor~cher in the French
translation of the Abstract is gratefully acknowledged.
1 owe a special debt of gratitude to my husband, Abd î
E1rahman, without whose support, encouragement and
understanding this work would have never been completed. A
word of love to my son Ibrahim whose presenc~,. has always
been a motivation to carry on with my studies. Special
( thanks are due to my family back home for their moral support.
I am indebted to the Sudan Government and the
University of Khartoum for sponsoring my studies,
appreciation should be made to McGill University and the r ' Department of Entomol~ for the oppjbrtunity they made
availab1e to enroll me. Thanks are due to Diana Lidstone • J who took the trouble of typing my thésis,in the best
possible manner.
I owe an apology to a11 those who he1ped and encouraged
me to start this study for not me~~ioning their names, a
( ward of thanks is reserved for them.
viii TABLE OF CONTENTS
Page
ABSTRACT ...... l
RESUME ...... i 1 1
ACKNOWLEDGEMENTS .... V 11
LIST OF TABLES .. Xlll
LIST OF FIGURES ..... xv
INTRODUCTION ...... l
.. LITERATURE REVIEW ...... 4
1. Cytology of the Acrididae:
A backg round ...... 4
2. Supernumeraries or B-chromosomes ... 7
a. Distribution of B-chromosomes ... 8
b. Inheritance of B-chromosomes .... 9
c. Structure of B-chromosomes...... 14
n. Polymorphism of B-chromosomes.... 15
e. Effects of B-chromosomes...... 16
f. Origin of B-chromosomes...... 22
_/
ix Page
3 ... Ploidy ...... 24
MATERIALS AND METHODS ...... 31
1. Bioloqical materials ...... 31
2. Chromosome preparation techniques ...... 31
3. Microphotography and chromosome
measurements ...... 35
4. Chiasma distribution ...... 36
THE CYTOGENETIC SYSTEM OF MELANOPLUS BIVITTATUS (SAY). 38
1. The complement and the structure of
t he chromosomes...... 38
2. The B-chromosome system ...... 39
a. Frèquency and morphology ...... 39
b. Ch la sma frequency ...... 42
c. Ch iasma dist r ibution ...... 47
3. The fragments ...... 48
a. The fl fragment ...... 48
b. The f2 fragment ...... 48
4. Missing chromosome•...... 52
5. Meiosis of polyploid cell •...... 53
x Page
THE CYTOGENETIC SYSTEM OF MELANOPLUS FEMURRUBRUM
FEMURRUBRUM (DE GEER)...... 59
1. The str~cture of the chromosomes ...... '" 59 \ 2 . The B-chr~osome system ...... 59
a. Frequency and morphology...... 59
b. Behaviour of the B-chromosome...... 62
c. Chlasma frequency and distribution. .... 66
3. Changes in chromosome structure and number. 71
THE CYTOGENETIC SYSTEM OF MELANOPLUS BOREALIS
BOREALIS (FIEBER)...... 75
1. The karyotype ...... 75
2. The 3-B karyomorph ...... 76
3. Chiasma frequency and distribution ...... 81
4. Missing autosomes and other
irregulari tiea...... 83
THE CYTOGENETIC SYSTEM OF PmI...AHOPLUS SAlIGUUTIPES
SAliGUINIPES (FABRICIUS)...... 86
1. The standard complement ...... 86
xi / (
Page
2. The B-chromosome syste•...... 90
3. Chiasma frequency and distribution in
relation to the 8-chra.osome...... 94
4. Karyomorphs with irregularities ...... 98
DISCUSSION ...... •...... , 101
1. Karyotypic differences between the four
Melanoplus species ...... 101
2. The Supernumeraries: A Comparison ...... 103
3. Chiasma conditions of the four species .... . 108
4. Chromosomes and taxonomy ...... 113
SUMMARY AIID CONCLUSIONS. . . • ...... • • • . • . • ...... • • . . •• 11 7
REFERENCES •...... ••••..••.••..•.•.•.• , 121
xii 1 LI ST OP TABLItS
Table Page
1. The lengths of the chromosomes of Me1anop1us
bivittatus ...... 41
2. The chiasma frequency data in M. bivittatus ... 44
3. Inter-follicu1ar variation in the nwnber of
B-chromosomes of M. bivittatus ...... 46
4. Inter-follicular variation in fhe absence of
an autosome from the complement of ~.
bivittatus...... 54
5. A between-generation comparison of B-chromo-
some frequency in three populations of
Melan~1us femurrubrum femurrubrum ...... 64
6. Type and frequency of X-B associations at
diplotene-diakinesis in 10 males of ~. f.
femurrubrum with a supernumerary ...... 65
7. B-chromosome segregation patterns with respect ~ to the X-chromosome in M. f. femurrubrum ..•... 68
8. The c~asma frequency data in M. f. femurrubrum
with and without a supernumerary ...... 69
xiii Table Page
9. Chromosome measurements of Melanoplus borealis
borealis...... 79
10. The variations in centromeric positions of the
chromosomes of M. borealis borealis •...... 80
11. Chromosome measurements of Melanoplus
sanguinipes sanguinipes...... 88
12. The variations of centromeric positions of the
chromosomes of M. s. sanquinipes ...... 89
13. A bet~een-generation comparison of B-chromosome
frequency in three populations of ) ~ . .!_ sanguinipes ...... 91 (---
14. The chiasma frequency data in ~. ~. sanguinipes
~ith and ~ithout B-chromosomes .•••.....•...•••. 96),
xiv ( LIST OP PIGUllBS
Figures Page
1. Idiogram of M.lanoplu8 birittatus mal ••••...... 40
2. First meiotic Metaphase in M. bivittatu8
to show the difference in pycnosity betwe.n the X and B-chromosome...... 43
3. Meiotic division in M. bivittatus to show the " different configurations of chiasmata •••••.•... 49
4. The structure of the telocentric fragment (fl)
at first metapbase of M. bivittatus •••••.••.••• 50
5. Translocation of the fragment (f2) in the
medium bivalent (Me) of M. bivittatus •••••••••. 51
6. Second division cells with missing X-chromosome
and showing heteromorphic bivalents in
M., b i vitta tus...... • . . . . • . • • . . • . . .. 55
7. Second meiotic division in M. bivittatus to show
ploidy...... • • • • • • • . . • . . • . . • • • • • • • • • • • • • • • •. 56
8. Second meiotic division in M. bivittatus showing
different degress of ploidy within the same
individual. . . . • • • . . • • • . . • • • • • • • . • • • • • • . • • • • • • •• 57 ~
xv ( ,
FIGURES Page
9. Po1yploidy in M. bivittatus second division.... 58
10. Idiogram of Melanoplus femurrubrum femurrubrum
male...... 60
11. Meiotic division in M. f. femurrubrum to
show types of associations between X and B ..... 63
12. Melotic division in M. f. femurrubrum to show
different chiasmatlc configurations ...... •...•. 70
13. Second meiotie metaphase in M. f. femurrubrum showing heteromorphlc bivalents and chroma tin
strands between X and M6 bivalent .••...... •.•.. 73
14. Idiogram of Me1anoplus borealis borealis .••.... 77 ( 15. Meiotic division in M. b. borealis...... 78
a. The complement of M. b. borealis •..•...... • 78
b. M. b. borealis with 3 Bs...... 78
16. Melotic division in M. b. borealis with
missing autosomes...... 85
17. Idiogram of Melanoplus sanguinipes SangUi~es. 87
18. Melotie division in males of M. 8. sanguini~s. 92
a. Positive heteropycnoaity of X and B ...•..... 92
xvi
( FIGURES Page
b. A structural variant of the ba.ic
B-chrCll108C1D.8. • . . • . . • • • . • • . • . • . • • • • • • . . • • . . • 92
c. Position of the 8-chromosome in relation ta SI O· ...... 92
19. Second meiotic divisions in ~. s .•anguinipes
to show different chiasma configuration •...... 97
20. Second meiotie Metaphase of ~. ~.
sanguin! pies...... 99
a. A cell from an individual with the M3
autosOIDe mi seing...... 99
b. Heteromorphie M3 and sites of breakage
a t LI and L2 ...... 99
"
xvii IR~RODOC~IOR
The large grasshopper genus Melanoplus Stal is one of
the Most characteristic components of the North A.erican
Acrididae. The genus is interesting in that it contains a
large number of species (at least 18) which show
considerable diversity both in morphology and habitat.
Melanoplus species have been grouped into a number of
sections or series, using characters of wing length, colour
of hind tibia, shape' of prosternaI spine, and the general
shape of the male and female genetalia. Previous groupings ( do not agree with groupings based on the aedeagus, and at present, the exact relationship of the Many species is
unknown.
MelanopluB bivittatus (Say) -the two-striped
grasshopper-, Melanoplus femurrubrum femurrubrum (De Geer)
-the red-legged grasshopper- and Melanoplus sanguinipes
sanguinipes (Fabricius) -the lesser migra tory grasshopper-
share the distinction of being the Most destructive species as of grasshoppers in Canada. Though notAeconomically
l • ( 2
important as the above three species Melanoplus borealis
borealis (Fieber) is of taxonomical importance.
The working hypothesis of this study is that
investigation of chromosome morphology and behaviour would
yield essential and original information of Melanoplus
species, in order to establish an efficient and sound basis
for a revision of the classification of the taxa.
The approach chosen for this thesis is the study of the
variability of the endophenotypes of the four species
through the investigation of chromosome structure,
supernumerary chromosome polymorphisms and chiasma
frequency and distribution. The main themes of this study
are in order of appearance: the structure of the
chromosomee~ the supernumerary chromosome system~ chiasma
frequency and distribution~ the effect of supernumeraries
on chiasma frequency~ and irregularities of certain
genomes. These objectives are used to determine the
adaptive, taxonomie a~d evolutionary aspects of chromosome
variation in the genus Melanoplus.
For a full understanding of the cytogenetic systems in
( 3
Melanoplus epecies it would be neceesary to have
information of the cytogenetice of FI hybrids between at
least some of the taxa. The limitations of such an
investigation are that many epeciee exhibit diapause giving
one or two geaer~tions per year or one generation may
require two years. Phenotypic eensitivity to factors such
as temperature, humidity and population deneity is common. '/ The large amount of rearing space required represente
another dlfficulty. It would also be necessary to carry
out banding studies on the karyotypes of the various
species. Since most of this information le not likely to
be available for a long while, the present study is being
done as an Interim contribution.
( (
4
1. Cytology of the Acrididae: A Background
The use of cytology in systematics of Orthoptera is not
new~ McC1ung (1908, 1914) studied chromosomes of Orthoptera
and Robertson (1916) produced a good treatise on the \ chromosomes of many species of Orthoptera. White (1974, • 1977) has worked extensive1y on the Morabine grasshoppers
of Australia. This enab1ed Key (1976) to complete work on
the classification of this group. One interesting aspect ( of Key's work is the use of karyotypic characters in ~
numerical analysis, together with dimensional and meristic
characters.
In the Orthoptera, the degree of karyotypic
conservatism varies fram one family to another (White,
1954, 1971, 1973). Fami1ies 1ike the Eu~stacidae are
morpho1ogically and cytologically very heterogeneous, while
on the other hand, families like the Pamphagidae and the
Acrididae are karyotypically more stable and un~f9~. In 1
these fami1ies, it has been possible to establish a
-primitive- or -typical- karyotype. l'
5
The,. r'u1es governing chromosome rearrangements and
numerica1 changes in the katyotypes of the acridoids have
been discussed by White (1951, 1954, 1973). It has been
he1d for a long time that the rod-shaped chromosomes of
grasshoppers are invariab1y acrocentric in nature (i.e.,
the çentromere occupies a eub-terminal position). Within a
particular group, numerica1 deviations from the "primitive"
karyotype arose mainly via three types of reciprocal
translocations: (i) centric fu(ion~ (ii) tandem fusions~
and (iii) dissociation. Thue, for example, within the
Acridoidea, where the primitive karyotype of 2n Ô = 23 acrocentrics, has been retained by the majority of
contemporary genera, there are "divergent" groups with (a)
lower chromosome numbers, and (b) higher chromosome numberé
(White, 1973). These are believed to be derivative
karyotypes which have arisen in evo1ution as a result of
successive fusions in the case of numerical reâuction, or
dissociation in the case of numerica1 increase (White,
1951, 1954, 1969, 1974).
