Journal of Genetics Votume 63, Number 2 Dec. 19'77

SEED FIBRE COLOUR IN GOSSYPIUM AND ITS POSSIBLE SIGNIFICANCE IN THE EVOLUTION OF DOMESTICATED COTTONS

S. G. Stephens

Department of Genetics, North Carolina State University Raleigh, North Carolina 27607

A central element in the study of crop plant evolution is a comparison of the

cultivar with its nearest wild relatives. This not only iDvolves comparative

morphology, cytogenetics, taxonomy and other disciplines, but also consideration

of the geographicalranges of the wild and cultivated forms. I f the wild form

has all the characteristics of a "relic", i.e. existence in very small popula-

tions, occupying widely disjunct areas, then its present geographical range may

bear l i t t l e relation to the area occupied in the past. When wild forms and

cultivars belong to the same species, the evolutionary problem becomes magnified.

I t is fair ly easy to develop morphological and physiological criteria that

distinguish the wild form from the cultivar, but the same criteria are usually

not useful to distinguish authentic wild from feral types. In order to survive,

both wild and feral types must possess "wild" characteristics. These usually

involve efficient means of seed dispersal, capacity to produce large numbers of

small seeds rather than fewer numbers of large seeds, methods of delaying germi-

nation (hard seedcoatsor physiological dormancy), and perhaps also mechanisms

for regulating the flowering cycle in accordance with local habitat requirements

(photoperiodism, thermoperiodicity). Most of these characteristics contrast

sharply with those in a cultivar, grown directly or indirectly for seed production.

The nature of the problem is well illustrated by the two New World cultivated

species of cotton, G. barbadense L. and G. hirsutum L. All the primitive forms

of these species are perennial shrubs or trees, not tolerant of low temperatures

and therefore confined to tropical and sub-tropical regions.. G. barbadense

63

64 S e e d f i b r e c o I o u r in C o t t o n

occurs as a primitive cultivar in northern South America, extending through the

Antilles to Central America. G. hirsutum is found as a primitive cultivar in

Mexico, Central America, the Antilles, Venezuela and Colombia, with an outlying

pocket in northeast Brazil. "Wild" forms of both species are known: these

always occur in small localized populations in areas that seem to be well

removed from habitats associated with present human activities. They have a

curious geographical distribution. "Wild" forms of G. barbadense have been

found only in the Guayas region of northern Peru and Ecuador, and on the Galapagos

Islands. "Wild" forms of G. hirsutum, in contrast, occupy an extremely wide

geographical range. They occur in small localized populations from Venezuela

through several Caribbean islands to the Yucatan Peninsula, the Gulf Coast of

Mexico and the Florida Keys. They are also widely scattered on Pacific islands,

from the Revillagigedos in the east, Wake Island in the north, to Polynesia in

the south..Are these truly wild or feral? The question is important, because

i f one supposes them to be feral, i t is l ikely that their present distribution

has been influenced to a large extent by the migrations of human cultures. I f

truly wild, there is no necessary connection with human activities. Although

these alternatives have been studied at length (Hutchinson, 1951; Stephens, 1958,

1965, 1971; Fryxell, 1965) bearing in mind the possible relevance of the diverse

information provided by ocean currents, trade routes, prehistory and early

historical documents, there seemed to be no satisfactory way of deciding between

them. In these earlier discussions, the possible significance of seed fibre

colour in wild and cultivated cottons had been overlooked. I t is the purpose

of this paper to supply the deficiency.

S.G. Stephens 65

SEED-FIBRE COLOUR IN CULTIVATED FORMS

In cul t ivars of G. hirsutum and G. barbadense, the seeds bear two kinds of f i b re :

the long l i n t f ibres that can be removed from the seeds and spun, and an undercoat

of short "fuzz" f ibres, f i rmly adherent to the seedcoat. The fuzz may cover the

seeds ent i re ly , as in many forms of G. hirsutum, or be reduced to a small t u f t

at the micropylar end of the seed, as in most cul t ivated forms of G. barbadense.

In both species, mutants occur in which the fuzzy undercoat is absent (naked

seed types).

