Directory UMM :Data Elmu:jurnal:A:American Journal of Botany:Vol88.Issue1.Jan2001:

American Journal of Botany 88(1): 92–102. 2001.

EVOLUTION OF THE FAD2-1 FATTY ACID DESATURASE
59 UTR INTRON AND THE MOLECULAR SYSTEMATICS
OF GOSSYPIUM (MALVACEAE)1
DON

QING LIU,2,5 CURT L. BRUBAKER,3 ALLAN G. GREEN,2
R. MARSHALL,4 PETER J. SHARP,4 AND SURINDER P. SINGH2

CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia;
Centre for Plant Biodiversity Research, CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia; and
4
University of Sydney, Plant Breeding Institute, Cobbitty, PMB11, Camden, NSW 2570, Australia
2

3

The FAD2-1 microsomal v-6 desaturase gene contains a large intron (;1133 bp [base pairs]) in the 59 untranslated region that may
participate in gene regulation and, in Gossypium, is evolving at an evolutionary rate useful for elucidating recently diverged lineages.
FAD2-1 is single copy in diploid Gossypium species, and two orthologs are present in the allotetraploid species. Among the diploid

species, the D-genome FAD2-1 introns have accumulated substitutions 1.4–1.8 times faster than the A-genome introns. In the tetraploids, the difference between the D-subgenome introns and their A-subgenome orthologs is even greater. The substitution rate of the
intron in the D-genome diploid G. gossypioides more closely approximates that of the A genome than other D genome species,
highlighting its unique evolutionary history. However, phylogenetic analyses support G. raimondii as the closest living relative of the
D-subgenome donor. The Australian K-genome species diverged 8–16 million years ago into two clades. One clade comprises the
sporadically distributed, erect to suberect coastal species; a second clade comprises the more widely spread, prostrate, inland species.
A comparison of published gene trees to the FAD2-1 intron topology suggests that G. bickii arose from an early divergence, but that
it carries a G. australe-like rDNA captured via a previously undetected hybridization event.
Key words:

cotton; FAD2-1; fatty acid desaturase; Gossypium; intron; Malvaceae; polyploidy; reticulate evolution.

All higher plants contain one or more microsomal v-6 desaturase(s) that insert a double bond between carbons 12 and
13 of monounsaturated oleic acid to generate polyunsaturated
linoleic acid. This enzyme is mainly responsible for the production of the polyunsaturated fatty acids that are integral
components of plant cellular membranes and of storage lipids
in many vegetable oils (Shanklin and Cahoon, 1998). In Gossypium, the microsomal v-6 desaturase family comprises at
least two distinct members in diploid species and perhaps as
many as five in the allotetraploid species (Liu et al., 1996). Of
particular interest is one member of this gene family, FAD21, which is encoded by at least two copies in the tetraploid
cotton species G. barbadense and G. hirsutum (ghFAD2-1),

and by a single copy in the diploid cotton species G. arboreum, G. raimondii, and G. robinsonii (Liu et al., 1999).
FAD2-1 is highly expressed and seed-specific and, therefore, is probably the main contributor of the polyunsaturated
fatty acids in the seed oil of cultivated cottons (Liu et al.,
1999). In addition to the three histidine boxes that are typical
of all membrane-bound desaturases, the ghFAD2-1 gene contains a stretch of six contiguous glycine residues in the Cterminus of the open reading frame. Moreover, comparisons
of genomic and cDNA clones encoding the ghFAD2-1 gene
revealed a single large intron (;1133 bp [base pairs]) in the
59 untranslated region (UTR) located 9 bp upstream from the
putative translation start site (Liu et al., 1997). Preliminary
examination of the FAD2-1 gene from five species (G. arboreum, G. barbadense, G. hirsutum, G. raimondii, and G. robinsonii) revealed that the size and position of the intron were
conserved. Sequence comparisons also suggested that the

FAD2-1 intron may be evolving at a quick enough rate for
inferring evolutionary relationships among recently diverged
lineages and, in this regard, could be particularly useful for
elucidating evolutionary pathways among the 17 Gossypium
species indigenous to Australia, a group whose evolutionary
history remains unresolved (Seelanan et al., 1999).
The current evolutionary understanding of the 17 Australian
Gossypium species is based on morphological and cytological

comparisons, and the phylogenetic analyses of three nucleotide
sequences derived from the rpl16 intron (1155 bp), the 18S–
26S rDNA internal transcribed spacer (ITS: 688 bp), and a
portion of an alcohol dehydrogenase gene (AdhD: 1600 bp)
(Seelanan et al., 1999). These analyses confirmed hypotheses
regarding the basal divergences on the Australian continent but
provided little resolution of the evolutionary relationships
among the 12 species indigenous to the Kimberley plateau of
northwestern Australia. The nuclear gene topologies were also
incongruent regarding the evolution of G. bickii, which has a
biphyletic ancestry. Gossypium bickii captured a G. sturtianum-like chloroplast earlier in its evolutionary history, but to
date there is no evidence that this was accompanied by nuclear
introgression (Wendel, Stewart, and Rettig, 1991). The topological incongruencies may point to the first evidence that hybridization and introgression also altered the composition of
G. bickii’s nuclear genome, or that G. bickii experienced a
second and heretofore undescribed evolutionary reticulation.
To provide an evolutionary context for investigations into
the regulatory role of ghFAD2-1 intron and to refine our understanding of the evolution of the Australian Gossypium species, the FAD2-1 intron was cloned and sequenced from 31
Gossypium species. Each major geographic region within the
indigenous range of Gossypium (Africa/Arabia A and E genome; New World D genome) is represented, including all of
the Australian C, G, and K species and all the AD genome

Manuscript received 3 September 1999; revision accepted 3 March 2000.
This work was supported by grants CSP78C and CSP85C from the Cotton
Research & Development Corporation.
5
Author for reprint requests (qliu@pi.csiro.au.)
1

92

January 2001]

LIU

ET AL.—FAD2-1 INTRON EVOLUTION IN

New World allotetraploids. The resultant data allowed us to
address the following questions: (1) Does the intron occur in
all the Gossypium FAD2-1 genes? (2) Are the two copies in
the allotetraploid species orthologs, inherited from their A and