However, John and Hewitt (1966, 1968) have demonstrated
that such a direct derivation of one structural condition
from another may be an ove~simplified inference, and that
karyotype relationships within and between related groups ( 6
<ë\re considerably more complex than had previOllsly been
supposed. It lS becoming increas1ngly evident that the
cl~lmed karyotypic stability in certain orthopteran
fami~ies ls more apparent than real, and that
p n,lophenot ypi c un i f0rm l t Y 0 ften does concea l a cons i dera b le degree of genetlc dlvergence or even maJor changes in
('hr0mÎlsomp 0rg<ë\nlz~ti()n (John and Lewis, l<}65; John ~nd lIewltt, lQ66, 1<}68; Font~n~ and Hogan, 1<}6<}; Fox, 1<}70;
Klknë\dzp ~nrl Vysotskay<\, 1<}70; Shaw, 1<}70, 1<}71, Fontan~
ë\ n d V l (' k e r y, 1 Q 74 , F () n t ë\ n ~, 1 g 7 '); Vic k e r y , 1 g 7 7)
('"refll! ('omparl'30n nf karyotypes ln the acri(ll Inv01vlng accurë\te chromosome measllrements, Identi fic<ë\tlon 0f heterochromatlc segments, determination of DNA values and experimental hybri~lz~~ion, has revealed a wealth of dlfferences between karyotypes o1F many species, even where no major structural rearrangements such as fusions or inversions can be demonstrated. The most extreme and widesprea(j source of chromosome variation, in the otherwise c0nservative karyotypes of the Acrididae, is iound in the heterochromatic material which lS especially liable to structural chçnge by saltatory repllcation and deletion. 7 2. Supernumeraries or B-chromosomes Supernumerary or B-chromosomes have been reported in a l~rge number of plant and animal species, and the extensive literature ~hich has accumulated about them has been rpvie~ed by Rutishauser (1960), Battaglia (1964), John and LeWiS (1965,1968), White (l973),Muntzing (1974), Jones A-chromosomes ore freguent1y heterochromatic and lenpr~l ly exhihlt no homoloqy with any of the A-- ch romosomes They may he mltotica11y stable or unst~hle MltotlC instabi1itiy can lead to on accumulatlon of B-chromosomes ln the gametes (Nur, 1963, 1969). The effects of A-chromosomes on the phenotype are under diScussion. At the exophenotypic level, there is usually no detectable effect although there are sorne exceptions. In the plant Haplopappus gracills (Nutt.), the colour of • the achenes lS changed by the presence of Bs (JacKson and Ne~an, 1960). In Plantago coronopus, the single B found by Pali~al and Hyde (1959) induced complete male sterility in the plants in which it ~as carried. In the mealy bug, Pseudococcus obscurus Essig.,the presence of B-chromosomes 8 increased the length of the tibiae (Nur, 1962). Finally, ln the grasshopper Cammula pellucida(Scudder), individuals with B-chromosomes have fewer follicles in the testes, more abnormal sperm and shorter femura than those without supernumeraries (Nur, 1969). At the level of the endophenotype, there is considerable support for an effect of A-chromosomes on the frequency of chiasma formation, on A-chromosome instability and on cell and nuclear metabolism (Jones, 1975). Recent evidence suggests an effect of supernumerarles on gene expression. Dlstribution of B-chromosomes B-chromosomes have been reported in over flve hundred species of plants and animaIs but data on the distribution of B-chromosomes within and between populations are available for very few species (.Jones, 1975). In animaIs, they occur main1y in the Co1eoptera and Orthoptera. Their discovery in mammalian species lS recent (Hayam and Martin, 1965) . Many chromosome counts are based on but a single individual of a species, and thus many B-chromosomes have probably been overlooked. 9 B-chromosomes are rarely found in inbreeding species. Information assembled by Moss (1969) suggest that there is a strong correlation between the occurrence of B-chromo- somes and a tendency to outbreeding. The reasons for this have never been adequately understood, however it is known that forced inbreeding of a naturally crossing species, with Bs, rapidly leads to a decline in their frequency. b. Inheritance of B-chromosomes (Somatic cell division, Melosis and accumulation mechanism) In many plants and animaIs, the B-chromosomès are completely stable during the cell cycle and are inherited in a constant and unchanging form along with the A-chromosomes. In many other species, this is not the case, and they are less stable than the A-chromosomes. They are mechanically less efficient, succumbing to loss through anaphase-lagging and mitotic-nondisjunction. The latter event leads to numerical variation within the individual, or part of the individual, and may be associated with a net gain or loss of 8s,' depending on the stage of development at which it occurs, and the way in which the dividing cells are subsequently distributed 10 (White, 1954). In Many species, it is not known ~hether mitotic stability or instability of B-chromosomes extends to the soma tic tissues or to the female sex since Most studies have been based on spermatogenesis. In sorne species of grasshoppers, different follicles in the sarne testis contain different numbers of Bs suggesting that rnitotic-nondisjunction occurs in the germ line (Carroll, 1930: Itoh, 1934: Nur, 1969: Rothllels, 1950: Sannomiya, 1962: Sannomiya and Kayano, 1969; Stephens and Bergman 1 1972). Nondisjunction seems to be the Most plausible mechanism to account for the type of intra individual variation in the number of Bs encountered in some species of grasshoppers. Nur (1963) believes that a tendency towards mitotic-nondisjunction ~ould probably be selected against because it hinders the adjustment of the individual and the population to an optimal number of supernumeraries. Whatever the actual mechanism of this intra-individual variation (by mechanical or physio1ogica1 10sses, by nondisjunction, etc.), B-chromosomes with a tendency towards mitotic instability are expected to be eliminated. The only unstable Bs which are likely to be maintained in the population are those in which the instability led to an Il increase in the frequency of the Bs among the gametes, i.e., an increase in the likelihood of transmission to the progeny by a mechanism of "accumulation" (Nur, 1963, 1969). Nur suggests that the Most plausible mechanism of accumulation is a form of preferential nondisjunction at embryonic divisions in which future germ-line cells are segregated from future somatic cells. An accumulation mechanisrn may produce a very sensitive increase in B-chromosome frequency. However, Hewitt and John (1972) and Sannomiya (1974) have reported three species of grasshoppers with small B-chromosomes showing a cytological picture which appears to be in disagreernent with Nur's (1969) conclusion, which states that: "In aIl grasshopper species from which enough data are available the mitotic instability of the B's is apparently always associated with a tendency of the B's to accumulate." It i9 likely that certain unstable Bs May be retained in the population by "non-mitotic" accumulation strategies operating, for example, at male and/or female meiosis, or later in gametogenesis. Alternatively, they have some beneficial effects on the individual or on the population, at least when present in low numbers, in spite of their mechanical instability. In this case, there could ~ 12 be a si tuation where from intrinsical.,1y unstab1e enti ties at their inception, they evo1ve into stable Bs through selection of genotypes possessing or promoting higher mitotic efficiency. The transition could be slow and undetectab1e by recurrent samp1ing over a 1imited period of time (Hewitt and John, 1972). This assumption may a1so explain those cases where B-chromosomes are stable in 10w numbers in an individual, but become unstab1e when present in high numbers. The B-chromosomes are structurally homo1ogous with one another and polysamie. They pair strictly "inter se" and have the capacity to form multivalents when more than two are present, although they seldam pair with the sarne efficiency and regularity as do A-chromosomes. B-chromosomes are generally heterochromatic and positively heteropycnotic during first meiotic prophase. As a consequence, they show a strong tendency to engage in non-specifie associations either with the terminal heterochromatic b10CKS present in some of the autosomes, or more frequently, with the X-chromosome (Rothfe1s, 1950: White 1973: Hewitt and John, 1971, 1972). These associations being non-chiasmate in character, end prior to metaphase-anaphase l and the B migra te a randomly to either 13 pole at anaphase 1. Should they persist into first metaphase-anaphase, as in certain types of heterochromatic associations (Southern, 1967, 1968), they could easily lead to a distortional segregation of the B-chromosome. In Tettigidea lateralis (Say) (Tetrigidae), however, there is a persistent heterochromatic association between the Band X, which 1eads to preferential migration of the B with the X to the same pole in males with a single B (Fontana and Vickery, 1973). When B-chromosomes are present in pairs, their behaviour as bivalents ls very regular; they show normal orientation, congression and disjunction. In many instances, however, their pairing efficiency, as judged from the percentage of bivalents at MI, ls not as good as in the As. Not aIl bivalent behaviour is regular in two B-containing individuals. The acrocentric bivalents of . Trimerotropsis sparsa (Thomas) (Locustinae:Acrididae) frequently orient themselves on the spindle at MI with both centromeres close to the sarne pole. This leads to nondisjunction, and one secondary spermatocyte receiving both members of the bivalent (White, 1973). When more than two Bs are present, multivalent associations are frequent: although there is still a preference for bivalents, many ( 14 univalents also occur. In some species, there are no associations higher than bivalents, regardless of the number of Bs. Complications of pairlng relationslps of Bs also occur on account of their polymorphisme What frequently happens ls that a standard subterminal B type undergoes centromere misdivision, giving rise to telocentrics, or small and large isochromosomes formed from the two arms of the standard B from which they derive. c. Structure of B-chromosomes When B-chromosomes are first discovereo in a species, they are generally recognized by their small size and lack of homology with the normal set at meiosis. However, sorne species have Bs as large as the As or exceeding them (BaverstocK et al., 1977: JaCKson and Cheung, 1967: John and Freeman, 1974). In addition to this, of course, they are variable wlth respect to their presence or absence within individuals of a population. B-chromosomes are frequently heterochromatic along their entire length. In sorne species however, they are euchromatic as in Atractomorpha bedeli(Boliver) (Sannomiya, 1973), or contain regions which are euchromatic as in Gonista bicolor (deHaan) (Sannomiya, 1974). In certain 15 cases, they exhibit segments which do not undergo heteropycnosis at the sarne time as the rest of the chromosome as in Podisrna pedestris (Linnaeus) (Hewitt and John, 1972). Different types of Bs can exist within a population or a single individual. In Podisrna pedestris, for example, certain populations were polymorphie for a large B, while others contained individuals with very minute Bs (Hewitt and John, 1972). Stephens and Bergman (1972) found three morphologically different types of Bs in the sarne individua1 of Melanoplus fernurrubrurn(De Geer). d. Polymorphisrn of B-chromosomes Apart from their size, and sometimes heterochromatic nature, another characteristic feature of B-chromosomes is their polymorphism. The most extreme case of B-chromosome polymorphisrn occurs in Aster ageratoides, in which a standard morphological type and up to 24 derivatives may occur (Mastuda, 1970). Supernumerary heterochromatin polymorphisms may arise from the presence in the population of individuals carrying one or more B-chromosomes. In Most 1 l cases, these extra elements are entirely or partly 1 heterochromatic. Although the presence of B-chromosomes 16 hae been reported in a large number of plant and animal speciee, extensive polymorphie conditions seem to be particulary frequent in the Orthoptera. In almost all species of Orthoptera, cytological polymorphism of aupernumerary segmente involves the smaller members of the complement (White, 1973; John and Lewis, 1965, 1968) but the causes or the significance of this association are not known. The most thorough etudies on the structural and dynamic aspects of supernumerary segment polymorphism in species of Acridoidea are those of John and Hewitt (1966, 1969), Hewitt and John (1968, 1970) and Westerman (1969, 1970), who studied European populations of Chorthippus parallelus (Zetterstedt); and of Shaw (1970, 1971) who investigated European and North American populations of three species of Stethophyma. Fontana and Vickery (1973, 1974), who studied two North American Stethophyma species, discovered that polymorphism for supernumerary segments involves the medium chromosomes (Mg). e. Effects of B-chromosomes In species of plants and animaIs that carry ( 17 B-chromosomes, those individuals of a population with or without Bs cannot generally be distinguished fram one another phenotypically. The variation produced by Bs is continuous, like that due to polygenes (Mather, 1945), and direct cytological observation is the only sure way of determining whether B-chromosomes are present or absent. In fact l they are most frequently discovered by chance during cytological studies and their presence betrayed only when the chromosomes are properly counted and classified. As far as outward appearances are concerned, B-chromosome effects are either neutral or more commonly, negative. At the level of the endophenotype, the story has a positive side as weIl. Important results have been obtained concerning the effects of Bs on A-chromosome behaviour at meiosis, and on sorne fundamental cellular processes governing growth and development. Firstly, as chromosomes, Bs change the genotype. Secondly, aside fram their elusive genetic propertiee, the y modify the immediate environment of the As themselves, that is, the nucleus. Thirdly, they have long been known to "interfere" with Many other gene-controlled aspects of growth and development in both plant and animal species. 