In both species, var iet ies in modern cu l t iva t ion have white, less frequent ly,

cream coloured l i n t . Under low magnification (circa I0 X) and with ref lected

l igh t , the individual f ibres have a character ist ic s i l ve ry ref lect ion. On the

contrary, the fuzz in such white l in ted 'forms may be drab or bright green in

colour. Green l i n t is known only as a rare mutant in Upland cottons (annual forms

of G. hirsutum). The bright green colour in the newly opened boll is retained i f

the l i n t is harvested immediately and placed in cold storage. Otherwise the

colour changes rather quickly to a dull greyish brown, par t i cu la r ly under f i e l d

exposure. Green l i n t is always associated with green fuzz, though as noted

ea r l i e r , many white l inted forms have green fuzz. In a l l pr imi t ive cu l t ivars

the green colour is confined to the fuzz, except in certain strains of G. barbadense

in which the green colour extends a short distance into the bases of the otherwise

white l i n t f ibres. These are known as "green halo" types.

Primit ive cul t ivars of both species often have brown l i n t , which is always

associated with brown fuzz. The brown colour may vary from a deep chocolate or

mahogany, through reddish brown to a l i gh t tan. Under low magnification, individual

brown l i n t f ibres show considerable variat ion both in density and d is t r ibu t ion of

the pigment. Chocolate coloured f ibres tend to be most intensely and uniformly

pigmented. Lighter coloured f ibres may show only patches of pigment that have a

66 S e e d f i b r e coLour in C o t t o n

F i g u r e 1. T h e l o c a t i o n s of b r o w n and g r e e n p i g m e n t s in the l i n t f i b r e s of two c u l t t v a r s .

(l) G. barbadense: Piura "chocolate" fibre mounted in ZO percent sodium hydroxide.

(z)

(3)

T h e s a m e , wi th f i b r e w a i l p a r t l y d i g e s t e d in s u l p h u r [ c a c i d . No te the s o l i d c o r e of p i g m e n t .

G. hirsutum: Upland "Green Lint" fibre. Transverse section mounted in ZO percent sodium hydroxide. In the photograph the green pigment appears as a broad grey zone in the wall. The lumen (black in the photograph) is devoid of pigment.

S.G. Stephens 67

brassy reflection, as contrasted with the silvery reflection of a white fibre.

Development and location of the pigments. Brown pigment is deposited in the

lumen of the fibre, while the green pigment seems to be interfaced between the

cellulose layers of the fibre wall (Kerr, 1936). The location of the brown pigment

can be seen most readily in whole mounts of chocolate coloured fibres (Figure l - l ) .

I t is preferable to make cross-sections of the fibres to locate the green pigment

(Figure I-3).

The chemical nature of the pigments is presently unknown. They only become

clearly visible in the fibres a few days before the boll opens. Younger fibres,

irrespective of their colour at maturity, are colourless'and contain large

quantities of leucoanthocyanins. Levings (unpublished) was able to identify two

of these as leucocyanidin and leucodelphinidin, by acid hydrolysis and chromato-

graphic matching with authentic samples of cyanidin and delphinidin,, respectively.

At maturity, the leucoanthocyanins disappear from green and white fibres but are

retained, at least in part, in brown fibres. I f the leucoanthocyanins are

precursors of one or both pigments, i t is possible that the lat ter may be polymerized

tannin-like substances. Further analysis is hampered by their insolubi l i ty and

d i f f icu l ty of extracting them from the fibre.

Genetic basis for seed-fibre colour. Dominant genes at a minimum of three loci

are individually capable of producing the brown pigment. Details have been

published elsewhere (Hutchinson, 1946a; Stephens, 1955; Rhyne, 1957; Endrizzi

and Kohel, 1966) and wi l l not be reviewed here. I t is worth noting, however, that

although multiple loci are involved, the usual situation is for any particular

brown linted strain to carry only one of the alternative genes that have been

identified. Thus, in crossing white and brown linted strains, as they occur in

nature, monofactorial inheritance is the rule.

In contrast to the multiple loci involved with brown pigment, only one is

known to control green colour. The L_9_gene, that produces green pigment in both

68 Seed fibre co[our in Cotton

TABLE I. Allelism of genes controlling the development of green pigment in

seed fibre of Upland cottons.