D genome progenitors, respectively? and (3) Does this intron
contain sufficient phylogenetic signal to resolve the evolutionary pathways among the Australian Gossypium species and the
ambiguities regarding the evolution of G. bickii?
MATERIALS AND METHODS
The microsomal v-6 desaturase FAD2-1 intron was amplified and cloned
from 39 accessions of 31 Gossypium species (Table 1). Multiple accessions
of G. australe, G. bickii, G. nelsonii, G. robinsonii, and G. sturtianum were
assayed to strengthen inferences regarding the basal divergences among the
Australian Gossypium species. Pairs of putatively orthologous clones were
sequenced from each of the five tetraploid species. The two A-genome species
and five representative D-genome species were included to confirm the inferred subgenomic origin of the clones of the tetraploid species, including the
two D-genome species nominated as most likely to be sister to the D-subgenome (Endrizzi, Turcotte, and Kohel, 1985; Wendel and Albert, 1992; Wendel, Schnabel, and Seelanan, 1995).
Total genomic DNA was extracted following Paterson, Brubaker, and Wendel (1993) and further purified by CsCl gradients following Sambrook,
Fritsch, and Maniatis (1989). The entire 59 UTR intron was amplified using
primers that flanked the predicted splice site. The upstream primer (S1: 59CCTGGCGTTAAACTGCTTTC-39) is located at 44–63 bp downstream of
the transcription start site in the 59 UTR and the downstream primer (A1: 59GCATAGGTCATGGACCACGT-39) is located at 239–258 in the coding region (exon2) of ghFAD2-1 (EMBL accession X97016). The 50-mL polymerase chain reactions (PCRs) contained 200 mmol/L dNTPs, 1X PE Applied
Biosystems (Scoresby, VIC, Australia) PCR buffer, 20 pmol of each primer,
10 ng genomic DNA, and 1 unit of Taq DNA polymerase. PCRs started with
a 2-min denaturation at 948C, followed by 30 cycles of 948C for 1 min, 568C
for 1 min, and 728C for 1 min, and finished with 10-min final extension at

728C. PCR products were purified with Wizardt PCR Preps DNA Purification
System (Promega; Annandale, NSW, Australia) and cloned into Tt-vector
(Promega) according to manufacturer’s instructions. Plasmids were isolated
following Sambrook, Fritsch, and Maniatis (1989), and the DNA sequences
were determined using the PRISMy kit (PE Applied Biosystems) on an
ABI373 DNA Sequencer.
Sequence analysis—Sequences were initially aligned using GCG-pileup
(Wisconsin Package Version 9.1, Genetics Computer Group [GCG], Madison,
Wisconsin, USA) and then adjusted manually. Individual sequences have been
submitted to EMBL (Table 1); the sequence alignment was also submitted to
EMBL (Accession DS41945). Mega 1.01 (Kumar, Tamura, and Nei, 1993)
was used to characterize the sequences and compute pairwise Jukes-Cantor
distances (Jukes and Cantor, 1969).
Topologies were inferred heuristically using the GCG implementation of
PAUP (Wisconsin Package Version 9.1, Genetics Computer Group [GCG],
Madison, Wisconsin, USA) using parsimony or distance (minimum evolution)
as the optimality criterion. In both cases, starting trees were acquired by stepwise addition (simple), ten trees were held at each step, and the TBR algorithm was used for branch swapping using steepest descent. Gaps were treated
as missing data, and potentially informative indels were recoded as binary
characters and included in some analyses. Distance-optimized topologies were
initially inferred using the Jukes-Cantor (Jukes and Cantor, 1969) model of

nucleotide substitution, however, because transition/transversion ratios were
generally ,2 but the frequencies of the four nucleotides deviated substantially
from equality, the Tajima-Nei (Tajima and Nei, 1984) estimator was also used
(Kumar, Tamura, and Nei, 1993). Sites containing gaps and regions of ambiguous homology were ignored. Negative branch lengths were set to zero.
The model for substitution rate variation across sites was determined by the
gamma distribution (shape parameter set equal to 0.5). As a measure of clade
‘‘strength,’’ Autodecay, in association with PAUP 3.1 for Macintosh (Eriksson

GOSSYPIUM

93

and Wikstro¨m, 1995; Swofford, 1991), was used to determine the length of
the shortest tree in which each clade failed to appear (Bremer, 1988; Donoghue et al., 1992). Relative rate tests followed Tajima (1993) using Tajima93
(see Seelanan et al., 1999).

RESULTS
Comparison of the cDNA sequence of the G. hirsutum
ghFAD2-1 gene and its corresponding genomic clone confirmed the presence of a single large intron in the 59 untranslated region (UTR) (Liu et al., 1997, 1999). The intron is
;1133 bp and is located 9 bp upstream of the translation initiation site in G. hirsutum. This is strikingly similar to the

microsomal v-6 desaturase in Arabidopsis thaliana, which has
a 1130-bp intron located 4 bp upstream of the translation initiation site (Okuley et al., 1994). In contrast, the Glycine max
FAD2-1 has a much smaller intron of 320 bp located 4 bp
downstream of the translation initiation site (Liu et al., 1997).
Attempts to align these three intron sequences revealed numerous sequence dissimilarities and no obvious regions of
conservation.
Using the primers developed for G. hirsutum, the intron was
amplified from the 39 Gossypium accessions (Table 1). The
diploid Gossypium species contained a single copy of the
FAD2-1 gene, and in each case the gene contained the 59 UTR
intron. The five allotetraploid species contained two distinct
FAD2-1 genes and each contained an intron (subgenus Karpas; Table 1). All of the introns started with GT and ended
with AG, consistent with the plant consensus 59 and 39 exon/
intron boundaries (Simpson and Filipowicz, 1996). The introns
had a mean GC content of 24%, and the 154 bp of exon 2
had a mean GC content of 55%. The length of the introns
ranged from 1065 (G. australe-2) to 1166 bp (G. gossypioides). The G-genome species, G. raimondii, and the tetraploid
A- and D-subgenome orthologs had mean intron lengths below
1120 bp, while the A-, C-, D-, E-, and K- genome species had
mean intron lengths greater than 1130 bp. The introns contained 14 simple sequence repeat regions with more than five