18 Barker (1960) was the first to discover the effects of Bs on A-chromosome chiasma frequency,in the grasshopper Myrmeleotettix maculatus. The analysis was limited to Mean cell chiasma frequencies and took no account of possible changes in variance. John and Hewitt (1965) subsequently showed that the Bs in Myrmeleotettix could affect the distribution of chiasmata among cells as well as increase the mean. There is no way of predicting precise1y how B-chromosomes are likely to modify the regulation of recombination in As. The Mean chiasma frequency can be raised (Myreme1eotettix) or 10wered (Lolium perenne), and this effect upon the Mean May or May not be accompanied by changes in the cell and bivalent variances. In the case of variances, which are a measure of the regularity of chiasma distribution among and within celle, the effect ie ueually one of increasing the variation. Forcible arguments are now being advanced for an important and adaptive ro1e for B-chromosomes in the genetic systems of outbreeding plant and animal species, namely, that of boosting and regcilating the release of ( , , 19 variability. The co~trol mechanism operates at three levels of organization: (1) within populations through adjustment in mean chiasma frequencies of individualsi (2) within individuals at the cell level; and (3) within cells at the level of individual chromosomes. A novel B-chromosome effect has been discovered in Zea ~ ~ mays (Rhoades and Dempsey, 1972, 1~73). Certain members of the normal A-chromosome complement possess large heterochromatic knobs at specifie sites in the e~romosome arms. When two or more Bs are present in a plant, the arms, or parts of the arms carrying these knobs, are frequently eliminated at the second microspore division. Little or no 1088 occurs in the microspores with one B, and the rate is not increased with more than two Bs. A-chromosomes that do not possess heterochrmatic knobs are stable in their inheritance. Appropriate methods of quantitative analysis reveal that B-chromsomes have wide-ranging effects on the phenotype, especially in plants. B-chromosomes contribute extra DNA ta the nucleus. In rye, each additional B increases the OB DNA amount by approximately 5%, sa ~at over the range 0-8B there is a considerable diversity of ( 20 nur-le"r [INA valueH (,Jones and Reeq, 1967) Th 1 B quantItative nuclear DNA v"rlatlon haB reper,llsBlons on the met rt h n 1 1 r- i'I (; t l V 1 t Y n f the n u c 1 P \l R One effect 1'3 the pxtenqlOn nf thp mltnt le cycle In terms of A-chromosome lenp "r't lVlty, qnme nf the Cnn'3e'lllencpc; i,f the extra n-,'hromnsome [INA i'lre 'llllte fllnclamental rtnd reqlllar (,Jones, 1 q 7 r) ) heh"Vli)llr 1'3 thelr '11fferentl,,1 i'l.ctlVlty ln reli'ltlon tel ()d~ ;ln,l P'Jf~11 numhered ,'Omhlr1i'1t I()ns The effel't lS most freqllent ly pn('()untered fnr l'hara,ter" of the nllî'le"r _phenntype, hllt lt ha!'> "lso heen ohserved for the whole- plant phenotype ln rye One Instance has heen recorded ln "lnlmals (Shcherhakov, 1966). Here it is found that "adaptlve value" than those with odd numbers. In the plant mat_erlal also, It 18 the even numbered combinations that have the 1east effect upon VIgor and aspects of nu,lear metaboll sm, Including recomblnation. It was proposed, when thlS phenomenon was flret demonstrated by Jones and Rees ( l 969 ), t ha t the e f f e c t ma y r e sul t f r om a .. con t i gUI t Y effect" between B-chromosomes; paired combinations in the nucleus act more favorably than unpaired ones. After all, 21 the whGle system of B-chromosome inheritance IS adapted, through the nondis]unrtlon mechanism, toward malntalning a preponder an ce 0 f even- number ed comb 1 na t ions in the population. Furthermore, there la some recent and ronvlnclng eVldence to show that the A-chromosomes themselves have a close homologous aSSOCiation durlng lnterface of the rell rycle (Feldman et al., 1966) Such a mp(~hanlsm of somatlr aSSOCiation couid .... eil apply to R - (' h rom 0 S om e s i'1 n"l ev en h a v e som e fun c t i G n a l sig nif 1 r li n c e .... hl 'h 19 expressed as the o In Vlew of the above account of B-chromosomes effects ln flo .... erlng plants and anlmals, one coui (1965) has suggested, and eVldence has now been found, that B-chromosomes have a constructive role to play ln 22 short-llveo, outbreeding plants (and the same reasoning can be applled to animaIs) in regulating the release of genetic vi'irlablllty. The evidence on recombination has now been tlsed to establlsh the case that B-chromosomes are, in fact, i'i ~evice for controlling recomblnation through aOJustment of chIasma frequency and dlstribution at melOS1S (Hewitt, 1q79) The questlon of the orlgln of the B-chromosomes 1S one that has sa perslstently naggeo cytologlsts over the years that many have felt obl1ged ta present qome scheme or other, no matter how fragile or speculatlve.· The nucleolus organlzer reglon, a particular pOlnt of weakness ln a chromosome complement, has been repeatedly selzed upon as a likely slte at WhlCh breakage could occur and ultimately glve rlse to Bs. Evidently, there are many dlfferent ways ln which B-chromosomes might have arlsen. This is reflected in the heterogenelty of B-types, which range from true Bs showing a complete lack of homology or structural relationshlp to any of the As, through those of sorne insects wlth affinltles for the X-chromosome, to the minorlty of cases 11ke those ln Phlox and Chironomus ln WhlCh the Bs represent centric fragments of certain of the A-chromosomes. The origin of true Bs remains an enigma. 23 B-chromosomes break most of the laws of classic genetics, lncluding those of Mendelian heredity and apparently those too of natural selection. Yet, Darlington (1965) has seen fit ta incorporate them into what he calls the genetlc system - "the organization of the reproduction and heredlty of a group of organisms aB a unit in evolutlon" - and even ascribe to them an important role as agents for boostlng the varlablilty, of Bpecies. Ostergren (1945) hn9 taken the opposlte positlon and labelled them parasitlc, malntalned in populations only on account of thelr ~evious mechanlBms of accumulation and infiltration. The arguments for and against both points of Vlew have been discussed by many authors (Nur, 1969); Darlington's case appears ta be the most convincing. One of the strong points however, in favor of the latter case, ls the nature of B-chromosome DNA (Rhoades and Dempsey, 1972). It is speclalized in sa far as it does not appear ta have any proper funct10nal cistrons, and may not even be transcribable at all. In this sense, it's mere existence can be expected ta account for Many, if not all, of the physiological consequences of B-chromosomes. The properties of B-chromosomes and of repetitious DNA do not seem ta be all that different in many respects, and the 24 same May weIl be said of their consequences. The one major difference is that within species, individuals do not dlffer with respect to their repetitious DNA, like they do with Bs, so it has not been possible to study its consequences with the same facility. The argument amounts to saying that B-chromosomes and B-chromosomes effects shoulo not be considered in isolation but in the wider context of genome organization. 3. P101dy It 18 an axiom of genetics that the synapsis and crossing·-over 'Which characterize meiosis are exclusively confined to "homologous segments of chromosomes" (Darlington, 1973). While the concept of homology and those conditions which allow its expression are fundamental aspects of cytology, the underlying mechanism of homologue recognition, pairing and crossing-over still remain unresol ved. Riley and Flavell (1977) have 8uggested that "homologoU8 chromosomes or chromosome segments would usually have a common origin in evolution from the same 25 chromosome of an ancestral species and overall morphological similarity, fOr exarnp1e in arro lengths, distribution of secondary constrictions and distribution of heterochromatic or other discernib1e bands or regions. Homo1ogous chromosomes would probably have the sarne structural and regulatory genes arranged in the sarne linear arder." They point out, however, that the homology necessary for meiotic pairing May be quite different from the nucleotide sequence homo1ogy which they consider is necessary for recombination to occur. Thus meiotic pairing May only require a limited number of sites te initiate the pairing process. The pairing of chromosomes, by itse1f, is not necessarily a good indicator of homology. Gi1lies (1973) has shown that normal synaptonemal complexes can forro between non-homologous chromosomes of maize. Simi1ar situations have frequently been observed in several monohaploid plants where non-homoloqous chromosome pairing is prevalent, but crossing-over is considered to be absent (Kimber and Riley, 1963: John and Lewis, 1965a). The critical factor in any definition of homology between chromosomes or chromosome segments i8 that they must show, or be capable of, cro8sing-over. 26 In the normal eup10id state, the dip10id genome contains two haploid sets, and each chromosome, within one of these sets, pairs and crosses over on1y with its equivalent partner in the complementary hap10id set. This primary homo1ogy between pairs of chromosomes can be reduced or a1tered during the process of divergence from a common ancestor in two ways. First1y, the original or ancestral homologous chromosomes can lose their affinities for each other after periods of isolation without any obvioU8 changes in their overall morpho1ogy or appearance. If the divergent ~axa are then brought together, either artificially or natura11y and still retain the abi1ity to hybridise, it is a common feature to find unpaired chromosomes during meiosis in such hybrids. Mu11er(1977) has shown that in the genus Xenopus, the number of univalents per... meiotic cell in different hybrids is positive1y corre1ated with the taxonomie distance between the species. The under1ying causes of such a "loss of homo1ogy" during divergence are not understood. Alternatively, the primary homology between pairs of chromosomes can be redistributed within the genome by the interchange of chromosome segments between two non-homologues. In the heterozygous state, four 27 chromosomes now share partial homologies which often leads to multivalent formation at meiosis. ~gain, these evants May be accompanied by little or no obvious change in the overall karyotypic pattern of the two parental species and, in fact, can only be deduced from studies of hybrid meiosis as was evident in species of the genus Triturus (Callan and Spurway, 1951) and in the subspacies of Eyprepocnemis plorans Charpentier (Locustidae) (John and Lewis, 1965b). Multivalent formation in hybrid meiosis was a feature of both these cases and it was proposed that the parental species and subspecies were homozygous for interchanges which had accumulated during or after species or subspecies divergence. However, certain anomalies in the nature and frequency of non- homologous chromosome associations in the two cases prompted White (1961, 1973) to advocate an alternative mechanism to explain the formation and maintenance of at least some of these associations. He proposed that during the course of evolution, minute duplicated chromosome ends have been translocated amang different members of the genome resulting in "homologies" between the terminal regions of otherwise non-homologous chromosomes. White aiso considers that these examples illustrate what may prove ta be a more widespread phenomenon in evolution. 28 Shaw and Wilkinson (1978) presented an alternative hypothesis based upon similar observations to those listed above. They found that meiosis is consistently characterized by abnormal patterns of synapsis and reveals a clear example of meiotic exchange and crossing-over between non-homologous chromosomes, including the X-chromosome. The abberant pattern of diploid meiosis is correlated, in certain cases, with the production of tetraploid meiocytes within the same individual which show 100% bivalent formation with regular orientation and segregation patterns. This system of boosting the ploidy level in hybrids is very similar to that proposed by Muller (1977) for ploidy increases in inter-specifie hybrids of Xenopus and may represent a more general phenomenon relevant to the evolution of parthenogenetic species via interspecific hybridization. In Most species of grasshoppers, polyploid spennatocytes are occasionally found in the testis. They are not, as a rule, present in every testis, and one May have to examine material from a number of individuals before encountering such cells (White, 1951). These polyploid cells are probably of no functional significance, there being no evidence that they ever give rise to ( 29 functional sperms. They do, however, provide important information on some problems of chromosome behaviour. Some polyploid spermatocytes are pathological and undergo degeneration during or shortly after the meiotic divisions, but others appear to be physiologically normal and pass through meiosis without any apparent difficulty. There are two main types of polyploid spermatocytes in grasshoppers. The first type arise through failure of anaphase separation at one of the spermatogonial divisions. Such cells are always tetraploid and may occur singly or in groups of 2, 4, B, 16 ... according to whether the failure to divide effectively occurred at the last spermatogonial division, or the penultimate one, etc. The second type of polyploid cells arise through fusions of 2, 3, 4, 5, or more neighbouring cells at any stage in spermatogenesis. The two categories are not always distinguishable, but in the second case, there may be pentaploid, hexaploid, heptaploid, etc. cells, as well as the more common tetraploid type. Where fusion of the cells has ta ken place after the zygotene stage, the chromosomes of the originally distinct nuclei will have already undergone pairing before the 30 establishment of polyploidy. Under 8uch circumstances, no associations of more than two chromosomes will be present at meiosis (White, 1951); in XO species all the X's will be present as univalents and all the autosomes as bivalents. Where polyploidy was established before the zygotene stage, some trivalents, quadrivalents or higher associatibns of chromosomes are found at meiosis. Very little information is available regarding polyploidy ln natural populations of non-parthenogenetic grasshoppers. / 31 MATERIALS AND METRODS 1. Bio1ogical Materials The specimens studied were collected from the Morgan Arboretum, Ste. Anne de Rellevue, Quebec. Collections were made on a weekly basis for a perlod of an hour durlng the summers of 1981, 1982 and 1983. Collections were made ln aIl three years between May and August. Each lndividual yleldlng a cytological preparation was glven a label bearing the same code number as the flxed tissue to provide a permanent record for routine taxonomlC ldentification and future reference. 2. Chromosome Preparation Technisues In aIl individuals col1ected in 1981, testes were removed by vivisection and fixed immediately in fresh 1:3 acetic acid (glacial) :ethanol. Squash preparations were made later in aceto-orcein or lactopropionic orcein (Dyer, 1963). 32 Testes f~om individuals collected in 1982 and 1983 we~e ~emoved by vivisection immediately after collection. An air-d~ying technique was used which inco~po~ated elements • of both the ai~-drying techniques of C~ozier (1968) and Imai et al. (1977) and the imp~oved squash technique-- developed by Imai and Kubota (1972) and Imai (1974). It yie1ded high-qua1ity metaphases. The steps we~e: ( 1) Testes We~e transferied immediately to a colchicine-hypotonic solution (0.005% w/v colchicine in 1% sodium cItrate solution) on a cavity slide fo~ two minutes. Fat body, tracheae and epithelial membranes we~e removed. (2) The testes were t~ansfe~~ed to a fresh colchicine-hypotonie solution on anothe~ depression slide using a Pasteu~ pipette. ( ..... 33 the material was left for 20 minutes at room temperature. (3) The material was tranaferred ta a freshly wiped, pre-cleaned slide (waahed in detergent solution, rinsed in distilled water, and stored in absolute ethanol) using the Pasteur pipette. With one end of the slide on à damp absorbent plastic sponge, the slide was tilted 80 as to ~rain off most of the hypotonie solutIon. (4) With the slide inclined at 10-13° into the sponge, several drops of freshly-prepared fixative l (60% acetic ethanol, i.e. 3:3:4, glacial acetic acid: absolute ethanol: distilled water) were applied so that the fixatIve flowed over the organs and drained off the end of the slide. (5) The alide was placed under a dissecting microscope. Two further drops of fixative l were added directly onto the material. After a few seconds, the organs were macerated as \4 ''';1" ~n,i tlpf lr p 'hp 'P 1 9 119 pp "1 9 1 ln i r 1 P l ' '-', lr, pl" • ~ p q r J. y ç'r P,lci r p x ~ t .je> 'l tl S l' P pt i r 1 ~ pt ..... ' 1 h~rl P -1 (- ~r 'l 1 j l t P P , >,~ ') .Jt " \.Ipr~ ~ l ri p l A. t • P" '1 ') , '1 P l ; r-i ,- 1 r'I) ~ C p r 1 - ~ (- 1 'wIP r p r'I d i p l ci n d li f t p r F rf)p flXii'lVP '-'r'l9 lr'llned hy The 9 j ip '-'d q "'1er c l( .9n 'Jrlnn ) 4 1 n "" 'i C;'ren.gen'q Pf-I F,.R hutter f'}r tpn mlnutp~ ~t room temperatur p • ""i'ltpr i'lnr1 thpn rlri'llnerl. Preparat Inn~ wpre i'l l l ) Wf>' -j t \' i r y, r 0 ver e d w 1 t h Cl \") V p r "; l I~' Cl n <-j AF", a t ta\"hmpnt Cinrl a \')mm campr~ fIt tpd ,r] , rt !