Parental phenotypes

green l in t white l in t x

green fuzz white fuzz

F 2 phenotypes

green l in t white l in t white l i n t Total green fuzz green fuzz -white fuzz

56 14 70

green l in t white l in t x

green fuzz green fuzz 207 62 269

white l in t white l in t x

green fuzz white fuzz 20 8 28

S.G. Stephens 69

l int and fuzz fibres, has only been found inUpland cottons. Earlier work on

the inheritance of green fuzz is conflicting (Harland, 1939) and the relation

of green fuzz to green l int does not seem to have been reported previously. The

data given in Table l show that in Upland cottons green l int and green fuzz are

allel ic: Lg and Ig F, respectively.

As far as I know, combinations of L~with any of the brown l in t genes are

not found in nature. In Upland cottons the experimental combination of green

and brown l int genes produces a brown phenotype which becomes a dull, dingy brown

in storage. The dingy appearance is due, presumably, to the fading and colour

change of the green pigment. In the absence of the green pigment the brown

colour remains clear and relatively bright.

SEED FIBRE COLOUR IN WILD FORMS

The seeds of wild forms of G. barbadense and G. hirsutum, like their cultivated

relatives, bear both l int and fuzz fibres, though differentiation into adherent

fuzz and non-adherent l int is not so clear-cut. Mutants which reduce or remove

the fuzz are very rare in wild forms. A type lacking both fuzz and l in t is found

in some forms of the wild Galapagos cotton, G. barbadense vat. darwinii. Hutchinson,

and in the Polynesian wild form of G. hirsutum the fuzz is very sparse or absent.

In the Asiatic cottons, G. arboreum L. and G. herbaceum L., only the latter species

is known in a wild form.

No other species of Gossypium has cultivated forms. With one exception, the

seed fibres are undifferentiated and more or less strongly adherent to the seedcoat.

The exception is the Brazilian species, G. mustelinum Watt (= G. caicoense Aranha

et al . ) , recently described by Pickersgill et al. (1975). In this species

differentiation of the seed fibres resembles that found in wild forms of

G. barbadense and G. hirsutum.

A casual inspection of the seed fibres of all wild forms of Gossypium gives

the impression that they vary from dull brown to a greyish white in colour,

70 Seed fibre cotour in Cotton

TABLE 2. Distribution of brown and green pigments in the lumen (L) and

wall (W) of the seed fibre in certain wild species of Gossypium which have

undifferentiated fibres.

Species Genome Brown pigment Green pigment L W L W

barbosanum B ++ 0 O? -

sturtianum C ++ 0 0 +

armourianum D ++ 0 0 +

9ossypioides D + 0 0 +

klotzschianum D ++ 0 0 +

areysianum E ++ 0 0 -

bicki i E ++ 0 0 -

somalense E ++ 0 0 -

longicalyx F + 0 O? +

tomentosum AD + 0 0 ++

Key to symbols:

Brown

++ Pigment forming solid core in lumen

+ Pigment forming diffuse patches in lumen

Trace only

0 Pigment absent

Green

++ Pigment strongly developed

+ Light pigmentation: intensif ied with ammonia

- Trace only with ammonia treatment

0 Pigment absent

S . G . S t e p h e n s 71

depending on the species. For crit ical examination i t is necessary to examine

microscopically the fibres from newly-opened bolls. Samples obtained From

original collections in nature are frequently faded and discoloured through

isolation and weathering. Further loss of the original colour occurs during

storage. In some wild species, e.~. G. tomentosum Nutt. (= G. sandvicense Parl.),

G. mustelinum, G. raimon~ii Ulb., G. aridum Rose & Standley, G. armourianum Kearney,

and G. harknessii Brandegee, both green and brown colours are clearly visible

without microscopic examination. Hutchinson (1946b) extracted types with a very

dilute green l int and bright green fuzz by backcrossing G. armourianum to white

linted G. barbadense. The transfe~ed gene was shown to be linked with "crinkle"

(cr) known to be linked with Lg C When G. armourianum, G. mustelinum, G. tomentosum

and G. barbadense yar. darwinii are backcrossed to white linted G. hirsutum, green

fuzz (not. green lint~ is transferred as a simple dominant gene (Stephens,

unpublished). Thus i t is l ikely, though unproven, that the green Colour of

several wild forms is controlled by an allele at the L~locus.