repeat units in at least one accession: 12 (T)n, 1 (CT)n, and 1
(AAG)n (individual accession data available from corresponding author).
Phylogenetic analysis—The final aligned length of the analyzed matrix was 1406 bp. The analyzed sequences start with
the first nucleotide of the intron and end with the 154th nucleotide of the second exon. Twenty-eight of the insertion/
deletion events (1–57 bp) inferred from this alignment were
potentially phylogenetically informative and were coded as binary characters. The 154 nucleotides of exon 2 aligned without
gaps. Simple sequence repeat regions were excluded. Homology assessments in several other short regions were ambiguous
and also excluded. Of the final aligned length of 1406 bp, 221
bp were excluded from phylogenetic analyses. Considering
only the 1185 nucleotide positions used in the phylogenetic
analyses, 344 were variable, of which 169 were parsimony
informative: 319 variable sites occurred in the intron, of which
158 were parsimony informative; 25 variable sites occurred in
154 bp of the 59 end of the exon, of which 11 were parsimony
informative.
Parsimony-optimized topologies were inferred with and
without the coded indels. An heuristic search without the coded indels returned 108 equally parsimonious trees with consistency indices (CI) of 0.692, excluding uninformative char-

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TABLE 1. Gossypium accessions assayed. Full provenance details available from C. L. Brubaker. Genome designations follow Stewart (1995);
taxonomy follows Fryxell (1992).
Taxa

Subgenus Sturtia (R. Brown) Todaro
Section Sturtia
G. robinsonii F. Mueller

G. sturtianum J. H. Willis

Genome

Accession no.

Locality/Cultivar

EMBL no.a

1 C

CAT #1364

EMBL-AJ244884

2 C

GOS-5170

1 C

s.n.

2 C

GOS-5297

3 C

GOS-5076

Nindethana Seed Service, WA,
Australia
WA: Australia (MillStream National Park)
NT: Australia (Hermansburg; Collected by P. Abell)
NT: Australia (Glen Helen Station)
NSW: Australia (Narrabri)

K
K
K

GOS-5378
GOS-5309
GOS-5176

EMBL-AJ244889
EMBL-AJ244890
EMBL-AJ244891

K

GOS-5347

Yampi Peninsula (WA: Australia)
Cobourg Peninsula (WA: Australia)
Mitchell River Basin (WA: Australia)
King Edward River (WA: Australia)

EMBL-AJ244893

Section Grandicalyx (Fryxell) Fryxell
G. costulatum Todaro
G. cunninghamii Todaro
G. enthyle Fryxell, Craven, &
Stewart
G. exiguum Fryxell, Craven, &
Stewart
G. londonderriense Fryxell, Craven, & Stewart
G. marchantii Fryxell, Craven, &
Stewart
G. nobile Fryxell, Craven, &
Stewart
G. pilosum Fryxell

GOS-5191

G. bickii Prokhanov

G. nelsonii Fryxell
Subgenus Houzingenia Fryxell
Section Houzingenia
Subsection Houzingenia
G. trilobum (Mocin˜o & Sesse´ ex
DC) Skovsted
Subsection Integrifolia (Todaro)
Todaro
G. klotzschianum Andersson
Subsection Caducibracteolata
Mauer
G. turneri Fryxell
Section Erioxylum (Rose & Standley) Prokh.
Subsection Selera (Ulbrich) Fryxell
G. gossypioides (Ulbrich) Standley
Subsection Austroamericana Fryxell
G. raimondii Ulbrich
Subgenus Gossypium
Section Gossypium
Subsection Gossypium
G. arboreum L.
G. herbaceum L.

EMBL-AJ244886
EMBL-AJ244887
EMBL-AJ244888

EMBL-AJ244892

K

GOS-5193

K

GOS-5196

K

GOS-5203

K

GOS-5210

Drysdale River Mouth NE of Kalumburu (WA: Australia)
Bougainville Peninsula (WA: Australia)
Carson River Station (WA: Australia)
Northern end of Mitchell Plateau
(WA: Australia)
Augustus Island (WA: Australia)

K

GOS-5204

Vansittart Bay (WA: Australia)

EMBL-AJ244898

K

GOS-5030

EMBL-AJ244899

K

GOS-5223

16 km North of Broome towards
Beagle Bay (WA: Australia)
Cape Talbot (WA: Australia)

1
2
3
1

G
G
G
G

CAT #1363
GOS-5041
PI-499756
GOS-5338

EMBL-AJ244901
EMBL-AJ244902
EMBL-AJ244903
EMBL-AJ244904

2
3
1
2

G
G
G
G

PI-464843
GOS-5048
GOS-5024
PI-499783

Nindethana Seed Service, WA
Carawine Gorge (WA: Australia)
Ormiston Gorge (NT: Australia)
Urandangie Road S of Barkly
Hwy (QLD: Australia)
Supplejack Station (NT: Australia)
Alice Springs (NT: Australia)
Richmond (QLD: Australia)
Ormiston Gorge (NT: Australia)

D

s.n.

Mexico

EMBL-AJ244909

D

GOS-5400

Gala´pagos Islands

EMBL-AJ244910

D

GOS-5403

Mexico

EMBL-AJ244911

D

CPI-138644

Mexico

EMBL-AJ244912

D

GOS-5330

Peru

EMBL-AJ244913

A
A

GOS-5264
GOS-5380

India Type 9
Wagad

EMBL-AJ244914
EMBL-AJ244915

G. populifolium (Bentham) G.
Mueller ex Todaro
G. pulchellum (C. A. Gardner)
Fryxell
G. rotundifolium Fryxell, Craven,
& Stewart
G. species novum (see Stewart,
Craven, and Wendel, 1997)
Section Hibiscoidea Todaro
G. australe F. Mueller

EMBL-AJ244885

EMBL-AJ244894
EMBL-AJ244895
EMBL-AJ244896
EMBL-AJ244897

EMBL-AJ244900

EMBL-AJ244905
EMBL-AJ244906
EMBL-AJ244907
EMBL-AJ244908

January 2001]
TABLE 1.