\Ilk'n ml\"r<'.9\"npp. 'rt Ir,,; ILKe \"(Jndensor i'lnrl nLI JhlE"'lVP9 T'le Kndak Panat'Jmlr X anrl Ilf)rd Pan-F fllm9 JSP F , r r h r ,) m r ) S n m pme a sur 1" men t 9, P h Cl t r) q r a phi \" P r 1 n t s ,f well-spread metaphase platf's at a final magnlflcatlon )f Tr,e chr )mr)somes were dl i'ferent lated as pr)pnSe1 hv Levan et al , l q64 36 A flldp-scrp", nlvlner and a trlanqular metrlc scalp (The "., Montreal) .... ere used as tools t'i measure ,'hr lmCl t Il Clrm )f pa,h chr'imnsomp, a"l ",plI afl nf PClrh 'hr,mJsomp paIr ln pvery rell. Idl'iqrams ,f thp " fl r 'm ( , '" ( J m p " 4. ~hldsmd 01strlbutl'Jn :"'ells Clt eClrIy and mld-dlplotene were selected. ~lvClIpnts werp 1rawn from pro]ected photographlc negatlves 3t Cl maqnlflratlnn of approxlmatply X 2500, the posItIons if thp cpntr0mere marken, and the posItIon ,f each chIasma measurpd. T~ 'vprcome 'he pr'Jblem if varIation ln absolute blvale'l' length between cells, the posItIon ,f each ~hlasma W.;lf< rer')rded afl '~e mpan d1stance, averaqed ')ver bath hnmnlnquf>~, trom the centrQmere tn the chlat'lma, pxpre8ged ~S a percentage nf the length of the chromosome arm. -h1~t'!mata '.Jf>rp scored ln the rl~sse8 ciescrlbed hy F1ptrhpr an~ He'.J1tt (1980) w1th sllght variation to SUIt 1 n, ~ l '/1 rj U d: ') p f> C 1 @9 a scie S c r 1 b e d 1 n the Res U 1 t s. r: h 1 a El m d t a wprp sc)red dt'! "tprmlnal" If no free ends ,)f rhrGmGsomes wprp Vl.91hlp beynnd thf> ,ilstal rh1at'lmata, and fJnly th@ 1np~ltahlp fln@ thread was vIsible het'.Jeen the centromere "Suhtermlnal" chla9mata '.Jprp cln')e t 1 thp pnds, effp,t-lvely ln the tprmlnal 10% ')f the )nqerhlv~lpnt~, ')r terminal 2'1% nf shorter blvalentR. n P r ) t p r min al" 1 S II se d t 0 des cri be h () t h ter m 1 na 1 and suhtermlnal rhlasmdt~ lo1ntly. -hlasmata '.Jere alsn scored as proxlmal (near the rentr0mere\ or "31stal (near the telomerel. The crlterla Jsed '.Jere arb~trary and much more accurate measurement of ,hlasma [)OSl~lnn lS pGsslblp' however, the methods used were qUlte sufflclent to 8ho'.J dlfferences between specles w1thout aS8umlng unreallst1r accuracy. 38 THE CYTOGENETIC SYSTEM OF MELANOPLUS BIVITTATUS (SAY). 1. The lomplpment and the Structure of the Chromosomes The Rtanciard male complement of MelanopluB blv1ttatus lncludes eleven palrs of autosomes and d slngle X- ThlS 21 ~ karyotype lS typlcal of the famlly A (- r ul l'id e a n ri 1 S f () un ci 1 n a pp r 0 x 1 ma tel y gO % 0 f the In thls study, the ~iJt()s<)mes ..... ere class1f1ed lnto three Blze groups lnclucilng fGdr large IL), SlX merllum (M), and one small (5) chromosome palrs. ThlS breakdown seemB sUltable Slnce there lB a clear rllscontinulty ln length bet ..... een the smallest chromosome of the medlum palr (MIO) and the sma Il (S 1 1) pa 1 r . The dlfference ln length bet ..... een the medlum Blze group and the large Blze group IS not as sharp aa that bet ..... een the medIum and the small group. However, there 18 dlBcontlnulty ln length between the 8mal1e8t chromosome of the large group (L4) and the largeat chromosome of the medlum group (Mc.;), (Plg. 1). 39 ~e X-chromosome is non-synaptic, poeitively heteropycnotic at dlplotene-diakineeia and negatively heteropycnotlc at Metaphase I. AlI eleven autosomes and the X, the flfth largest chromosome, are telocentrlc. It shoulrl be noted here that three of the large palrs (LI' L2' anrl L4) carry mlnute arms which are Indlcated by a small clrcle ln flg. 1. These short arme do show complete ,onslstency ln thelr appearance ln aIl cella and ln aIl IndlVlduals. The ratlo for the three chromosomes that carry the short arms 18 more than 8 WhlCh put them ln the relocentrlc group wlth the rest of the chromosomes in the complement (Levan et al., 1(64). The relatlve length of each member of the complement lS glven ln Table 1. 2. The B-chromosome System a. frequency and morphology The phenomenon of an addltional chromosome in the complement wae not noticed in the 1981 and 1982 samplee, probably due to the small sample size. In the 1983 populatlon, out of the 51 males examined cytologically, four (7.8\) were carrying a supernumerary. The B-chromosome was found to be telocentric and approximately 40 FIgure 1. Idlogram of Melanoplus bivittatus male (2n 23) based upon the percentages of total complement length. The smali circles represent the short arms on chromosomes LI, L2 and L4' Idiogram represents the hapiold set. 6- ! 1 1 4- .....J ....U ~ 2J ... J 1 A 1 TABLE 1. The 1ength of the chromosomes of Me1anop1us blvlttatus expreased as percentages of the total complement length (TeL) as seen in spermatogonial metaphase plates. The mean (X), the standard deviation, (0) and coefficlent ot varlation (CV) are given for each chromosome pair. LI L2 L3 L4 MS M6 M7 M8 M9 MIO SIl x TOTAL x* 14.56 13.18 10.72 10.18 7.70 7.29 6.62 6.28 6.~8 6.20 2.74 8.24 99.99 o 0.31 0.24 0.50 0.16 0.24 0.36 0.45 0.11 0.61 0.22 0.22 0.72 CV 2.13 1.82 4.66 1.57 3.12 4.94 7.19 1.75 9.71 3.55 8.01 8.74 * Values represent the means of measurements trom 10 males. ~ ~ 42 three-fourths the Slze of the smallest A-chromosome AlI four males with the B element had a single supernumerary. The B-chromosome had no X-association and it acted like the autosomes in respect to heteropycnoslty (Flg. 2). The B ln M. bivittatus appeared to have an autosomal nature and was lndistlnguishable from the normal autosomes except by Slze. It showed no synaptlc association with any of the chromosomes of the complement except ln the case of a polyplold lndivldual WhlCh wlll be rllscussed separately. b. Chiasma frequency To ascertaln whether chiasma frequency is affected by the presence of B-chromosomes, the number of chiasmata ln A-chromosomes were scored ln males wlth and wlthout supernumeraries (Table 2). Chiasmata were scored in 10 cells from each of 20 males without the B-chromosome and in 10 cells from each of the 4 males carrying the B-chromosome. The chiasma frequency data were analysed to determine whether the B-chromosome ln M. bivittatus affects the amount of variation ln chiasma frequency between lndividuals and wlthin individiuala. The between-individual variance, an estimate of the variation among the lndividuals of a group, ia given for the OB and the 43 Plgur~ 2. Plrst melotic metaphase in Melanoplus blvittatus male showing the negatlve heteropycnosity of the X-chromosome. Note, the B-chromosome lS not dlstingulshable from the rest of the autosomes except by Slze. • x ( ,.-. . tABLE 2. Th@ chla~ma fr@qu@ncy data ln M. hlvlttatus males wlth and wlthout B- chromosomes. Ce Il Types No. of Chlasma frequency for Mean t Between Indlvi- Mean of the logs Scored Males each male s . e . dual varlance of the within lndivldual variances OB 20 * 12. 1 13.0 15. 2 13.91t 1 . 143 -0.2296 13.9 13.9 13. 1 0.24 13. 2 12.0 12. 1 15. 1 15. 1 14.8 14.6 15.0 14.8 13. 2 14.6 1 "3.9 13.8 14.8 B+ 4 13.6 14. 1 15. 2 14.101: 0.607 -0.0993 13. 5 0.39 * Each flgure represents ten chlasma scores per lndlVldual. ~ ~ 1 45 B-containing groups in Table 2. There is no significant olfference among the between-individual varIances of the OB and B-containlng groups (OB mean 13.91, variance 1.143: B+ mean 14.10, varIance 0.607). In order to ~nalyze the within-lndividual varlation, or varli~tlon among cells, the variancp of the 10 chiasma scores in each male was first converteo into a log varlance. The means of the logs of the wlthin-lndivloual variances are given for the two groups ln Table 2. The log variances of the OB and the B-containing groups werp compareo in a one-way analyses of varIance (Hewltt, 1964). The difference between the mean log variances for the two groups is signlflcant (t = 2.146, 0.02..:: P-oo::.O. 0'), 23 d f ) . Slnce the mean log variance for the B-containing group 13 greater than that for the OB group, there may weIl he a higher within-indlvldua1 variation in B-containlng males. The lack of significance in the analysl3 of variance may b~ due to the small sample size in ( the B-containing group. .. Two of the four males carrying the B element exhibited variation in the presence of the B-chromosome among follicles (Table 3), although when the B-chromosome was present it was found in aIl the cells within the follicle. The mean number of Bs per individual in the two males i3 ( 0.76± .0.00. However, in the other two males containing F = : :: 1 cr cr : C- " '1' 1 \ 'T .7 ~ t'" :.>-. : cr: '1: C. 0« :f" F f-- 1 , . ... . ' ... pc; . ' ... . "' .. " . • t . .... p :.. ; , • c; c;' ; -1 • • d' 1 1 l j" • ," 1 . , .... 4 f .. ~ .. ,1-' • '1 J r, rm '.'l)" J t • 'lP 'l1P'1, JfT' ,P99 • p'l 'r t 'IP , J"I • P n t,<; "1 ~ n l '1 Therp ,1 r 'il r' 1 ln t'lP 1 ) n 'J h 1 J rt p n t 9 . • hp l' hl Rmrt t n ln hnth iR dnd B-r'lntrtlnln'J h )'Wpvpr i 1 f f 1 r Il 1 t 1 p " n !'Ir')!"- 1 ng p'lSltl ln", ln Ruch d sh0rt hlVrtlent make ('ljmparlB'Jn q i 1 f fI' 1 J 1 t »0 s h n r t blvlttatiJ.9 .... • • --1 " .. • , p ~ . , . - L .. rr .• f--' , , m .;pr<> l'JP" hp,...- " • p X.md·p.v ''lf''''.ZP .,.., p pa" p r " • v f r ~qme '1' • ha t 4 .... f hpha'-./' )ur rliOmp'l· .9h~WE'rj '1 ~~ 2 f ragmE'n t - ~ BE' 1 ~€'l\)tl(' ilV151Jn ln i'I ~e_la~~l?lils hlvlttatus 'Tl ale s h n .... 1 n q i 1 f fer en t c n n f l '1 u r i'I tin n" n f 'll "i'll ent S .... 1 t h m()re ~hléi6mata per " 1 val "" n t t", a n m~d 1 \l m h 1 v i'I 1 ~ nt" :'1il'J111i'l' ~c\s d sup~rnumerary '",l,'\sma)f the SI: '"malI hlvéilpnt" 17 1 rst metéiphase ~~ll shO .... lng t .... n ')f the l(Jng hlvalents (LI, L2) .... lth -..,0 -~lasmati'l each, ..,hlle the r~Bt nf the hl'Ji'llents ~as ,ne ('hlac;ma per hlvalent J \ o c 1 Flgure 4 The strùcture nf the telocentrlc fragment (fI) at flret metaphase 'jf ~ male lnrilvlliual of Melan_<"2.e1us ~lvl~atus lnrilvlriual ~ag ~ B-chromosome • • ( FI q IJ r p r, MfO'lannrlus hlvlttatus mil)'" 9h<)lo/lfl'l merllum hlVdlf'nt n p '1 d t 1 / P ) y h p t p r -l P Y Î n "t 1" n il' J r .., 'f • h p ( .~lnqlp polyplnlri mal~ Indlvldual )f the lq83 populatlnn Jf M. blvlttatus. P~rt ,f the melncytes 'jf that malp C n n t a 1 n e Ô a 5 mal 1 ., ete r 'j c h r 'J ma t l (" f r a q men t ,- h r n m0 A 0 mpin arlôltlnn t, thp standarô complement. The fraqment ·ransl()C'aterl ln the 1'1/1 ~LJtnsome (F'11. ')\ T h ~ f r a q me nt' '1 X-chromosomp It resembles the auto30mes ln thelr lHln~ Anaphase II ;F'lq. ')1. l t l "1 n, t p) s S 1 hie ~) "A S U mf> t'"!,,' 1· 1"1" pr,ôuct lf fraqmentatl)n ,f aut ,some"'A \Jl.'l "" hl," hl' 1 sas An (" 1 d te d "1 l n (" Pit car r 1 P 3 n') s pe (" 1 f l (" 4. 1'113s1n9 :hromosomes Sorne In~lvlduals ,f M. blvlttatus ("nntain cells WhlCh lack. a medium autosome, brlng1ng the 2n à' tn 22. The perC'entage )f Inôlv1duals lacklng an autosome was fnund to be qUlte hlgh (20.')%). Ho we ver, no t a 1 1 the cel l 3 W i t h 1 n the fnllicie lack. an autosome. The in ter - fol l 1 cul a r var1at1on for the ahsence of an autosome is given ln Table 4. Slnce the mlsslng chromosome lB not Identified ln thlS study, the result lB a product of the pooled miBBlng autosomes ln aIl the Indivlduals. 1 H .we 'If> r lt IR 'ln t Therp 1 R '" caS4=> lf "91n'llp mal., lnrll'11rludl ~dcklnq the 1(-cnr)mnsome ,., el) SIS ) f P'} 1 yp 1 () 1 ci 'e Ils In thl9 9tu,iy )f th., ')] ma!P9 9t,Jeiled frJm the 19B, sampl p , t'1r"'E' mal",g ') . q %' wp r p f "U n d t l '1 a ve po 1 y pIn 1 d - p ] l s . )f the fnlllc1es hut nnt Io/er'" tetr polyplnld relIs had trlpl'lld and tetraplOld cells ln the same f'}llirle. ThlS lndlvlr1ua1 carrleri a supernumer (PlgS. 7 13, and g). .;,..:1' The chro~osomeg u8ually assoclateri dB blvdlents, though there Io/ere trivalents present. The Xe behaved as unl- valents. ( -, ... TABLE 4. Inter-folllcular varlat10n ln the absen-p -'f Males No. of folllcles % ,f f,lllrlef\ .... lth Mean/MdJe Normal A karyotype kary()type • Melan'lplus blvlttatus malp shnwlnq cnmplpmen~ wlth ~lpvpn hlv~l@nts ~nd ~ ml~slnq x- (-hromnsnme. MIl Nntl'-p h. MIl· -in Inrllvlrlual wlth ri ml~'31nq x '-hr )mnsomp and heter')mnrphlc LI hlVrilent. a • ,... '. - •" b ., ( 1 , , i' .... \ .... - ~ , ... rr ~ 1 ." rr ... , , ~ \.'& a - ~, b .... -1 • " . , ,.. r m q lm' .. q a . !, , c ( 1 , " Pl) l Y pl 1 1 li rel l '2 n q 2 1 n '1 t 1 (- p { b ( THE rYTOGENETIC SYSTEM OP MELANOPLUS PEMURRUBRUM FEMURRUBRUM (DE GEER) 1. The Structure ()f the ,~hr()mosomes The malp karyatype nf Melanoplus femurrubrum femurrubrum IncllJrleA elpven palr.9 of telacentric "iutoBomes anrl ~ 9Inqlp, telocentrlc, heterachromatic X-chromosame (Hearne and HusKlns, 1935). The ~utosomes can be subrllvlrlerl Into t .... n large palrs (LI' L2)' SlX palr" of merllum Blze (Mj ta MR)' and three short pairs (Sq t0 SIl). The t .... o large paIrs can be consistently dlstln- gUlsherl, though nat fram pach other. The AiX palrs of merllum size sho .... d grarlatlan in length anrl are nnt easily distlngulshed fram earh other. Althallgh t .... o af the three short pairs (SIO and SIl) are very small and cannot be d19tlngulshed fram each other, the remaining pair (S9)' being sllghtly larger, can ~ dlstingulshed (Stephens anrl Bergman, 1972). The relative lengths of the chromosomes are shawn ln Fig. la. 2. The B-ChroffiÇsome System a. Frequency and morphology Of the 46 males examined from the 1981 population, each j,il ''lrrtm )f Melnn')plus f~murrl1hrum femurrubrl1m shn .... lnq the hap!'nrl 9pt lf thp mal~. T h ~ rt 1 J t 1 fl n me fl d r p n r r ii n q p ri rt C l 'i r ci 1 n 'l to thplr de,reii'llng lenqth9. Trll lenqth (T('L). 6- 4- III x 1 hl 1 \ . () 4 % ) • The 19A2 population harl a B-rhromosome frequenry 'lf 11.11% '10 mnle9 'lut nf <)0 carlled a R-rhromosome). F. 1 g h t Y- f 1 ve ma 1 e s Iole r e e x am 1 ne rl 1 n 1 98 :3 , e 1 ev en') f 101 h 1 C" h had a 9upprnumeraly (12.94%). In ail three populations the B-chromosome loIas found ta he telacentrlc and approxlmately the Slze of the X-chulmoRomp (Flg. Il a, c, dl. One of the prlnclpal questlons that needs to be anslolered ln attemptlng ta eluc1date the role of a supernumerary chromosome system lS whether the polymorphlsm lS temporarlly stable, 1.e. whether the frequenc1es of the ~lfferent karyomorphys are ln genetlc equlllhrlum. In orUer ta obtaln eVldencf> fOL this Important nSpect, the three rllfferent pOpUlntlons (1981, 1982, 1983) were compared. Table 5 shows that there is no signlficant dlfference between the three generatlons when the G-test (Sokal and Rohlf, 1969) was applied. The frequency of B-carriers ranged between Il.11 ta 13.04 percent. Although the study period is brief and the samples were small in ( 91ze, lt "emnn~trate9 that no dramatlc flllctu<'\tl,)nS ln R -chr lmnsome rontpnt l)ccur between c)ne generat lon and thp npxt anrl that there lS no marked dlfferentlal mortallty p f f e (' t ln - R a n cl + B l n '11 v l du a l 9 ci url n g the long h l be r n a t l n '1 pprlod. b. Behavlour of the B-chromosome At me10S1S, ln A-carriers, the lntimat~ pairing between the X and the B-chromosome becomes vlslble from early l pptotene" The average frequency of thlS heterochromatlc X/B assoclatlon at flrst melotic prophase was found to be greater than 82% (Table 6) and showed very llttle inter-lndlVldual varlatlon. Three maln types of as~oclat1ons were observed: (1) side-ta-side' (li) end-tn-f>nd" (111) end-to-side. Of these, side-to-end ~~soclatlons are the least common and the slde-to-side the most (1"1<]. lIa to rj: Table 6). Although non-chlasmate in character, the X/8 associations can perslst not only into fiLst metaphase, but also throu~hout first anaphase. No intimate association of the X and the 8 was observed ln second division cells, however, their strong tendency to lie in close proximity at thiS stage is indicative of previous somatic association (Fig. Ile). ( FI q llrp )). Mf>l,)tl, ,ilV1Slnn ln M. f. femurrubrum ma)"",. a. M II t-" show '01de-t)-SlOf> F\9.s')'latl,n ':if Rand X. h. M Ir t'l shnw· 1. pnd-t,)-pnd a'ORnC lat Inn nf Rand X· Il hptprnmorphlC M, wlth dlffen'nt heternpycnoBlty (drrow)· III translncat lnn n f éI fraqmE'nt at Ma· lV. dflymmf>try and ("on9trlct lnn ln ('. M Rand X. d. Ea rI y AIl 9how 1 nq : 1. free X and B: 11 constrlcted L1: iil end of L2 (arrow) wlth dlfferent heteropycnoslty. . .'