In some wild forms the presence of one or both pigments is d i f f icul t to

verify without microscopic examination. The data to be presented were obtained

by mounting fibres from newly-opened bolls in 20 percent sodium hydroxide, and

examining them at c. 650 X. Sodium hydroxide expands the fibre walls and

intensifies the green pigment.

The distribution of pigments in the undifferentiated fibres of ten wild

species is summarized in Table 2, and in Table 3 similar data obtained from

eight wild forms, with fibres differentiated into fuzz and l in t , are presented.

In a rather crude fashion an attempt was made to record not only the location,

but also the intensity of the pigments, according to the key below Table 2.

Inspection of these tables leads to the following conclusions:-

7Z Seed fibre co[our in CoLton

(a) In a l l the forms examined, the f ibres contained both brown and green

pigments; whether the f ibres were undi f ferent iatea (Table 2) or d i f fe ren t ia ted

into fuzz and l i n t (Table 3).

(b) In most cases i t could be determined with confidence that brown pigment

was present in the lumen, and green pigment confined to the wal l .

(c) Both pigments tend to be more strongly developed in fuzz f ib res than

in l i n t f ibres (Table 3).

The variations in in tensi ty of brown pigmentation in a range of f ib re types are

i l lus t ra ted in Figure 2. The microphotographs were made with a yellow f i l t e r ,

and the dark areas indicate brown pigment.

TABLE 3. Distr ibut ion of brown and green pigments in the lumen (L) and

wall (W) of l i n t and fuzz f ibres in certain wi ld forms of Gossypium.

Al l forms l is ted belong to theAD genome group.

Species and type Brown pigments in Green pigments in l i n t fuzz l i n t fuzz L W L W L W L W

mustelinum - o + o o - o ++

barbadense

Guayas wild - o ++ o o + o ++

Tumbes wild - o + o o - o

vat. darwinii - o ++ o o - o ++

hirsutum

Venezuela wi ld + o + o o - o +

Puerto Rico wi ld - o + o o - o +

Yucatan wild - o + o o + o ++

Marquesas wi ld + o + o o - o +

S . G . S t e p h e n s 73

F i g u r e 2. B r o w n

(i) G.

(z) G.

{3) G.

(4) G.

{5} G.

{6) G.

(7) G.

(8) G.

{9} G.

{I0) G.

~-I l)

{iz)

E a c h division

p i g m e n t s in a r a n g e of G o s s y p i u m s e e d - f i b r e s .

a r m o u r i a n u m K e a r n e y

k l o t z s c h i a n u m A n d e r s o n

g o s s y p i o i d e s (Ulb.) S t a n d i e y

s t u r t i a n u m W i l l i s

s o m a l e n s e (Gurke ) H u t c h i n s o n

longicalyx Hutchinson & Lee

tomentos tlm Nutt

mustelinum Watt -- fuzz fibre

hirsutum race morrilli Hutchinson -- fuzz fibre

hirsutum race latifolium Hutchinson -- lint fibre

G. muste[inum Watt ~ tint fibre

G. hirsutum race morrilli Hutchinson -- lint fibre

on the scale equals 10 microns.

74 Seed fibre co[our in Cotton

DISCUSSION

The presence of both green and brown pigments in the undifferentiated fibres of

all the wild species examined (Table 2), and in the fuzz and l in t fibres of all

wild T~rms of the cultivated species (Table 3), shows that coloured fibre is an

ancestral primitive t rai t . With the outstanding exception of the green l in t

mutant (Lg) in Upland cottons, green pigment seems to have been reduced or

eliminated completely from the l in t fibres of cultivated forms. Green fuzz,

often reduced to a small tuft at the micropylar end of the seed, has been retained

in modern varieties of G. barbadense, but is found only in primitive cultivars

of G. hirsutum. When green pigment is present in both fuzz and l in t , i t is much

more strongly developed in the former. One could interpret the loss of green

pigment in cultivation as a progressive sequence: f i rs t , the loss of the pigment

from the l int fibre (as in G. barbadense) and later, the loss of the pigment from

both fuzz and l in t (as in modern varieties of G. hirsutum~.