LIU

ET AL.—FAD2-1 INTRON EVOLUTION IN

GOSSYPIUM

95

Continued.
Taxa

Subgenus Gossypium
Section Gossypium
Subsection Pseudopambak
(Prokh.) Fryxell
G. somalense (Gu¨rke) J.B. Hutchinson
G. stocksii Masters
Subgenus Karpas Rafinesque
G. barbadense L.

Genome

Accession no.

Locality/Cultivar

EMBL no.a

E

CPI-138057

Somalia, Kenya, Sudan

EMBL-AJ244916

E

CPI-138058

Pakistan, Arabia, Somalia

EMBL-AJ244917

AD

s.n.

Pima S7

G. darwinii Watt

AD

GOS-5399

Gala´pagos Islands

G. hirsutum L.

AD

s.n.

Deltapine-16

G. mustelinum Miers ex Watt

AD

GOS-5402

Brazil

G. tomentosum Todaro

AD

GOS-5404

Hawaii

EMBL-AJ244918
EMBL-AJ244919
EMBL-AJ244920
EMBL-AJ244921
EMBL-AJ244922
EMBL-AJ244923
EMBL-AJ244924
EMBL-AJ244925
EMBL-AJ244926
EMBL-AJ244927

(A)
(D)
(A)
(D)
(A)
(D)
(A)
(D)
(A)
(D)

a The prefix EMBL- has been added to all the EMBL accession numbers to link the online version of American Journal of Botany to EMBL but
is not part of the actual accession number.

Fig. 1. Parsimony (A) and distance (B) optimized topologies of the FAD21 intron among 39 accessions of 31 Gossypium species. (A) Strict consensus
tree of 103 equally parsimonious trees (517 steps; consistency index 5 0.717,
excluding uninformative characters; retention index 5 0.912). Twenty-eight
indels included in analysis as binary characters, otherwise gaps were treated
as missing data. The number of unambiguous substitutions are indicated above
branches; the decay index for each clade is indicated below each branch. (B)
Single most parsimonious distance-optimized (Tajima and Nei, 1984) tree
(485 steps ; consistency index 5 0.688, excluding uninformative characters;
retention index 5 0.899)

acters, and retention indices (RI) of 0.901 (not illustrated).
With the 28 indels included, an heuristic search returned 103
equally parsimonious trees (CI 5 0.717, excluding uninformative characters; RI 5 0.912) (Fig. 1A). Strict consensus
trees from analyses with and without the indels differed only
in the resolution of the G. hirsutum-A/G. tomentosum-A/G.
barbadense-A/G. darwinii-A clade relative to the G. arboreum/G. herbaceum clade. When the indels were excluded,
these four A-subgenome introns collapsed to form a polytomy
with the G. arboreum–G. herbaceum clade. With the indels
included, the A-subgenome intron of these four species appeared as a sister clade to the G. arboreum/G. herbaceum
clade (Fig. 1A).
Distance-optimized topologies were inferred from the pared
sequence matrix using Jukes-Cantor (Jukes and Cantor, 1969)
or Tajima-Nei (Tajima and Nei, 1984) models of nucleotide
substitution. Both analyses returned a single and identical tree
(CI 5 0.688, excluding uninformative characters; RI 5 0.899)
(Fig. 1B). The distance- and parsimony-optimized topologies
are congruent except for the placement of the E-genome clade
and G. marchantii (Fig. 1).
In both distance- and parsimony-optimized topologies, all
the diploid genome groups are resolved as monophyletic lineages except the Australian G genome. Bremer support for the
monophyly of the A-, D-, E-, and K-genome clades is strong,
whereas the monophyly of the C genome clade is weakly supported (Fig. 1A). Because no outgroup was included, both topologies are midpoint rooted and no inferences regarding the
basal divergence within Gossypium obtain from the figured
topologies.
The relationship of the E-genome clade to the other genomes is ambiguous. In parsimony-optimized topologies, it is
sister to the A genome with a decay index of one (Fig. 1A).
This relationship, however, is inconsistent with the distanceoptimized topologies, where the E-genome species are sister
to the D-genome species (Fig. 1B). However, the significantly
unequal substitution rates among the genome lineages (discussed below) suggest that the parsimony topology may be
more reliable. This then would provide weak evidence that the
A and E genomes shared a common ancestor more recently
than either did with the D genome.

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TABLE 2. Relative rate test (2D; Tajima, 1993) of nucleotide substitution rates among the A-, D-, and E-genome species relative to G. costulatum,
calculated using Tajima93 (see Seelanan, Schnabel, and Wendel, 1997). (P 5 0.05 . * . 0.01 . ** . 0.005 . ***). Exon sequence, regions
of dubious homology, and indels were not included in analyses.
Taxon

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19

G. arboreum
G. herbaceum
G. barbadense
G. darwinii
G. hirsutum
G. mustelinum
G. tomentosum
G. gossypioides
G. klotzschianum
G. raimondii
G. trilobum
G. turneri
G. barbadense
G. darwinii
G. hirsutum
G. mustelinum
G. tomentosum
G. somalense
G. stocksii