--, , b d ( \ -- TABLE 5. A between-generatlons comparls1n nf A-chromosome frequency ln thp populatIons of M. f. femurrubrum from three succe9S1ve generatll)ns. ~ Collection GeneratIon Chromosome G df p* constltutl()n -8 +8 Totals 1981 l 40 f, 46 O. 175 2 0'.95 -O.Bn 1982 2 811 ln 90 l~ 3 '4 l l 85 '1' TOTA.tS 1':i4 Fe 221 * Values are not slgnlflcantly dlfferent. y, .;:,. - , TABLE 6. Type and frequency of X-A assoclat10ns at ilpl~tene-dlaklne81s in 10 males of M. f. femurrubrum with a supernumerary (1981 populat10n\. " , rndividual X and B dssociaterl % X and A assOclate(j X and B free Total no. e/s * e/e sie of cells l 14 0 lOO.OO 0 15 2 12 0 l 92.86 14 3 12 () 86.67 2 15 4 12 2 0 93. 33 l 5 5 10 2 () 80.00 1 15 6 Il • 0 l 100.00 0 12 7 12 l 2 100.00 0 l 5 ... 8 Il 1 l 86.67 2 1 5 9 14 1 0 100.00 0 15 10/ 10 l 0 73. 33 4 15 TOTALS 116 10 5 13 146 Percent 80.82 6.85 3.42 8.90 !fi VI * sis side-ta-side: e/e end-ta-end· e/e side-to-end: refers to the type of X-B association. 1 06 In order to determine whether such a prolonged association causee a non-random segregation of the B with respect to the X-chromosome, the pattern of X-B movement at first metaphase-anaphase was compared with the second dIVISIon metaphase counts to avoid a possible bias Introduced by unIvalent re-orientatIon. The X-B segregatIon pattern at metaphase-anaphase l ln 10 lndivlduals of the 1983 population is shown ln Table 7. There lS a Slight predomInance of cells in which the X and the B move together to the same pole (55-64%). In aIl ten Individuals, the small excess dld not yield a significant departure trom the expected ratio (Table 7). c. Chiasma trequency and distribution Chiasmata positions were scored as described in the ~ethods for 15 cells trom each of 10 males without a supernumerary and ln 100 cells trom 10 individuals carrying aB-chromosome. ~ales f~om both groups were chosen randomly. There was clearly a preponderance ot proterminal (terminal and eubterminal) chiasmata in the long bivalents In the medium length bivalents (~3 to ~8)' if randomly positioned, at least haIt of the ,1 chiasmata should have been s~ored as interetitial, but signi~cantly more were prot~rminal (X 2 (l)= 186, p~ , ( " , , 1 67 0.001). There were also significantly more distal than proximal chiasmata in the medium length bivalents (210: 118, X2 (1)= 28.9, P two chiasmata more commonly occurred in the 10ngest The medium length bivalents M3 and MR seemed to have particularly high chiasma frequencies but this could be a peculiarity of scoring. The three shortest bivalents (S9 to SIl) appeared to have distal chiasmata. Fig.12a to c shows the different configurations of chiasmata of a male. ( The chiasma frequency data (Ta~ 8) were analysed to determine the amount of chiasma frequency variation between- angvwithin-individuals with and without a supernumerary. Ten individuals without a B were chosen randomly from each population (1981, 1982, 1983). The six males with the B from the 1981 population, the ten from the 1982 population and ten from the 1983 population were examined for chiasma frequency. There are no significant differences among the between-individual variances of the OB and the three B-containing populations [OB mean 13.53, variance 0.2691: (81+ + B2+ + 83+) mean 13.99, variance 0.2607]. However, the difference between the mean log variances for the two groups is signficant (t = 2.1972, ( 0.02 TABLE 7. B-chromosome segregation pa t ter n w i t h respect ta the X-chromosome, at metaphase- anaphase 1, in 10 males of M. f. femurrubrum carrying a supernumerary (1983 population) . The expected ratio of the two segregational classes lS 1: l. i- p* Individual X à~ B ta X and B to Total cella df opp site same acored x~ po e pole \ ~ 1 9 11 20 1 0.20 0.75-0.50 2 9 16 25 1 1. 96 0.25-0.10 3 8 12 20 1 0".80 0.50-0.2S 4 Il 15 26 1 0.62 0.50-0.25 \ 5 12 13 25 1 0.04 0.95-0.90 • , 6 8 12 20 1 0.80 0.50-0.20 --./ 7 9 11 20 1 0.20 0.75-0.50 8 12 )- 13 25 1 0.04 0.95-0.90 9 12 13 25 1 0.04 0.95-0.90 10 Il 14 25 l 0.36 0.75-0.50 SUM 101 130 231 0\ Total 10 5.06 0.90-0.80 CIl * No values are siqnificant _ ..mntrll;" rnfemOUPIi*. ' ...... _...... , __A.~_ :.-...... --. --- ~ TABLE 8. The chiasma fr6quency data in M. f. femurrubrum males with and without a supernumerary for populationSlof 1981, 1982,-and 1983. \ Year Ce11 types No. of Chiasma frequency Mean± Between Mean of the 1098 Scored ",ales for each male s.e. individua1 of the within- variance individua1 variance 1981 OB 30 *12.9 14.4 13.2 13.53± 0.6291 -0.2604 1982 13.8 13.6 13.1 00.14 1983 13.1 13.2 15.1 12.4 13.8 15.2 12.5 12.1 14.7 "" 13.8 13.2 14.0 13.3 14.7 13.1 12.9 14.2 13.8 -/ 12.9 13.5 13.8 N' 12.3 14.1 13.2 1981 B+l 6 14.0 13.2 13.5 13.65± 0.1110 -0.1224 14.0 13.8 13.4 00.14 1982 e+2 10 13.6 14.2 13.8 14.11.± 0.3490 -0.991 a 13.8 15.3 13.7 00.19 13.6 14.2 15.1 13.8 ,1983 B+3 10 13.9 14.2 14.1 14.20± 0.4444 -0.1083b 15.3 15.1 13.8 00.21 13.6 14.1 14.1 13.8 e+l +8+2 +e+3 26 13.99± 0.2607 -0.1099 00.10 ... 0'1 1.0 * Each figure ia a Mean of 10 chiasma scores per male individual. a Sig~ificantly different from the value for the De group (t=2.206, 0.02 ~ ~ "'A..... ""~''''':'-'kF~.tIol'~-PI~..;tt.«> ..~,~~~,~...I.o.- ... ~,-''------70 .. Figure 12. M. f. femurrubrum meiotic division showing different bivalent configurations. a. M l to show that rings form more often in LI and L2. chiasmata each. c. B- containing individual with more than one chiasma per LI and L2 ( bivalents. ( l ( '-2 lx 1(5. 6 " '-.1~. ,.. ~c:r D . à ( • : '. c ", JJiii '&.. ..... - c .. , • - ~ ...... "" ... "\>~ - ~1/ri'r'~'-"'.l"~" 71 No inter-follicular variation was detected in aIl of the males with the B-chromosome, except in one case where the B ie missing from some of the cells of an individual in which moet celle have the B-chromosome. 3. Changes in Chromosome Structure and Number The chromosomes of ~. !. femurrubrum, in general, show a high degree of stability, howeverl there are a few cas.s of some structural changes. Most of the abnormalitles noticed were connected with cells of individuals possessing ( \r the supernumerary. In an individual o~ the 1982 population which carried a B-chromosome, one of the long bivalents fi" (L2) showed asymmetrical arms (Fig. lIb) as weIl as an indication of a break in one of the arms. The medium length bivalent (M3) of the same individual showed arm asymmetry as weIl as a clear difference in heteropycnosity of the two arms. This same individiual carried a smal1 fragment which was translocated on one arm of the Ma chromosome. Another individual from the same population which also had a supernumerary exhibited a heteroehrom&tie knob in the L2 chromosome (Fig. Ile). There was an ( 1 72 individual from the 1981 population which showed a peculiar phenomenon which was not repeated in any other individual. This male which had no B-chromosome showed a continuous connection between the X-chromosome and the M6 with strands of chromatin occurring between them (Fig. 13b). Apart from the change in chromosome number caused by the presence of the Bupernumerary, there were individuals which lacked one chromosome. The three populations showed a high percentage of individuals which lacked a chromosome. The 1981 population had 32% of the individuals ( lackinq an autosome~ 8% were lacking one of the small pairs (59 to 511), while 24% were lacking either M3' M6 or MS. The 1982 sample had a 22% of the indi~iduals , i without one autosome, 10% of which laçked one of the smal1 autosomes and 12% were deficlent of a medium autosome. In the third generation of 1983, 35% of the males lacked an autosome~ 14% lacked one of the smal1 autosomes while 20% had a missing medium chromosome CM3' "51 "6' or Ma). One male individua1 from this generation, which had a 8-chromosome, lacked the X-chromosome from some of the follicles. The loss of the X was always accoepanied with the loss of the supernumerary (Fig. l3a). As seen from the figures above, thera was no uniform pattern for the missing ( , \ (. 1 73 Figure 13. Meiatic metaphases in M. f. femurrubrum. a. M II cel1 ta show heteromarphic M3 and Ma. b. M II cell ta show strands of chromatin occurring between the univalent X and M6 bivalent. ( ( \' , ( ( • 1 74 a~tosomes of the three generations. Although it was expected that the missing chromosome would be one of the small autosomes, as is the general bellef, lt is interesting to find that M. f. femurrubrum had more medium pairs than small pairs missing from the complements. However, the absence of an autosome, whether medium or small, was not stable within the individuals i.e. sorne of the folliclea contained cells which lacked an autosome as weIl as cells which had the complete chromosome number. , ~. The vast majority of individuals sampled in the present ( study were karyotypically very stable and uniform. ( ·, 75 THE CYTOGENETIC SYSTEM OF MELANOPLUS BOREALIS BOREALIS (FIEBER) 1. The Karyotype The chromosome cytology of Melanoplus borealis borealis was studied on material from two populations (1982 and 1983). Due to the fact that sampling was done randomly and that this grasshopper disappeared from the collecting site by mid-July, only four individual males from the 1982 ( population and five from the 1983 population were collected. The standard karyotype of the male includes 23 èhromosomes (XO), the X-chromosome being the seventh in length. The relative length of each member of the complement is given in TabYe 9 and in the idiogram (Fig. 14). In the present study, the autosomes w~e classified in three size groups including sTx large (LI ta L6)' ) two medium (M7 and Ma), and three small (S9 to SIl). The chromosomes show gradation and consistent discontinuity in length between the three groups (Fig. l5a). The chromosomes were divided further according to ( 1 the posItion of thp cpntromen" to faCll\tatp romparlson 'Tahl!" 10). The first flVP chromosomes, LI to LC;' \,Iere r!assIflPd as suhtelorentrlc \,II th arm ratIos ranglnq from 4 . () t '1 f,. () . The slxth c-hr,mo90me, L6' had no short arm a n ci l S r 1 a SAI fi!" d as r'e 1 n r p n trI r . Th!" t\,lO medIum three Amal1 paIrs arp dlRtInqulshahle from each other· C;q 19 il tel,)rpntrlr, S10' a metacentrlc and SIl' è! sllhmetac!"ntrlr'. Th p X - c h rom 0 som PIS t P ! (1 r p n trI r and hptpror'hrnmat Ir. ( 2. The 3B Ka ryomor'ph Due tn the small .'lample Slze, lt \,las not pOSSible to maK!" an accuratp pstlmate of the B-chromosome frequency \,Ilthln thp populations of the 1982 and 1983. O'lly ,ne Indlvldual among the males of the 1983 population carrled S u pe r n ume ra rie B • Th1.9 unique Individual \,las found to possess three supernumerarles classlfied in the present study as RI' B2' and 83' according to their relative --' slzes. AlI the thref> A's \,Ierp found to be telocentrlcs. BI \,las t\,lice the size of the smallest A-chromosome ( 1 77 Flqurp 14. Tdlogram nf ~. b. borealls male (2n 23) based upon the percentaqes of total complement lenqth (TeL). The haplold set l~ arranged dccordlng ta decreaslnq lengths of whole chromosomes (long + short arms). / ( 1 1 2- 1 ~ « 2 x ( t 78 Flgure IS. Meiotic division ln M. b. borealis males. a. M II showing the complement with the apparent short arms. b. M l of an individual male with 3 B- chromosomes. Note the negatively heteropycnotlc X-chromosome. ( ( { _ •••______~~~~~~~""'-~)'nW'~r""'IIt f~~~~l'J:mSl,'IiP'~~-...,-- -- / i : j , : , ' 1 \ 1 Il ~, TABLE 9. Chromosome measurementa of Melanorlus borealls borealls expressed as percentages of total dlploid chromosome lengthTCL). The mean (X), the standard deviation (~), and the coefficient of variation (CV) are based on measurements from five metaphase cells (~.!. Materials and Methode). LI L2 L3 L4 L5 L6 M7 M8 59 510 511 x TOTAL x 15.65 14.26 12.19 11.21 7.86 7.74 6.36 5.86 4.23 3.67 3.30 7.66 99.99 tS 00.37 00.42 00.14 00.17 00.18 0.22 0.26 0.12 0.18 0.11 0.23 0.16 cv 2.26 3.43 1.15 1.52 2.29 2.84 4.09 2.05 4.26 3.00 6.97 2.09 '-..1 .~~ 10 , .. ., / TABLE 10. The variations in centromeric positions of the chromosomes of M. borealis borealls based upon ara ratios (Ils). Chromosome Arlll ratio· Nomenclature nu.ber Ll 6.0 Subtelocentric L2 5.5 Subtelocentric Ll 4.5 Subtelocentric L4 4.0 Subtelocentric 1.5 6.0 Subtelocentric L6 CD Telocentric "7 1.6 Submetacentric M8 CD Telocentric 89 CD Telocentric S10 1.3 Metacentric SIl 2.0 Submetacentric X CD Telocentric * Each figure represents the ratio of the averaged arm lengths ~f the live males. CP o '" . __ / , ( 81 (SIl)' B2 was approximately the size of 511' and B3 was half the size of that of the 511' There was no association of any of the B-e1ements with the X-chromosome during any stage of meiosis in this individual, i.e., the B's are X-free. Though a reduction in the number of A-chromosomes was expected, aIl the cells had the normal number of autosomes (Fig. 15b). The three B's were very stable in aIl the cells within the follicles examined. BI' B2' B) were aIl noticed to be negatively ( heteropycnotic during prophase 1 and positively heteropycnotic at metaphase 1 (Fig. ISb) and anaphase 1. The dissimilarity of size and centromere position, particular1y evident at certain stages, strongly suggests that the three B-chromosomes are not homo1ogous. The B's occurred as three univalents during prophase 1 and ana phase l. They do not pair in a conventional manner~ nor do they exchange a chiasma. 