The limited information presently available suggests that the green colour

of f~bres in several wild forms is determined by a single dominant gene. When

the gene is transferred to cultivated forms of G. barbadense, the colour is

expressed very weakly in the l in t and intensely in the fuzz fibres. Transference

of the gene to cultivated forms of G. hirsutum produces coloured fuzz only. The

gene responsible may be identical with l~ F of Upland cottons or an allele of the

same series. The reduced expression of the gene when transferred to cultivated

forms shows that the genetic milieu in which i t operates is different in wild

and cultivated forms, even when these belong to the same species. Is the

difference in genetic milieu a by-product of human selection? From the human

point of view, a green fibre is deleterious for two reasons -- (a) i t fades to

an unattractive grey colour (b) its presence in the fibre wall impairs the tensile

properties of the fibre (Neely, 1943; Richmond, 1943). Thus under human selection

there would be a cumulative tendency to select types with whiter and stronger l i n t

S.G. Stephens 75

fibres; types in which the green lint gene had a low expressiVity, or alternatively,

types in which it had mutated to white (]g). On this interpretation one would

regardS(only known as a unique mutant in Upland cottons) as a "super-green"

allele, capable of overriding the genetic milieu, h~seems to have no counterpart

in wild forms and may be a relatively recent mutant in cultivation.

The genetic basis for the development of brown pigment is more complex, in

the sense that several alternative loci are involved. Nevertheless, there is no

firm evidence thatmore than one locus is operating in any naturally occurring

type that has been investigated. In crossing brown with white linted types,

monofactorial inheritance is the rule, though the locus involved may differ from

race to race and species to species. From a modern point of view, brown l int is

as deleterious as green l int , since both have inferior fdbre strength. But

archaeological evidence (Stephens and Moseley, 1974; S~ephens, 1975) suggests that

white linted forms may not have been available during the earliest stages of

domestication. In several archaeological sites in coastal Peru, all cotton fibres

recovered in the form of raw cotton wads, seed cotton in mummy wrappings, or

textile scraps, varied in colour from light tan to dark brown. Today, dooryard

cottons with brown l int are common in Peru and Ecuador, and in living memory

naturally brown fibres have been used in texti le patterns. In contrast to the

fibres of wild forms, the colours of dooryard cottons occur in clear shades of

brown and reddish brown. The dingy brown colour of the wild fibre is missing --

presumably due to the loss of green pigment associated with wild type fibres.

I f the interpretation outlined above is substantially correct, one would

expect the fibres of authentic wild forms to contain both pigments. On the

other hand, there would seem to be no valid reason for both pigments to be

retained in feral forms. I f the green pigment was lost during the early stages

of domestication, all subsequent escapes from cultivation should be either

brown or white linted forms. All the wild forms of G. hirsutum and G. barbadense

76 S e e d fLbre coLour Ln C o t t o n

so far examined resemble the other w~Jd species of Gossypium in having fibres

that contain both pigments.

SUMMARY

Two genetic systems control the pigmentation of seed fibres in Gossypium. In

the New World amphidiploid cottons, and probably too in many of the wild diploid

species, the green colour is controlled by members of a series that includes a

minimum of three alleles: Lg (green l i n t ) , Ig F (green fuzz), Ig (white). Lg

as a mutant in Upland cottons, w h i l e ~ (or a similar al lele) is only known

is probably common to wild and primitive cultivars of G. hirsutum and G. barbadens(

G. tomentosum, G. mustelinum, and several wild diploid species. The recessive,

Ig, has not been found in any wild form of Gossypi~n.

At least three independent loci control the development of brown pigment,

but their homologies have not been analyzed systematically. No wild form of

Gossypium has been found to lack brown pigment completely.

All wild forms so far examined have fibres that contain both brown and

green pigments. I t is suggested that the loss of the green pigment and the

retention of brown pigment in the l i n t fibres of primitive cult ivars may have

been influenced by human selection. This favours the view that wild forms of

cultivated species are original ly wild -- not feral -- types.

S . G . S t e p h e n s 77

ACKNOWLEDGMENTS

Paper number 4403 of the Journal Series of the North Carolina Agr icu l tura l

Experiment Station, Raleigh, North Carolina. Work supported by NSF GB7769.

Permission to c i te the unpublished f indings of Dr. C. S. Levings, Genetics

Department, is gra te fu l ly acknowledged.

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