Genome
designation

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

A
A
A-AD
A-AD
A-AD
A-AD
A-AD
D
D
D
D
D
D-AD
D-AD
D-AD
D-AD
D-AD
E
E

NS
*
NS
NS
*
**
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS

NS
NS
NS
NS
NS
NS
*
*
*
*
*
**
*
*
**
NS
*

NS
NS
NS
NS
NS
**
*
***
*
***
***
***
***
***
NS
***

NS
NS
NS
NS
*
*
*
*
*
**
*
*
**
NS
*

NS
NS
NS
*
*
*
*
*
**
*
*
**
NS
*

N/A
*
***
**
***
**
***
***
***
***
***
NS
**

*
***
***
***
***
***
***
***
***
***
*
***

NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS

NS
NS
NS
NS
NS
NS
NS
NS
NS
NS

NS
NS
NS
NS
NS
NS
NS
NS
NS

NS
NS
NS
NS
NS
NS
NS
NS

NS
NS
NS
NS
NS
NS
NS

NS
NS
NS
NS
NS
NS

NS
NS
NS
NS
NS

NS
N/A
NS
NS

NS
NS
NS

NS
NS

NS

The Australian Gossypium species (C, G, and K genomes)
are also resolved as a monophyletic lineage in the parsimonyand distance-optimized topologies (Fig. 1). Within the Australian clade there is a primary divergence into four subclades:
(1) G. australe/G. nelsonii, (2) G. bickii, (3) the K genome,
and (4) the C genome.
Among the K-genome species, two well-supported clades
are evident (decay index of 3; Fig. 1). The first comprises G.
species novum, G. costulatum, G. cunninghamii, G. londonderriense, G. marchantii, G. nobile, G. populifolium, and G.
pulchellum; the second comprises G. enthyle, G. exiguum, G.
pilosum, and G. rotundifolium. Within the former, or K1 clade,
two subclades are evident, G. species novum/G. cunninghamii/
G. londonderriense, and G. costulatum/G. populifolium. The
relationship of G. nobile to these subclades is unresolved, and
G. marchantii is weakly supported as sister to the G. costulatum–G. populifolium clade. Gossypium pulchellum is weakly
supported as the single extant ancestor of one lineage arising
from the basal divergence in the K1 clade. The latter, or K2
clade, sees G. rotundifolium placed sister to an unresolved
trichotomy comprising G. enthyle, G. exiguum, and G. pilosum.
The topological placement of G. bickii was unexpected. The
two other G-genome species, G. australe and G. nelsonii, are
strongly supported as sister species (decay index of 9), but G.
bickii resolves as sister to the K-genome clade (Fig. 1). Because this implied that the G genome was paraphyletic, further
heuristic searches were undertaken to test the stability of this
result. In the first instance, the data matrix was reanalyzed
without the E-genome species (because of their own topological instability, discussed above), the C-genome species, or G.
australe and G. nelsonii. Subsequently, the E-genome species
in combination with the C-genome species or G. australe and
G. nelsonii were excluded. In all cases, distance- and parsimony-optimized searches returned consensus topologies congruent with those illustrated in Fig. 1. The same topology was
also recovered when the Australian species were analyzed with
only the A-genome, D-genome, or the E-genome species.
The topological placement of the tetraploid sequences is

consistent with the original assessment that each tetraploid
species contained a pair of orthologous loci. The putative Dsubgenome sequences from the tetraploid species resolved as
a monophyletic clade sister to G. raimondii within a clade of
other diploid D-genome species. The putative A-subgenome
sequences appear in a strongly supported A-genome clade (decay index of 13), but do not resolve as a monophyletic sublineage. Gossypium mustelinum appears as basal to two subclades: (1) G. arboreum/G. herbaceum and (2) G. barbadense/
G. hirsutum/G. darwinii/G. tomentosum.
Relative rates of nucleotide substitution—Because the basal divergence in Gossypium occurred between the ancestor of
the A-, D-, E-, and AD-taxa and the ancestor of the C-, G-,
and K-genome species (Wendel and Albert, 1992; Seelanan et
al., 1997, 1999), the relative substitution rates (2D test; Tajima,
1993) among the Australian species were evaluated using G.
somalense, G. raimondii, or G. herbaceum as reference taxa.
The pared sequences were used, i.e., areas of ambiguous homology were excluded, and regions with gaps in one or more
taxa were excluded from all comparisons. All three analyses
demonstrated that nucleotide substitution rates are largely homogeneous among the species. Only G. species novum, G.
australe-3, G. londonderriense, and G. rotundifolium returned
significant chi-square tests for some species combinations
(data not shown). These species were excluded from divergence time estimates (described below).
Conversely, homogeneity of substitution rates among the A-,
D-, E-, and AD-genome species were tested using G. costulatum, G. nelsonii, G. robinsonii, or G. sturtianum, respectively, as reference taxa. As above, the pared sequences were
used to eliminate ambiguous homology assessments and sites
with gaps were excluded from all comparisons. All four tests
consistently demonstrated that nucleotide substitution rates are
not homogeneous among these clades (Table 2). Particularly
notable was that the A- and D-genome lineages, except G.
arboreum and G. gossypioides, have accumulated substitutions
at significantly different rates.
To more fully understand the basis of these results, the mean

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97

TABLE 3. Mean Tajima-Nei distances (Tajima and Nei, 1984) between A or D species and the four Australian lineages. Exon sequence, regions
of dubious homology, and indels were not included in the distance estimation.
Mean Tajima-Nei distances (mean transition/transversion ratio)
Reference lineage

A

A subgenome

G. herbaceum

G. arboreum

D

D subgenome

D genome diploids

G. gossypioides

G. australe/G. nelsonii

0.0382
(1.4)
0.0358
(1.3)
0.0361
(1.8)
0.0434
(1.7)

0.0378
(1.3)
0.0353
(1.2)
0.0356
(1.7)
0.0430
(1.6)

0.0406
(1.5)
0.0381
(1.4)
0.0384
(1.9)
0.0456
(1.8)

0.0473
(1.4)
0.0462
(1.4)
0.0450
(1.9)
0.0538
(1.7)

0.0676
(1.6)
0.0659
(1.4)
0.0644
(1.6)
0.0748
(1.7)

0.0697
(1.8)
0.0677
(1.5)
0.0662
(1.8)
0.0767
(1.8)

0.0651
(1.4)
0.0637
(1.3)
0.0623
(1.4)
0.0725
(1.5)

0.0584
(1.2)
0.0574
(1.1)
0.0554
(1.3)
0.0645
(1.4)

G. bickii
C genome
K genome

Tajima-Nei distances between the A- and between the D-genome taxa and the four major Australian clades were calculated (Table 3). Gossypium arboreum and G. gossypioides
were considered separately, and G. australe-3, G. species novum, G. londonderriense, and G. rotundifolium were not included in the calculations. These comparisons demonstrate that
D-genome introns accumulated ;1.8 more substitutions per
site than did the A-genome introns. If these comparisons are
partitioned into tetraploid subgenomic and diploid components, the A-subgenome taxa had a lower mean distance from
the taxa in the four major Australian clades than did G. arboreum and G. herbaceum, in contrast to the D-subgenome
taxa, which have a higher mean distance from the taxa in the
four major Australian clades than is observed in the D diploid
taxa. This suggests that the differences in nucleotide substitution rates between the A- and D-genome lineages have been
magnified in the two polyploid lineages. These comparisons
are consistent regardless of which of the Australian lineages
are used as a point of reference.
TABLE 4. Estimated divergence times among the Australian clades. A
substitution rate of 0.721.7 3 1029 nucleotide substitutions per site
per year was calibrated using the estimated age of divergence
(10.5–21 mybp) between the C- and K-genome clades for AdhD
reported by Seelanan et al. (1999). Estimated divergence times estimated by dividing the mean Jukes-Cantor distance by twice the
nucleotide substitution rate.