3. Chias.a Prequency and Distribution chias.ata were scored in 15 cells from each of the 8 ( ( 82 males. The cell mean chiasma frequency was found to be 13.058 (variance 0.963). Bivalents of simi1ar length appeared to behave similarly, but the possibility of bivalent specific chiasma localization is difficult to exclude entirely. In some early diplotene spermatocytes where centromeres co~ld not be readily distinguished, third classes of terminal and subterminal chiasmata were scored which could not be designated proximal or distal. Diplotene chiasmata scored only as terminal or subterminal have been divided in the ratio of distal to proximal chiasmata in the long and medium length bivalents. If ( randomly positioned, at least half of the chiasmata should have Deen scored as interstitial, but significantly more were proterminal (X 2 {1)= 31, P<.O.OOl). There were a1so significantly more distal than proximal chiasmata in the medium length bivalents (X 2 {l)= 8.69, P<.0.005). (The number of proterm~nal chiasmata not allocated to ei ther end was larger than the di fference ~ however, th.ese were probably related to the meiotic stage rather than biased for chiasma position.) l, III i ~ j In the long bivalents (LI to L6)' 79' of single chiasmata were interstitia1, whereas in 45 rings there were 32 (70') protermina1 chiasmata and 13 interstitial. In the ( 1 1 1 83 ~ • î i11 shorter -medium- length bivalents ("7 to M8) with lower , ~ chiasma frequency and hence more sinqle chiasmata, the ~ :i proportion and frequency of proterminal chiasmata was increased. This decrease in the proportion of interstitial chiasmata between the long and medium length bivalents is not significant. However, this reduction could be due in part or entirely to an artifact of scoring, since a smaller proportion of the shorter bivalents would have been scored as interstitial. ( AlI the chiasmata in the three shortest bivalents (S9 to SIl) appeared to be extremely distal~ however, comparisons were difficult because of the difficulties of scorinq other chiasma positions in such short bivalents. The three short bivalents were never noticed to torm more than one chiasma. 4. "issinq Autosomes and Other Irregularities One male individual of the 1982 population possessed some follicles which had cells lackinq one of the small autosomes bringing the diploid chroeosome number down to 2n ( ( 84 5 = 22. Another individual, trom the 1983 population, had two of the autosome8 mi88ing (SlO and 511) (Fig. l6a). In this same individual, some of the follicles were ~acking one of the long autosomes (L6) (Fig. l6b). 80th males' exhibited variation among the follicles (inter-follicular variation), however, the number of autosomes was stable in aIl cells within the follicle for the first male. This individual had 89.7' of the follicles lacking the 510 autosome. The second male had Il.6' of the follicles with a missing L6' 26' were 510 and 511 deficient and 60\ of the follicles lacked 510 only. Apart from the presence of the supernumeraries and the absence of autosomes, aIl the rest of the individuals examined showed -normal- meiotic cells vith one exception. This exception vas one male vith a heteromorphic long autosome (LI) in some of its cells (Pig. l6c). There vas no evidence of translocation or 8 presence in this individual. 1 1 85 Figure 16. Meiotic division in males of M. b. boreali8. a. M II cell with missing autosomes (SlO and SIl). Note the negatively heteropycnotic X-chromo8ome. b. M II cell missing an L6. c. M l showing heteromorphic (H) LI" ( " ( ( 1 J j , x. )t«# +. fI · " a .• \ 1 ,. \ , 0 b "- , .",- J cl, x - "'"'-,e fi c ~,~ 1 . , ( ( a6 THE CYTOGENETIC SYSTEM OF MELANOPLUS SANGUINIPES SANGUINIPES (FABRICIUS) 1. The Standard Complement Like other species of Melanoplus so far examined cytolpgically, M. ~. sanguinipes has a diploid number of 236. The haploid number can be arranged into t~ long (LI' L2)' six medium (M3 to MS) and three short (S9 to SIl) sizé groups of autosomes with the telocentric X-chromosome, when isopycnotic, slightly larger The relative length of each individual chromosome is given in Table Il and in the idiogram (Fig. 17). The autosomes can be divided further according to their size and position of the centromere (Table 12). Though both long autosomes (LI' L2) are considered to be telocentrics, L2 has short arms which do not appear in any stage of meiosis in LI. The six medium autosomes can be grouped into two different categories; M3' M4 and MS are larger in size and aIl three possess the minute arms which distinguish them from the other smaller group (M6' M7 and Ma). Of the short group, S9,can easily be distinguished, being a subtelocentric, from SlO and ( 1 p,7 percpntn'1PR nf tntal ,nmplement length (TIL) wlth the chromosomes ~rran'1erl accnrrllnq tn rlerrea!H> ln lenqthR of t"trll rhrnmnsnmp (1 ,nq + shnrt arm) ( 1 2- OJ 1 1 2~ III 1 ....J 1 ....U ~ X 4 TABLE Il. Chromosome measurements of ~. ~. sangulnlpeB exp~eBged as percentages of the total dip10id complement length (% TCL). The mean (Xl, the standard devlation (6), and the coefficient of variation (CV) are based on measurementB of ten metaphase cells. LI L2 M) M4 MS M6 Mf 1'18 S9 S10 sl1 x TOTAL x 15.1214.1511.7010.)110.19 7.30 5.97 5. 7 9 4.47 4.39 3.20 7.39 99.99 6 00.28 00.19 00.14 00.22 00.12 0.11 0.24 0.14 0.19 0.12 0.21 0.33 cv 1.85 1.)1 1.20 2.13 1.18 1.51 4.02 2.25 3.18 2.f) 6.56 4.47 (J) (J) T ABLE l 2 . 'r h e var i a t ion 0 f c ~ n t rom e r- lep 0 S 1 t i 0 Il tl ') t tri.,.. chrllmùsomes ùt M. oS • sanguinipes based upon arm ratios (l/s)) after Levan e~ dl. ( 1964) . Chromosome Arm ratlo * Nomencldture number ------LI Telocentrlc L2 9.31 Subtelocentrlc M1 3. 57 Subtelocentrlc M4 4.95 Subtelocentric M5 Il . 39 Subteloc~ntric M6 CI) Telocentric ",7 CI) Telocentric M8 CI) Telocentric S9 4.44 Subtelocentrlc S10 CI) Telocentric SIl CI) Telocentric CI) X Telocentrlc * Each figure represents the ratlo ot tri t:' ,l V ,J r Cl g e ci a r m Iengths (long/short) of 10 cells. ..0 o i'.",~~~~~.",.",~ -- -~- ~" f ( 90 SIl . The latter two differ sligbtly in size from each other. 2. The B-chromoaome System Two populations of ~. ~. sanguinipes were studied ta determine the frequency and behaviour of the B-chromoaome found in the species. Three males out of the 26 examined cytoloqica11y in 1981 had a B-chromosome: i.e (11.54%). Of the 73 male individua1s examined in 1982, 14 carried a supernumerary (19.19%). The population of 1983 had a frequency of 8.57% (3 individua1s out of 35 examined possessed a supernumerary). The three populations (1981, 1982, 1983) were compared ta detect if there was any fluctuation in the B-chromosome content between the different generations. When the G-test waa applied (Table 13), it was found that there is no significant difference in the B-chromosome frequency between the three generàtions (it ranged from 8.57% to 19.19\). This imp1ies that the ~upernumerary did not fluctuate dramatically between the three generationa. l' ~ .-.., TABLE 13. A between-generations comparison of B-chromosome frequency in the populations of M. s. eanguinipee from three successive generations. Collection Generation Chromosome G df p* constitution -8 +8 Totals 1981 1 23 3 26 2.50 2 0.50 - 0.20 1982 2 59 14 73 1983 3 32 3 35 TOTALS 114 20 134 * Values are not significantly different. '"~ ( 92 Figure 18. Meiotic divisions in M. s. sanguinipes with supernumeraries. a. A cell showing the positively heteropycn~tic X and B. b. A structural variant of the basic B-chromosome: note the interstitial -vely pycnotic region and the two terminally sited isopycnotic sections. ( c. The position of the B-chromosomes in relation to the SlO autosome (no real association). ( ,~~ ---- ( . ~. a ( b ., • fi. r; 1 , ~ ,'f~ • • l' •. • " , c "" \ . ,; ( , i ( 93 AlI of the twenty individuals which carried the supernumerary possessed only one single B at a time. The B-chromosome was found to be approximately the same size as the 89 autosome. Though the B-chromosome followed the X-chromosome in pycnosity during the first meiotic stages (Fig. l8a), it showed no intimate association with the X at any stage. The segregation of the B and X to the same and/or to different poles was at random. Although the B was detected to lie in close promximity to the 810 autosome during the first and second divisions in three individuals, it showed no synaptic association with any of ( the autosomes (Fig. 18c). There was one individual from the twenty carrying the supernumerary which showed a different supernumerary from the ·standard· one described above. The supernumerary of this individual behaved in the same manner as the standard B. However, the structure of this B was different from the standard one. This B-chromosome had an interstitial region which differed in pycnosity from the two terminal sections of the chromosome (Fig. 18b). ( 94 AlI males with the B element had a telocentric B-chromosome. The B-chromosome appears to be meiotically stable as aIl follicles contained one B. 1 3. Chiasma Frequency and Distribution in Relation to the B-Chromosomes The chiasma frequency data for the OB and the B-containing groups are summarized in Table 14. Cell mean chiasma frequency was 12.73 (varlance~ 0.2165) for the OB ( group and 14.22 (variance 0.1635) for the pooled B- containing individuals. There was a significant difference between the two groups (t= 15.4l7.P<0.001, 48 dt). Bartlett's test of homogeneity of variance indicates that the OB group and the B-containing group differ significantly from one another (X 2 (3) = 75.785, P< 0.001). There was higher within-individual variation in • B-containing males than in the standard individuals (ti+: -0.05833: OB: -0.2649). In the long bivalents (LI' L2) there was a higher proportion of interstitial chiasmata. In the LI 1 bivalents, 85\ of single chiasmata were interstitial, 1 ( 1 j i ( 95 whereas in the rings only 29\ were interstitial and 71\ proterminal (either terminal or subterminal). The medium length bivalents (M) to Ma) had lower chiasma frequency and hence more single chiasmata. In this group (M3 to Ma), the proportion and frequency of proterminal chiasmata was larger than in the LI' L2 group. This decrease in interstitial chiasmata is not significant. Most of the chiasmata in the small group of bivalente (59 to 511) appeared to be extremely distal (Fig. 19a and b). However, there were sorne cases where interstitial chiasmata were detected (Fig. I9c). No rings were noticed in the short group (59 to 511). In the B-containing individuals chiasmata were scored in 10 celle from each of la males. There was a clear ten- dencyof the long (LI' L2) and medium (in particularly, M), M4' and MS) bivalents to acquire rings more fre- quently than in the OB individuals. The M6' M7 and Ma rarely showed more than one chiasma per bivalent. There was no evidence in this atudy of any chiasma effect on the emall group bivalents. In both long and medium groups of bivalents, there was a significantly higher proportion of interetitial chiasmata in the B-containing males than in , the OB individuals (X2 (1) = 29.2, P<0.991~ x2(l) = i - TABLE 14. The chiasma frequency data in ~. 8. sanguinipes males with and without B- chromosomes. Year Cell types No. of Chiasma frequency Mean.± Between Mean of the 10gs Scored Males for each male s.e. individual of the within- variance individual var~nce * 1981 OB 30 13.0 12.2 12. l 13.0 12.73.± 0.2165 -0.2649 1982 12.2 13.1 13.8 13.6 0.08 1983 13.0 12.4 12.2 13.0 12.8 12.6 13.0 12.5 12.3 12.1 13. 1 12.6 13.0 12.6 12.8 12.2 12.8 13.2 12.5 12. 1 13.6 12.6 1981 a+ 3 13.8 14.2 14. 1 14.03± 0.0434 -0.0346 0.12 1982 8+ 14 13.8 14.3 14.0 13.8 14. 1 li: 0.2002 -0.0628 13.6 13.8 14.2 13.5 0.12 14.2 14.6 14.6 13.8 .. 14.6 14.8 1983 a+ 3 14.2 14.8 14.6 14.53± 0.0934 -0.0776 0.18 1981 s+ 20 14.22.± 0.1635 -0.05833 1982 0.09 \0 1983 0'1 * To analyze the within-individual variation among cells, the variance of 10 chiasma scores in each male was tiret converted to a log variance. ~."jlVhf"''t~',..,lü. .... ,- .." ... 1 97 Figure 19. Second meiotic metaphases of ~. ~. sanguinipes showing different chiasma configurations. a and b. ama11 group bivalents with distal chiasmata, long bivalents with interstitial chiasmata. c. LI with two chiasmata, S9' SlO' and SIl with interatitial chiasmata ( l ) . ( '" ( ( a ( b " { ,; ~ c ( :#!." I n .?,P':: r) () () 1 l • l f 9 r 'l ,ln q . '4 V,., t Ï ) m, '(1) h:"" I,J 1 t h Ir, p ~ 1 n r ~ ~ 1 P ~ Of thp lq82 males, 12 141.'1%\ we,p lrtrklnq one or mo,p aut()l'1ome. !,'our male9 f,()m the IgRÎ pOpUl<'ltlon (11.4%) showed the same phenomenon. In 77% ~f those males, the mIsslnq autosome was a short one (5q, SlO' or 511) while ln 23% the mis8ing autnt'lome was a medIum one (M" M7' or 1"18) (fIg. 20a ). However, where there was more than one mlsslng auto8ome It was always the t'lmall group WhlCh waa lnvolved. ThlS lack of an autosome waa not a stable phenomenon 8lnce there was a notlceable inter-folllcular varlatlon l.e. sorne 1 3angulnlpes. a. A êpll fr0m an lnrllvujui'll .... lth h. A hptpr0mnrphlê /'l, ~nrl 91 t p9 lf brp~k.a'1P at LI ~nrl L? ( ,. , / /' i. ,'" a b ( 100 follicles had 22 autosomes while others had 23 within the same individual. Stlll, there were cases where aIl of the cells of the Indivldual were deficient of an autosome. In thlS study, the misl'Ilng autosome was correlated with the presence of the supernumerary in the JpUlationl but in fact there was no direct relation betw~en the two ln th i B J specles Slnce none of the lndlvidl~~~S wlth a deficient autosome acqulred a supernumerary ln any of the folllcies. Whenever there was more than one autosome misslng from a follicle, they were accompanled by a mlsslng sIngle ~ut0somp 0n other folllcies within the same lndlvldual i.e. thlS was not a stable sItuatIon. One male lndivldual which had a mosaic of follicles with 23, 22, 21 and 20 autosomes possessed a heteromorphic 1'17 (Flg. 20b) and showed sites of incomplete breakage in the LI and L2 autosomes (Fig. 20b). In this indi- vidual, the missing autosomes were two of the smaii group and the M3 and/or M7 when the three autosomes were ml8s1ng. 101 DISCUSSION 1. Ka~yotypic Diffe~ence Between the four Melanoplus Species The four Melanoplus species studied here have 2n ~ = 22 A + XO ln the male but the complements of the four a~e not 1dent ica l. M. f. femu~~ubrum has aIl ch~omosomes as telacent~ics with no indication of small arms, while M. b. b1vittatus, M. b. bo~ealls and M.~. sanguipies have short a~ms p~esent. Though the latte~ three passess sho~t arms, they d1ffer g~eatly in the ratios of these a~ms and the1r p~e8ence in d1fferent chromosomes. A main feature of the differing ka~yotypes is the lack of the thi~d small ch~omosomes from the complement of ~. ~. bivittatus and the presence of a medium length chromosome instead. Measurements of the chromosomes making up the karyotypee show differences between the four apecies in lengtha of comparable chromosomes. M. b. borealis is the only species amongst the tour which has metacentric (510) and submentacentric (M7 and 511) chromosomes: also it has two medium and 6 large chromosome pairs which ia a different feature from the other three. Al though aIl of ___ -Â... 102 the chromosomes of the four species show gradation in length, there is a remarkable di fference bet..,een one species and the other. The four complements show structural differencea and feature no stability of comparable autosomes. Even the length of the X-chromosome ..,as not constant: it ranged bet..,een the fifth and seventh compared ta the autosomes in the four species. 1 t i B weIl kno..,n that careful measurements of the chromosomes making up the karyotypes show differences bet..,een species in lengths of comparable chromosomes (Vickery, 1977). One useful character that has been found to vary great1y even in close1y related species, is the length of the small second arms of acrocentric (subte1ocentric) chromosomes. In the four Nelanoplus speciee, there are major differences bet..,een the single chromosomes beside this feature. Karyotypes of the four are clearly ditferent in contrast to the general belief that visible cytological differences bet..,een c1ose1y related species of Orthoptera are rare. White (1951) stated that -In some large genera such as Melanoplus it seems probably that aIl, or almost aIl, the species have chromosome complements ..,hich look indistinguishable at metaphase-. This etatement is no longer true considering the basic differences bet..,een the ( Melanoplus species studied here. ( 103 2. The Supernumeraries: A Comparison Although the four species of Melanoplus studied had suparnumeraries, the meiotic behaviour of the Bs and their sizes ara quite different. No marked differences are present in the frequency of the Bs in the different generations. In M. b. borealis, M. bivittatus and M. s. sanguinipes, the B-chromosomes do not assoclate with the X-chromosome or with any of the autosomes, while the B-chromosome of M. f. femurrubrum is associated with the X-chromosome. This X-B association has been described in a numbar of 8~acies of graBshoppers. The two elaments at first meiotic prophase are frequently engaged in a close and persistent association, which iB occasionally protracted until first metaphase. Since the X and B are hetarochromatic, and positively heteropycnotic at first prophase, they do not appear to form chiasmata, neverthe- less their synaptic association may wall be dua to some degrae of structural homology. In Podisma pedeatria (Rewitt, 1973), the relationship, although it does not appear to be chiaamate, ia claimed to be too regular to be dismissed as -heterochromatin stickiness- and la indicative ( 104 of some homology betveen the two elements. Rowever, there are also certain discrete differences in structure and/or a1locylic behaviour betveen the X and B-chromosome which indicate that the supernumerary element is not a direct product of non-disjunction and that it must have undergone evolutionary changes since its inception (Gibson and Hewitt, 1970, 1972: Gal1agher et ~., 1973: Fox et ~., 1974). In fact, the biochemical data (Gibson and Hevitt, 1972) indicate that there are discrete differences in base composition of the B-chromosome DNA of Myrmeleotettix maculatuB (Thunb.) populations studied. ( Both in size and allocylic behaviour the B-chromosome of M. t. femurrubrum is strikingly similar to the X-chromosome. However, the degree of pycnosity indicates that the B-chromosome cannot be regarded as a simple non-disjunction product. Nor can it represent the proximal part of the X-chromosome vhich arose by deletion as in sorne ) ather grasshoppers. ~he preferentia1 movement of the B vith the X-chromosome towards the same pole observed in males of M. f. femurrubrum is expected to produce differential transmission of the supernumerary to the two sexes aince X-B gametes viII produce temale oftspring. J Rowever, since this study does nat incluàe females, it ia ~ 1 ( difficult to explain thia assumption. 105 Hovever, the origin of the B-chromosomes in M. b. borealis, M. ~. sanguinipes and ~. bivittatus cannot be related to the X-chromosome, because there is no association of the B-chromosome vith the X. The Bs in the three species appeared to have an autosomic nature, though no synaptic associations with other chromosomes of the complement could be detected. In the three speciea, there was no relation between the B-chromosome and the miasing autosomes, so this will rule out the iSssumption that they might have originated ae a result of non-disjunction of sorne of the autosomes. In fact, not aIl the missing autosomes were the small group autoeomes~ sorne long and medium bivalents ware involved and there were no constant criteria for the loes or addition of an autosome in the three epeciee. The B-element of M. bivittatus ie smaller than the smallest autosome, that of M. s. eanguinlpes ls approximately the size of the SlO and the three Bs of M. b. borealis range from halE to twice the size of the It le not possible to draw a direct relationship between the B-chromosome system of the three species except that they aIl have telocentric Bs. 106 Three possible modes of origin have been proposed for o B-chrom~omes: (a) from the smaller autosomal members of the complement via trisomie or polysomie conditions for (i) largely heterochromatic autosomes, or (ii) euchromatic autosomes which become heterochromatic; (b) from centric fragments of standard chromosomes (consisting of centromere and adjacent heterochromatic region); and (c) from extra X-chromosomes. The major structural and behavioural types of B-chromosomes encountered in the four species of Melanop1us studied can readily be explained on the basis of the above derivation. Indeed, we know that a variety of such de novo, spontaneous structural mutations or chromosome conditions, which could potentially give rise to a "neo-B", do occur in natural populations with a relatively high frequency (Southern, 1967). For example, centric autosomal fragments produced by spontaneous deletions or translocations which are capable of perpetuating themse1ves through a number of generationa have been found in a number of species (Southern, 1967; La Chance and Degrugillierl 1969~ Hoehn et al., 1970). Such fragments may then become converted into true supernumeraries and poasibly increase in size following duplications or translocations from other chromosomes. 107 The newly arisen fragments found in M. bivittatus provide further evidence that B-chromosomes are intrlnsically unstable and rapidly evolving entities, which can be added to and lost from a population with relative ease. They provide an additional source of genetic variability and a "disposable" system for genetic experimentation without any of the "risks" arising from modifications of co-adapted gene complexes in the basic genome. Many challenging problems remain to be solved in the 8-chromosome system of Melanoplus species, while new questions are raised by the evidence emerging from the present study. An analysis of controlled crosses under experimental conditions may provide some of the answers, while others will no doubt be found in the natural environment of the species. Whatever the nature or purpose of the investigative approach, the intriguing characteristics of the cytogenetic system of Melanoplus speciea, eapecially M. bivittatus, will certainly provide scope and reward in future research work. 108 3. Chiasma Conditions of the Four Speciea The methods of describing chiasma position used in this study were relatively coarse, but simple and reliable. Modern microelectronic and computer techniques have made it possible to locat@ chiasmata ta a very high degree of accuracy (Shaw and Knowles, 1976). However, such accuracy was not required to produce positive results in this 3tudy and it is clear that the distribution of chiasmata lS dlfferent in each of the species studierl. The possibility of chiasma movement or terminalization 13 small. There is no evidence for it in changed chiasma patterns in the dlfferent stages of male meiosis in grasshoppers generally (Fox, 1973), nor in this study. Chiasma localization was often procentric and was seen as soon as diplotene bivalents become visible; the considerable tension from the centromeres did not cause chlasmata to move distally. In the absence• of localization, chiasmata were found in most parts of bivalents, so that they were clearly not terminalized. Jones (1977) has observed the behaviour of differentially Iabelled chromatids in Schistocerca gregaria and haB shown that the exchange of pairing pattners expected from chiasma ( movement did not occur. This has aiso been shown by 109 differential fluorescence in Locusta migratoria (Tease and Jones, 1978). In the four grasshopper spec i es exam i ned above, the males have localized chiasmata. The chiasma frequencies are only slightly above the minimum required for normal segregation of one per bivalent in either speCles. These speClE~s with localized chiasmata appear ta be specially adapted to particular enviranments which are apparently stable sa that recombination lS likely to produce less IoIell adapted offsprinq.,· However, it lS difficult to define a s pe C i a l i z e d n l che, and a the r s p@c i e s w i t hou t chi a .!ftn a localization may be equally specialized. FurtheI"more, in response to long term climatic chaqges grasshopper species are likely to change their geographic distribution to remain in a suitable habitat rather than adapting ta a different niche to which other species are already weIl adapted. This is weIl documented in beetles (Coope 1 1970). Chiasma localization may be an adaptation ta facilitate ~eiosis and increase the rate of production of spermatozoa rather than as a primary means of modifying recombination. It has now been amply documented in many plant and animal species that the presence of 8upernumerary o chromosome material can produce variation in chiasma ( 110 frequency at different levels. In the systems of M. f. femurrubrum, M. bivittatus and M. b. borealis, there does not appear to be a positive correlation between the presence of B-chromosomes and mean cell chiasma frequency. However, the number of males studied with the B-element is relatively smail. Whlle several studies have indicated that B-chromosomes can Increase the chiasma frequency (Rnrker, Ig60; John and Hewltt, 1965; Ayonoadu and Rees, 1968; Zecevlc and Paunovlc, 1969), such an increase does not accompany B-chromosomes ln aIl systems (Jones anrl Reês, l 967 ) . The variation of chiasma frequency between IndividualA was not increased by the presence of supernumerarles. In fact, the Inter- Individual var-lance was higher (though not significantly sol in the OB group than ln any of the B+ groups. In M. s. sanguinipes a significant effect of B-chromosomes in chiasma frequency was detected. The overall picture on chiasma condition emerging from studies on a number of grasshopper species which are polymorphie for supernumerary chromosomes (and/or supernumerary segments) has been summarized and tabulated by John (1973) and Schroeter and Hewitt (1974). From these surveys, it appears that B-chromosomes influence genetic III r P (' n m t) 1 n ~ t l " n mil r hIe 'l ~ f r e q u p n t 1 Y t han 1 ~ t h f' r n ~ p f -) r hptf>r'H hrl)mrtt l' 9Pqmf'ntfl Ciirrled by the 9tdnddrri "'lnl1t 1 ln "Y ~hp R"I '1ii" hppn 'h 'r luqhly ~n~ "'Kten~lVf'lv 1 ~ h l thf' f'!!)'-Ilnq lmp't'-l'l' "1 n 1 h p t 'W P P n - PI, ,; "1 r l ,i n (' p li ."p ln1j,;11'ld:"I ",hl'" ~'99P9q 11 1 1 hpre 1~ '1 ~lmp!p p'lVlr-,nmpnt ,-hilriict er 19t 11-9 Thus, qenqraphlcdlly iic11acent pnpula' l ,n9 miiy 11ffer mdrkedly b0th ln R-rhr')mosomf' fre':juenrv anl1 ln H ,'-le vp t i:l9 lt has open r0qently arqued 1 n mea n rel! ~hlii8ma freqlJency ~ sp may not hf' neariy a~ lmp,)rtant ln the release H ')nservat ,n' )f genet le 112 r n M. "1 9an~lnlpe8, the B-carrlers show a sharp Indped rhl.9 phenomennn l~ not llnr-ommon ln natural Slmllar effects nn Chl"i9ma Incallzatlnn hy 1 ha n Cl tiC a Lllldceap :'arrhl f't '11., l ':1~4 Sur-h pffer-ts, whatever ,~mnunt if recomblnatlon betIJeen ·w" lr more genet'! ln the 9 éI m p ,- h r ") m 0.9 0 m p, ~ han t h r r) Uq h sel f' c t Ion 0 f SUI t ab 1 f' p'llyqenlr comblnatlJns. 1 () h n 19'"3' has argued that thf' val Je n f suc h ait e rat 1 0 n R 1 n p r og en y g en 0 type Ile 1 n t ~ e pntentlal they provlde f::n a population t'J respond t-:J, and drc,mmClrlate, sudden envlr,nmental changes, althouqh they rannot confer any selective advantage to the Indlvldual. In M. s. sangulnlpes, the markerl Increase ln lnterstitlal chiasma frequency accompanylnq the presence ,f Bs should strengthen linkage bet\Jeen t\JO genes ,r gene comblnatlons and thlS may weIl be fav0ured by natural selection under partlcular envlr'Jnmental conrlltlons 'Dobshansky, 1970). l l 1 4. Chromosomes and Taxonomy A parr Icularly Important reaaon for the utillzation of ka r y') type a 1 n t a x 0 nom y 1 S t ha t the y se r v e d sac he c k 0 n t h p The general - h r '~m 0 som e ~ t r u ("" t ure 1 a d r e f l p c t Ion 0 f a l a r q e pa r t 0 f the ~ p n ) type d n d t hua pe r mit s relia b l e c () n c l u a Ion a 0 n r pl d t l ,jn shi ps . Whlle kary0types are useful. they cannat he ~ 1 st Inqu 1 qh hetween known ')rthoptpriin generii . 1.P. JPry many have d (""omplement jf 2n ~ = 211. pxamlnat!')n ,t ~hE' chrJmosomes maklnq up these karyotypes ,ttpn will prJvlde vdluable lnfJrmatlon (Vlckery. 19 77 '. În the bdS1S jf chr0moaomal ~rqanlziitl,)n. Melanoplua specles studled here can be separated lnto four chromosomal qr)ups. .... t. femurrubrum has d complement 0f (terminal te10centrlc chromosomea representlnq the ·prlmltlve· kary0type 'Jf the famlly Acrldldae. Populations share a slml Ildr chromoaomal 0rganlzatlon WhlCh dlffer~ markedly f rom t ha t ') f the 0 the r t a x d . On the other hand. M. blvlttatuB has members ')f the complement (Ll' L2' L4) wlth the more advanced aubtelocentrlc chromosomes, I.e. they are clearly and 114 conslstently characterlzed by the presence of short euchromatic arms beyond the centromere. l n the p rima r y pest, M. s. sangulnlpes more members of the complement possess the advanced character (L2' 1"13' 1"14' M') and c; 9 ) . M. h. borealls have obvious karyotypic dlfferences fr0m any jf the above. l n t h 1 S S pe cie s, non e 0 f the autosomps has the ·prlmltive" telocentrlc appearance. Thus ~hp tplo- and subtelocentrlc chromosomes, characterlstl< Jf the ") the r s pe cie s, are h e r e r e coq n 1 z a hIe a s met a - a n 911hm€'tacpntrlr- r-hromosom€'s. The telo/metacentrlc ~lfferences between the four specles have unquestlonably resulted from perlcentrlc rearrangements durlng evolutlon Jf th€' North Amerlcan taxa. In the absence of eVldence from cyt0genetlc techniques (bandlng studles, trltiated thymidine, autoradlography, etc.) and wlthout naturally occurrlnq or laboratory produced hybrids between the varlOUs taxa WhlCh constltute the genus Melanoplus, conclusions on many aspects of chromosomal evolution are necessarlly tentative and lncomplete. Nevertheless, the outllne lS clear. M. blvlttatua, a specles widespread throughout Canada and the United States, has a maJor chromosomal difference represented by substltution of one of the amaller autosomes by a medlum-sized chromosome. Not only that, but this 115 species has 37% of the populatlon deviating from the "baslc" chromosomal structure of the taxon. The possession of about 6% of the karyomorphs of polyploid spermatocytes lS by no means a mere chance. rt is suggested that these polyplairl spermatocytes have appeared due ta fallure of anaphase separatlon (Whlte, 1951). ()n qeneral grounds, it lS posslble that the present taxon M. blvlttatus ls actually not a slngle blologlcal specles, or that sorne populations have dlverged genetically far enaugh ta represent specles. The degree of morphologlcal variaton between M. bivittatus and the other three specles lS sa great that lt could be considered as belonglng to a distlnct genus. Even lf the karyotypes Investlgated were not very dlverse, Its wide dIstribution, by comparison with other specIes, WIll give rlse to SUspiclon. On the other hand, with the lncomplete 1 n for ma t Ion a val la b l e a t the pre sen t t i me, i t i s b y no means easy to decide on the number and llmita of any specles that mlght be recognlzed. The eVldence presented in thlS study Buggests that the taxa included in M. blvittatus repreeent several stages in epeclation. Most of the taxa within thie ·species group· seem to dlffer by more than a chromosomal rearrangement. Should further 116 collectlons reveal consistent differences in size and ~itat preference, then the taxonomie status of M. bivittatus as a single species must be called into question, especially if any differences are confirmed by laboratory breeding experiments. An lnteresting point ~merging from the present study la that there is a clear case of divergence involving major chromosomal rearrangements in M. h. borealis and ~. ~. 