Nodes

K genome-1c,d
G. sturtianum
C genome
G. australe &
G. nelsonii
C genome
K genome
C genome
K genome
a

Mean
Tajima-Nei
distance

Estimated divergence times (mybp)
FAD2-1a
intron

rpl16b

AdhDb

K genome-2c,d
G. robinsonii
G. bickii
G. bickii

0.0247
0.0247
0.0258
0.0263

8.1–16.2 0.7–2
8.1–16.2 3–8
8.4–16.9
8.6–17.3

1.7–3
3–6
8–15e
7-14

G. australe &
G. nelsonii
G. bickii
K genome
G. australe/
G. nelsoniid

0.0288

9.4–18.8

8–15e

0.0292
0.0321
0.0368

9.6–19.1
10.5–21.0
12.0–24.1

10.5–21
11–22

This paper.
Seelanan et al. (1999).
c Basal divergence among the K-genome species (see Fig. 1): K genome-1 5 G. costulatum, G. cunninghamii, G. marchantii, G. nobile,
and G. populifolium; K genome-2 5 G. enthyle, G. exiguum, and G.
pilosum.
d Gossypium species novum, G. australe-3, G. londonderriense, and
G. rotundifolium were excluded (see text).
e Seelanan et al. (1999) calculated this figure assuming G. australe,
G. bickii, and G. nelsonii formed a monophyletic clade.
b

The comparisons in Table 3 also illustrate why G. arboreum
and G. gossypioides appeared as anomalies in the global relative rate tests (Table 2). Both the A-genome diploids have
higher mean Tajima-Nei distances relative to the Australian
species than the A-subgenome homologs, however this difference is most pronounced for G. arboreum, and it most closely
approaches the mean Tajima-Nei distances between the D-genome species and the Australian species. In contrast, the mean
Tajima-Nei distances between the Australian species and Dgenome diploid species are lower than those for the D-subgenome homologs, and among the D-genome diploids, G. gossypioides has the lowest distance to the Australian species and
thus more closely approaches the nucleotide substitution rate
of the A-genome species.
Divergence time estimates—Because substitution rates
among the Australian species were largely homogeneous, the
mean Tajima-Nei distances were used to estimate the time
since divergence among the major Australian clades. In each
clade, the mean Tajima-Nei distances were calculated from all
pairwise comparisons except for the taxa that returned significant relative rate statistics (viz., G. species novum, G. australe-3, G. londonderriense, and G. rotundifolium). The substitution rate was calibrated using Seelanan et al.’s (1999) estimate that the C genome diverged from the K genome 10.5–
21 million years before present (mybp). This returned an estimated substitution rate of 0.7–1.7 3 1029 substitutions per
site per year. Although the accuracy of this estimated substitution rate cannot be independently verified, it permits relative
comparisons of divergence times among the major Australian
clades derived from the chloroplast gene rpl16 and the nuclear
gene AdhD (Seelanan et al., 1999).
The estimated divergence times derived from the FAD2-1
intron mostly are congruent with estimates derived from rpl16
and AdhD (Table 4). Comparisons are complicated because
Seelanan et al. (1999) considered the G-genome species monophyletic, whereas G. bickii resolved as a clade in its own right
here. Nonetheless, the estimated divergence times between the
C-genome species and G. bickii (8.9–17.8 million years before
present or mybp) or the G. australe/G. nelsonii clade (9.5–
18.9 mybp) overlaps previous estimated divergence between
the C- and G-genome species of 8–15 mybp (Table 4). Similarly, the estimated time of divergence between the G. bickii
and G. australe/G. nelsonii clades (8.4–16.9 mybp) overlaps
the estimate of 8–15 mybp for the earliest divergence among
the G-genome species based on analysis of AdhD sequences
(Table 4). There are, however, two striking incongruities.
FAD2-1-based estimates for the basal divergence within the K
genome and between G. robinsonii and G. sturtianum in the

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C genome are five- to tenfold and threefold older, respectively,
than previously estimated (Table 4).
DISCUSSION
The A- and D-genome FAD2-1 introns are orthologs but
are evolving at significantly different rate—In an earlier paper, the size similarity between the two FAD2-1-specific restriction fragments in G. barbadense and in G. hirsutum and
the A-genome G. arboreum and the D-genome G. raimondii
fragments suggested that the two allotetraploid FAD2-1 genes
were A- and D-subgenome orthologs (Liu et al., 1999). The
topological placements of the ten intron sequences from the
five allotetraploid species support this interpretation (Fig. 1).
Five of the allotetraploid sequences, one from each species
resolved within a monophyletic D-genome lineage, sister to G.
raimondii; the other five resolved as part of a monophyletic
A-genome lineage. Support for these topological placements
is strong. The A-subgenome sequences share 13 unambiguous
substitutions with the two A-genome diploid species, and the
D-subgenome sequences share 33 unambiguous substitutions
with the D-genome diploid species.
Relative rate tests demonstrate that, except for G. arboreum
and G. gossypioides (discussed below), the D-genome FAD21 introns have accumulated 1.8 times the number of base substitutions than have the A-genome introns relative to the phylogenetically equidistant Australian Gossypium species (Tables
2, 3). This difference is reflected in the relative branch lengths
of the A- and D-genome clades in Fig. 1B. These differences
are even greater among the A- and D-subgenome species (Table 3). For example, the mean Tajima-Nei distance of the Dgenome diploids from G. bickii is 1.4 and 1.7 times greater
than it is for G. herbaceum and G. arboreum, respectively, but
the mean Tajima-Nei distance of the D-subgenome intron sequences from G. bickii is 1.9 times greater than for the Asubgenome intron sequences. This pattern is consistent regardless of the reference taxon used (Table 3). Small et al. (1998)
made a similar observation based on relative rate tests of AdhC
among all five Gossypium allotetraploid species, G. arboreum,
and G. raimondii relative to G. robinsonii. Although nucleotide substitution rates for the A- and D-subgenome AdhC introns are roughly equal, the substitution rate in the D-subgenome exons is nearly five times greater than it is in the A
subgenome. Overall, the D-subgenome AdhC sequences are
accumulating substitutions about 1.8 times faster than their
orthologs in the A subgenome (Small et al., 1998). The consequences of this differential rate of sequence evolution in
phylogenetic reconstruction are evident in Fig. 1. The A subgenome lacks even one defining synapomorphy and is completely unresolved except for one substitution that differentiates G. mustelinum from the other taxa. In contrast, the D
subgenome is defined by 11 substitutions and is more fully
resolved.
Small et al. (1998) suggest differential selection between the
two subgenomes may partially account for different nucleotide
substitution rates between orthologous genes in the same nucleus. While this certainly may play a role, the FAD2-1 intron
analysis, which includes both A-genome species and six of the
D-genome species suggests that the underlying mechanistic
differences were active in the A- and D-subgenome progenitors and that these lineage differences have been exaggerated
in the allopolyploids. In this regard it is notable that the G.
arboreum and G. raimondii AdhC sequences are also evolving

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[Vol. 88

at significantly different rates, relative to each other, and relative to their D-subgenome and A-subgenome orthologs, respectively (Small et al., 1998). More recent data suggest that
the assumption that the A and D genomes are phyletically
equidistant from the Australian species may be incorrect (J. F.
Wendel, personal communication). However, even if the Agenome and the Australian species share a more recent common ancestor than either does with the D genome, the phyletic
distance between the A-genome/Australian and the D-genome
lineages would have to be much greater than these data suggest it will be to account for the differences in the absolute
number of nucleotide substitutions between the A- and D-genome lineages (Wendel and Albert, 1992; Seelanan, Schnabel,
and Wendel, 1997).
The exceptions to this differential rate of nucleotide substitution between the A and D subgenomes are G. arboreum and
G. gossypioides. The substitution rates of these two species,
run counter to their genomic compatriots. Gossypium arboreum has the highest substitution rate among the A-genome
diploid and allopolyploid species, and G. gossypioides has the
lowest rate among the D-genome species. Gossypium gossypioides is accumulating substitutions only 1.2 times faster than
G. arboreum compared to a general difference of 1.8 observed
between the A and D genomes (Table 3). This observation is
intriguing because both of these species have unique evolutionary histories.
Gossypium arboreum is one of the four domesticated Gossypium species and is the only one for which a wild progenitor
has never been identified, existing only as a domesticated cultigen or feral derivatives of domesticated forms (Brubaker,
Bourland, and Wendel, 1999). Although it is tempting to attribute some proportion of G. arboreum’s anomolous sequence
substitution rate to the effects of human domestication, the
degree to which human selection has altered rates of gene
evolution in G. arboreum cannot be ascertained without the
wild progenitor as a point of reference. Nonetheless, it is plausible that human selection has altered rates of sequence evolution in G. arboreum, an hypothesis worth testing in crop
species for which clearly identified wild progenitors are available.
Gossypium gossypioides is distinct among other D-genome
diploid species, because it contains a mosaic genome resulting
from an ancient hybridization (Wendel, Schnabel, and Seelanan, 1995). Despite the fact that there are no A-genome species
extant in the New World, the G. gossypioides genome contains
A-genome-specific dispersed repeats and a mosaic nuclear ribosomal DNA repeat that combines features of the A-genome
rDNA repeat with those of the D-genome rDNA (Wendel,
Schnabel, and Seelanan, 1995; Zhao et al., 1998). Whether
there is a causal link between the G. gossypioides mosaic genome and the tardy rate of sequence evolution in its FAD2-1
intron remains to be determined.
D-genome evolution and the origin of the New World
allotetraploids—The topological placement of the D-subgenome intron sequences sister to G. raimondii, while G. gossypioides resolves as basal to the entire D-genome lineage, is
worthy of note. Gossypium gossypioides and G. raimondii are
traditionally considered to be sister species (Brown and Menzel, 1952a, b; Wendel and Albert, 1992; Wendel, Schnabel,
and Seelanan, 1995). Both species have been implicated in the
origin of the allopolyploids (Endrizzi, Turcotte, and Kohel,
1985; Wendel, Schnabel, and Seelanan, 1995), and G. gossy-

January 2001]

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ET AL.—FAD2-1 INTRON EVOLUTION IN