3 a n gui n i pe s . The numerous structural changes that have occurred in the autosomes of the two species - whatever thelr precise nature - have probably also been involved in speciatlon processes and, possibly, reached fixation during karyotype evolution of the species. For the time belng, it seems to be more accurate to divide the genus Melanoplus into -species groups- according to chromosomal organization. The present study raises the issue of the significance of chromosome differences between Melanoplus specles. 117 SÛMMARY AND CONCLUSIONS In the present study, various aspecte of chromosome variation in species of Acrididae have been investigated. This group of short-horned grasshoppers hae often been quoted as a classic example of karyotypic stability and conservatism. Despite the lack of major chromosomal rearrangements, it is clear that numerous minute structural changes have been extraordinarily frequent in the karyotypic evolutlon of thp group. In the large genus Melanoplus, aIl species so far investigated have 2n 0 = 23. A close comparison of the karyotypes of the four species studied show, however, that a large number of deletions and duplications of genetic material must have occurred and reached fixation in the evolution of the genus. These have modified considerably not only the length but also the atructural organization of the individual chromosomes. In M. t. femurrubrum, the B is likely to have derived from the X-chro.osome, but has clearly undergone numerous structural and behavioural changes during its genetie isolation and independent evolution trom the X. The 118 som~what shorter length of the B, compared to the similar X, do~s not seem to be due to del~tion(B) from the distal end, as beli~ved by Bome authors. The Bs in M. b. bivittatus, M. b. borealis, and M. B. sanguinipeB are of autosomal origin. In M. s. aanguinipes the presence of the B-chromosome Induces a slqnlflcant incrpase in the frequency at which the chiasma la formed. This is particularly eVldent in the lonq and medium blvalents. Recurrent sampling over a three-year period (three generations) of M. f. femurrubrum and M. s. sanguinipes ahowed that the frequency of B-chromoBome carriers did not change significantly. Structural and behavioural variants of the standard B-chromoBome were discovered in M. b. borealis and M. s. sanguinipes, and fragments of autosomal origin were discovered in a population of ~. bivittatus. The structure and variable behaviour of these extra elements provide further eVldence of the possible modes or orlgin and rapid evolution of this and other B-chromosome systems. 119 In the four Melanoplus species, the chiasmata are localized. The only detected effect of the B-chromosomes in chiasma frequency is found in M. ~. sanguinipes. The B-carriers show a sharp increase in the frequency of lnterstitial chiasmata. A look at the availa~le cytogenetic data on species of MelanopluB revealed obvious genetic heterogeneity withln the qeneric gr0up. ThlS iR a further example of the low predictive value of a classification which la based entirely upon a sUb)ective evaluatinn of exophenotypic characters. A new clasSification based upon cytogenetic evidence ia suggested. It 18 becoming increasingly evident that karyotypic uniformity in groups such as the Acrididae has been grossly exaggerated in the past, on the basis of relatively superficial etudies or inadequate techniques. Careful comparison ~ karyotypes in this group involving accurate chromosome measurements, identification of heterochromatic segments, determination of DNA values and experimental hybridisation, has revealed a wealth of differences between karyotypes of many species, even where no major structural rearrangements such dS fusions or inversions can be 120 demonetrated. It ie quite possible that such -minute- changes may have pIayed the same kind of role in apeciation as the more conspicuous types of rearrangementa seem to have done in the morabine graaahoppers. 12 l RBP'BRBNCBS Ayonoadu, U., and H. Rees. 1968. The lnfluence of 8- chromosomes on chiasma frequencies in Black Mexican sweet corn. Genetica 39, 75-81. Rarker, J.F'. 1960. Variation of chiasma frequency in and hf>tween natural populations of Acrldidae. Heredity 14, 211-214. Barlow, P.W., and C.G. Vosa. l Battaq11a, E. 1964. Cytoqenetlc~ of B-chr()mORom~s. Caryologia 17, 245-299. Baverstock, P.R., CH.H.S. Watts, J.T. Hoqarth, A.C. Robinson, and J.F. Robonson. 1977. Chromosome evolution in Austra1ian rodents 1. The Pseudomyinae, the Hydromyinae and the Uramys/Me1omys group. Chromosoma (Berl.) 61, 95-125. Callan, H.G., and H. Spurway. 1951. A study of meiosis ln interracial hybrids of the newt Triturus cristatus. J. Genetics 50, 235-249. Cameron, F.M., and H. Rees. 1967. The influence of B- chromosomes on meiosis in Lolium. Heredity 22, 446-450. Carroll, M. 1920. An extra dyad and an extra tetrad in the spermatogenesis of Cammu1a ~e11ucida (Orthoptera): numedca1 variation in the chromosome comp1ex within the individua1. J. Morph. 34, 375-455. Coope, G.R. 1970. Interpretation of quarternary insect fossils. Ann. Rev. Entom. 15, 97-120. Crozier, R.H. 1968. An acetic acid dissociation, air drying technique for insect chromosomes, .... ith aceto1actic orcein staining. Stain Techno1ogy 43, 171-173. 122 Darllngton, C.D. 1965. Natural populatIons and breakdowns of classica1 geneties. Proc. Roy. Soc., Ser. B., 145, 350-364. 1973. Recent Advanees ln Cytology, 2nd ed. London: Churchill. Dobzhansky, Th. 1970. Geneties of the Evo1utlonary Process. New York and London, Columbia University Pr~ss. Oyer, A.F. 1963. The use of 1acto-propionlc oreeln ln rapld squash m~thods for chromosome pre pa rat ion s. S ta l n Te c h n 0 log y 38, 8 5 - q 0 _ Feldman, M., 'l'.R. M~llo-Sampayo, and E.R. Sears. 1966. Somatic associatIon ln Triticum aestivum. Proc. Nat. Acad. SCl. !J.S. 56, 1192-1199. FIsher, R.A. 1930. The Genetlcal Theory of Naturi'll SelectIon. Oxford: Clarendon Press. Fletcher, H.L., and M. Hewltt. 1980. A comparlson of chiasma frequency and dIstribution between sexes in three species of grasshoppers. Chromosoma (Berl.) 77, 129-144. Fon tan a, P. G. 1 9 7 5 . Ka r y 0 t Y P est a b i l t t yan d heterochromatin variabi1ity in specles of Orthoptera (Cae11fera). Ph.D. Thesis. MCC;lll University, Montreal, Canada. Fontana, P.G., and 'l'.W. Hogan. 1969. Cytoqenetlc and hybrldization studies of geographic populatlons of Te1eogrf1lus commodus (Wa1k.) and T. oceanicusLe Gui1lou) (Orthoptera: Gry11idae). Aust. J. Zool. 17, 13-35. Fontana, P.G., and V.R. Vickery. 1973. Segregation-distortion in the B- chromosome system of Tettigidea lateralis (Say) (Orthoptera: Tetrigidae). ChromoBoma (Berl.) 43, 75-100. 123 P0ntana, p.G. and V.R. Vlckery. 1974. Heterochromatlc content and chiasma distribution in the megameric chromosomes of Stethophyma gracile and S. li ne a t um (0 r th 0 pte ra: Ac r i d i da e ). Chromosoma (Berl.) 46, 375-395. IQ75. The B-chromosome systpm of Tettlgldea lateralis (Say) II. New karyomorphs, patterns of pycnosity and Giemsa-banding. Chromosoma ( Be r 1 .) 50, 37 1 - 3 q 1 . pen, n.p. 1q 7 0. A non-doubllng DNA series ln somatic tissues nf the locusts Schistocerca gre aria (Forskal) and Locusta migratoria (Llnn. 1 Chromosoma (Berl.) 29, 446-461. IQ73. The control of chIasma dIstrIbutIon ln the locust Schistocerca gregarla (Porsk.al). Chromosoma 'Berl.) 43, 2AQ-328. Fox, n.p., G.M. Hewltt, and D.L. Hall. Iq74. DNA replication and RNA transcrlption of euchromatic and heterochromatic chromosome regions during grasshopper melOSlS. Chromosoma ( Be rI.) 45, 43- 6 2 . Gallagher, A., G.M. HewItt, and 1. Glbson. 1973. DifferentIaI Giemsa staining of heterochromatlc B-chromosomes in Myrmeieotettix maculatus (Thunb.) (Orthoptera: Acrididael. Chromosoma (Berl.) 40, 167-172. Glbson, 1. and G.M. Hewitt. lQ70. Isolation of DNA from B-chromosomes in grasshoppers. Nature 225, 67-68. 1972. Interpopulation variatlon in the satellite DNA from grasshoppers with B chromosomes. Chromosoma (Berl.) 38, 121-138. GIlles, C.B. 1973. U1trastructural analysis of maize pachytene karyotypes by three dimensiona1 reconstructlon of the synaptinemal complexes. Chromosoma (Berl.) 43, 145-176. 124 Hayam, D.L., and P.G. MartIn. 1965. Supernumerary chromosomes in the marsupial Shoinobates vo1ans (Kerr.) Auste ,J. Biol. SCL 18, 1081-1082 Hea rne , E.M., ann C.L. Husklns. 1935. Chromo~ome pa 1 rIng ln Melanoplus femur-rubrum. Cytologia (Tokyo) 6, 123-147. 1973. On the evnlutlon and maintenancp of R- chromosomes with partlcu1ar reference to Orthopteran systems. In Chromosomes Tj)day 4, 3')1-369. I1PW1~t, G.M. 1979. Grasshoppers clnd r'rlcket9. Animal cytogenetic8 vol. 3: Insecta l, Orthoptera: Berl in StllttC]i'\rt: \,phruder Rorntri'\eger. fjPWltt, (;."1., and B. John. 1967,,; The A-chromosome '3ystem nf Myrme1eotettlx macu1atus (Thunb.). TIl The stati9tic cl. Chromosoma (Berl.) 21, 140-162. 19nA. ParaI leI polymorphlsm for supernumprary segments ln Chorthl~pUS parallelus (Zetterstedt). 1. Aritish populations. Chromosoma (Berl.) 25, 319-342. 1970. The B-chromosome system of Myrmeleotettix macu1atus (Thunh.) IV. The dynamics. Evolution (Lawrence, Kans.) 24, 169-180. 1971. The cytogentic systpmB of grasshoppers and locusts. 1. Chortiocetes terminifera. Chromosoma (Berl.) 34, 302-323. Hewltt, ".M., and B. John. 1972. Inter-population sex chromosome polymorphlsm in the grasshopper Podlsma pedestrls. II. Population parameters. , Chromosoma (Berl.) 32, 23-42. Hoehn, H., H. Reinwein, and W. Engel. 1970. Genetic stucHes on a minute centric fragment transmitted through three generations. Cytogenetics 9, 186'-198. ; mil l , 1'.~4 R ,rr lm' ~)mp" :n th.,. mY''1l1 ~ ~~ rd x ~ 1 n ") 8 1 ") r ~hr lm '9 'ma 4') 411 444. R. y T'IV pv ,] Jt 1 ln .n A J,>rr'l c,q 14: ~H "il L"panp,>p -'ln'" "ympn l[,tpf-'l '~r)m'c;'ma ~pr' l' l, " -;j '>p<>rma> )(~@np~l '1 l r '1,>" h ,~pp r ". J '> • cl j;j " c. J-:J? ~p np" ] " Î 4 . , ~ ~ 1 H, ~ ~ f p ( . J il ' k r' '\nd ',jP\JlT\a" " ' SI J t)P r n Jmpf'lry . '1' 'm ,~ )m~ ~ 'rl ,l[ Il(1IJ . 'r' w -'l n l >'P 'Hl 'jf ,j ,,\ J pp f n lJ m p r d r f j l ' f p f p n' 1 a 1 f r P '1 \ J p n ,- 1 P q 9P X p '9 1 n s h 'r '1 'r n p l ~ r <'1 El El h 0 P pp r P~-'l U] 'l" r j 1 il m 'nr,m",»ma 'Qprl 2 1 , --~~-- - 24 r , , l 'h n, P "l' \ - v" )(1 P nt-~ t l 1 r -'l " "h ) fi ['P r" '1 n , f 9\JpprnùmPfclr.; 9PQmentR 44, ] AlI '.le,. J ,hn '1nrl M F'rppman '1P '1 cl 'J 1 l' J r 1 n Phi'\uldcrlrllum ''; 1 t ta t 'Jm 'h r )mnsnmi'\ Rp r ] 46, lAl-l'1'i J ,'ln an'i "'1 Hp .... l'· j4f)C,. Tf-Je R-,hrlm n 9nme '>y,>tpm )f MyrmelpotettlX macllatu'9. II Thp ,>tatlstl'.9 'hromosoma (Rer1 1"1 121-11R. '\ Rary,tYfiP 9 t d hl] 1 t Y ~'ff ï N A v d rIa h 1 1 1 t Y ln thp A,rl 1 g h R Pat t P r n fi i1 n d pa th .... a y '> 1 f rhrom<)somp p v r,j il t l 'J n \J 1 t 'lIn t h p ') r • f-J n r t pra Chromo!'loma 'Rer] 2'), 4!l--'4. ]7{-, hn I,.M. H"\Jltt. Parilllp] pnJym n rph19m , r Sllpernumerary segments 1 n --: h 0 r t h 1 pp U S para11eJus Zptter9terlt rl1. Thp Ashurst population. and R.R. LpWI9. l cH:,C,. Thp mPj ,r II' 9yc;tpm. Prn t ~asma t"l ~ VT fI VIPnn~ ~nrl ~PW y rk "prlnqer-Ver1aq ]'H..,",. ~repC)cnpml_~ Li HilnG 16, lnR-144 Imf,:pm"n' ~r--,-,~')plasmat '1,~ V: y 1 p '1 f1 cl "rr lnqpr ·Vpr lilq. '1 l'l'''''. ,f "ilrlv , p r mina 1 1 7 ~. 1 ln " h 1 ~ "m a t il 1 n ~lf'l ,tPr1P ., pp r mat l,' V t p., 'f Sch lsto("prcCi ':.L~ PïCi r 1 ~ . -hrOm(lSI,ma Rer 1 7R' -)Q4 1'1''', ~-chr 'm(191)mp 9y9tpm9 ln fI 'wprlnq pl and H. RpP9 :qf,'. ,~pnl.,typl(· ,"lntrd "f " h r lm C) Rom p h p h cl VI" il r 1 n r y p X r . Th P lnfluencp lf R-chr)mos')mp.'l )n mplfî91.'l. Heredlty 22, nl-l47. 19f,'l An an0mal ""l3 varlat l'In ';uP t" R-chr lm'l9()mp ln rvp. Hererllty 24, 2/)')-}'l Kp'Jan, fi. K. i"\rE. lQ-'L Thp place Gf c1a"lslcal taxonomy 1 n modern 9y~tpmiltlr I>nt0mo10gyl wl~h part leular rpfprenee t" ,rthopter')l(l In.geet9. '~a n. En t . 105, 1211-1)22. Kpy, K.H.L. 1'17(-,. A genprl(' ilnd supragenerl(- classlflcatlon If the l"'Iorablne gra8shopper9 IOrthoptera: Eumastaclrlae) 1oI1th descrlptlon ,f the type .9 pe C les and b 1 b 1 1 0 q ra ph Y 0 f the 9ubfamlly. ~ustr J. ZooI. Supp. Ser. 37, 1-18'5 Ir Klknadzp T • , ~nrl L.V Vysot skaya. Measurpment9 ,f ONA mass per nucleus ln the '1 rd S .9 hop pp r 9 pe, 1 <> S w 1 ~ f"I ., 1 f fer e n t n u m hp r ,f rhromnsnm€'9. TSltologld 12, 110()-110-'. F' 1 mhp r .F Il Frprlqa, "ln A A. "ii1ndhlJ[1 "l" m p n ,la t u r p f J r , e n t r () m p r l" ÇJ n 9 1 • l ., n 1n r~r)mnqnmp9 Hpredl~aq 'i2, 2 n 1 ))n T l ,~ ''1 R-rhr lmn.90mp fi,)1 ym0rph l "lm ln A.'~tpr age rat '11 des. J SCI Hlr0shlma lJnlv. C;pr R, 1 - fA .------ni v. 2 'A"l t . 13 , 1 q 4 'i . The,~ p n p t l ,- a 1 i\ r" 1 vIt Y J f ~etpr')('hr0mdtln. Prjr. ~ Sn, 132, 208-n2. II'!, ' 1 'J n'l ' '1" 1 qnA. (~yt,lr)qy and ~ax,nnmy, C; r l . Bu Il. 4 '7\ l'1'1-21'i. 1<:)14. A rnmpnratlvP stddy "f thp ,hr)mnS<1mpq ln Jrthnptpran spermat"lgen p 9Is. II'!orph. 'i, 6'>1-749. -hromosomes Today 2, p. 268. W.P. lQ'""'-'. Dlp10tene chr')mnQ"me8 )f Xenopu.9 hybrIda ,)0cytes -:hromosoma 'Berl.\ S9, }'""'1-2A2. Mc.lntzln(l, A. lQ"4. Ac r p8s,ry ~hr)m0.90me9. Ann. Rev ;e net 1 cs 8, 24 3 - 2 6 f, . Nur ,<:)62. P0pulatl'Jn studle9 ,f supernumerarv ,hr'Jmosomes 1 Cl a mea 1 y bug. Genet les 4 7 , 16 -, '1 - 16'1() UR NI J r , . J. 1 9 n 1 . A m l t r"J t l Cd 1 1 Y un R t ab 1 e ~ u pe r n ume ra r y chromosome wlth an dccumulatlon mechanlsm ln ci qrasshopper. Chromo8oma (Berl.) 14,407-427. 196q. Mlt0tlC lnstablllty leaillnq tr"J an acrumulat10n of R-chrnmoRomes ln lra9Rhoppers. rhromosoma (Berl.) 27, 1-19. Î,. 194<;. Para.9ltu- lf extrn frnqmprl t rhromosomes. Aot 2, [')'-161. Pi\:l../ël p.r., dnrl R.R. Hyde. 19'i9. The a.'lSOrlatlnrl nf il Rlngle B-chroml1some wlth male Rterll lty ln Plantago coronopus. Amer. J. Bot. 46, 46n-4hh ,.,. J. 19 7 '.] R-chr'muAomp9. '3<>r. Monogr. Dpt n . Ge net l ca, Pile. B l 0 1 ., Un i v . Complutense riE' M'ad r i rj. 2, 'S4 p~ R ~ 'n' ~ .. q, ,.,. M ., il n ri F Dp m p fi P y. 1 97 2 . 'l n t h P m pc han l R m , f chromntln 10.9R ln~uced by the B-rhromofl0me 0f mallP. GenetlcR 71, 73-9f). 19'3. 'hr0matln ellmlnat10n lnduced hy the R------rhrnmosomp 'if malze. J. Hereri. 64, 13-18. R. , dnd R.B. Plavel!. 1977. A flrst vlew of the mp lot l c pr0cPs8. Ph 11 . Tran.9. ~. S,)c. Land. A. 277, 191-199. R)hpr~q,n, W R.A. 1916. rhrCJmo.9ome studles. I. TaxonomlC relatlonshlps shown ln the chromosome of Tetrlqldae and Acrldldae: V-shaperl chromosomes dnd thelr slgnlflcance ln Acrldldae, Locustldae and t,ryll1rlae: rhromoaomea and varlatlons. J. Morph. 27, 1 7 9-331. Rnthfel9, T<.H. 19')0. ~hromosome complement. polyplo1dy an~ supprnumerarl€9 ln Neopodismops18 abdomlnal18 (Acrldldae). J Morph. 87, 287-316. Rut 1 9hau.9pr A. 1960. Telocentrlc fraqment chromosomes ln Trll1um grandlflorum. Heredlty 15, 241-246. Sann0mlya, M. 1962. rntra-Indlvl 19-'4. 'ytr1qenet le Atunles nn natural populat l0n9 "f ']rasshopper9 wlth spe,lal reference t::l B-chromosome~. 1. Gonlsta ---- olco1or. Heredlty 32, 2C,1-26C,. ë'i'lnn1mlya, "1., i'lnn H. Kayanu 196A. L~cal varlatlonanrl yE'ar-t0-year ,han']e ln frequencles ,f R ,hrrJmosomp9 ln natural populi'ltlons nf 90me ,~ra38h()pper speeles. Pr0c. Xllth Int. Genet longr. 2, 116-lP. <";,hr ''''er. ',.L., i'lnrl ,~M. H., .... ltt supernumerary chromatln ln t h r e e s pe cie .9 ) f 'jr.'ls9h0pper 1 i'I n. J. Ge net. ryto1. 16, )A"i-796. c, _n 19 7 0. The BupernUmpri'lry seqment 9y"ltpm nf Steth0~hyrna 1. "ltructura1 basls. Chromusoma rBerl. 31, 4Jl-411. 1971 The supernumerary seqment 'lystem rd Stetho~hymd. TT. Hf?tprochromatln po 1 ymorph 1 sm ano chiasma varlatlon. ~hromosoma ( BE' rI.) 37, 297-308. 1972. r-;enetlC i'lnd envlr0nmenta1 c0mponentt'l ,f ch IcHHÀ ,ont rrd . T 1. The response t 0 se1eet10n ln Schlstoeerca. 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