pioides experienced introgressive hybridization with the progenitor of the allotetraploid A subgenome, either directly or
indirectly via the nascent allotetraploid (Wendel, Schnabel, and
Seelanan, 1995). Traditionally, G. raimondii is considered to
be the extant D-genome species sister to the D-subgenome
progenitor. This is based on leaf developmental patterns, seed
hair type, vigor of F1 hybrids with A-genome species, chromosome homologies inferred from multivalent frequencies in
synthetic hexaploid hybrids, and genetic segregation in synthetic allohexaploids (reviewed by Endrizzi, Turcotte, and Kohel, 1985). As comprehensive as this sounds, G. raimondii is
a geographical outlier relative to the other D-genome diploid
species. It is indigenous to Peru while the other species are
found in Mexico (except for G. klotzschianum in the Gala´pagos). Thus some question the assumption that G. raimondii is
the sister taxon to the D subgenome. This doubt is largely
based on the suspicion that the allotetraploid lineage arose in
Mexico (specifically the Isthmus of Tehuantepec) rather than
South America (Wendel, Schnabel, and Seelanan, 1995). If this
is true, G. raimondii is indeed an unlikely candidate despite
its genomic and genetic similarities to the D subgenome, and,
conversely, its putative sister taxon, G. gossypioides becomes
a strong candidate, lying as it does closer to the Isthmus of
Tehuantepec than any other D-genome species and carrying
genomic evidence of introgression with the A-subgenome progenitor (Wendel, Schnabel, and Seelanan, 1995).
This reasoning benefits from parsimony as it assumes a single A 3 D interspecific hybridization that resulted in the mosaic G. gossypioides rDNA internal transcribed spacer and the
evolution of an allotetraploid lineage rather than two hybridization events between two geographically separated D-genome species and a single A-genome taxon. Wendel, Schnabel,
and Seelanan (1995) are also correct in questioning the reliability of multivalent frequencies and segregation ratios in
synthetic hexaploids, the strongest but uncorroborated evidence rejecting G. gossypioides as the sister taxon of the Dsubgenome progenitor. However, hypotheses that the allotetraploid lineage arose in Mexico and that G. gossypioides and G.
raimondii are sister species may be untenable.
Ano et al. (1982) nominated northeastern Brazil as a probable site for the origin of the allotetraploid lineage. Fryxell
(1979) noted that all the wild populations of the tetraploid
species occupy littoral or littoral-derived habitats. This observation, in concert with a proposed divergence during the Pleisotocene (Fryxell, 1965; Phillips, 1963; Wendel, 1989), a period of rapidly changing ocean levels and the fact that Gossypium seeds are salt-water tolerant (Stephens, 1958; Fryxell,
1979), suggests that prevailing ocean currents would be a primary determinant in the direction of migration of the nascent
allopolyploids. The prevailing marine currents move from
northeastern Brazil along the northeastern coast of South
America passing through the Caribbean Sea into the Gulf of
Mexico. Ano et al.’s (1982) hypothesis suggests that the allotetraploid lineage arose in northeastern Brazil and that subsequent northerly movement of germplasm with the prevailing
marine currents along the coast of South America led to the
colonization of northern coastal South America, Gulf coastal
Mexico, and the Islands of the Antilles. Under this scenario,
G. mustelinum represents the resident descendent of the nascent allopolyploid, consistent with its basal position in gene
topologies, while the colonial populations diverged into the
other four allotetraploid species (see also Wendel, Rowley, and
Stewart, 1994).

GOSSYPIUM

99

The convention that G. gossypioides and G. raimondii are
sister species is based on gross comparative morphology and
the interfertility of the two species (Brown and Menzel, 1952a,
b). Although this conclusion is supported by phylogenetic
analyses of chloroplast restriction site mutations (Wendel and
Albert, 1992; Seelanan et al., 1997), it is not unassailable. In
the first place, gross morphological comparisons are subject to
contradictory interpretations. Although Brown and Menzel
(1952a, b) concluded that G. gossypioides and G. raimondii
are closely related, they also noted that G. gossypioides is
intermediate between G. thurberi and G. raimondii in flower
size, shape, and color, leaf shape, and stem texture, having ‘‘a
marked superficial resemblance to the F1 hybrid’’ between the
two (Brown and Menzel, 1952a, p. 120). Hutchinson, Silow,
and Stephens (1947) interpreted the situation differently and
classified G. gossypioides with G. thurberi and G. trilobum in
section ‘‘Thurberana.’’ They did this on the basis that the section ‘‘Thurberana’’ species were glabrous (or mostly so) and
had lobed leaves and entire or three-toothed epicalyx bracts,
in contrast to the section ‘‘Klotzschiana’’ species, including G.
raimondii, which were characterized by pubescent leaves and
stems, entire leaves, and laciniate epicalyx bracts.
In contrast to the equivocal conclusions afforded by gross
morphological comparisons, phylogenetic analyses of the 5S
ribosomal DNA (Cronn et al., 1996) and the FAD2-1 (Fig. 1)
intron both resolve G. gossypioides as the sole descendent of
one lineage from the basal divergence in a monophyletic Dgenome lineage. In the case of the 5S ribosomal DNA, a topology rejecting the basal position of G. gossypioides would
require another six steps. Although the basal position of G.
gossypioides in the FAD2-1 topology is supported by decay
index of only 1, collapsing this branch still would not resolve
G. gossypioides and G. raimondii as sister taxa. Conversely,
the FAD2-1 topology resolves G. raimondii (decay index of
2) as basal to the D-subgenome monophyletic lineage. Although not all the D-genome diploid species are included in
this analysis, G. gossypioides and G. raimondii are the only
probable candidates (reviewed by Endrizzi, Turcotte, and Kohel [1985] and Wendel, Schnabel, and Seelanan [1995]). Consequently, the weight of the evidence promotes G. raimondii
as the most likely extant taxon that is sister to the allotetraploid
D-subgenome progenitor. Furthermore, Stephens (1944a, b),
based on a genetic analysis of leaf shape alleles in Gossypium,
concluded that genetic control of leaf shape in the allotetraploids most likely reflected the interaction of the leaf shape
genes in the Old World A-subgenome progenitor in combination with the leaf shape genes from a D-genome diploid with
entire leaves, of which there are only four, viz., G. aridum, G.
armourianum, G. klotzschianum, and G. raimondii. Thus, it is
worthy of note that in the FAD2-1 topology, G. klotzschianum
resolves as basal to the combined G. raimondii-allotetraploid
D-subgenome lineage (Fig. 1).
These considerations, however, fail to resolve the evolutionary relationship of G. gossypioides to the other D-genome diploids. Setting aside the mosaic rDNA ITS (Wendel, Schnabel,
and Seelanan, 1995), G. gossypioides contains a chloroplast
genome sister to G. raimondii (Wendel and Albert, 1992) but
contains a 5S ribosomal repeat and a FAD2-1 intron sequence
that resolve as basal to the other D-genome diploids assayed
(Wendel, Schnabel, and Seelanan, 1995). Cronn et al. (1996)
suggest that the 5S ribosomal repeat may also be a mosaic
gene and therefore should be expected to resolve as a basal
taxon in a phylogenetic analysis but could not identify a single

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A-genome-specific base pair change. This reasoning may also
be applicable to the FAD2-1 intron, but given that it resides
in a single copy gene, it is unlikely. Particularly notable are
the 33 unambiguous base pair substitutions and indels the G.
gossypioides FAD2-1 intron shares with the other D-genome
taxa and that differentiate them from the Old World E- and Agenome lineages (Fig. 1). The G. gossypioides FAD2-1 intron
contains none of the 13 A-genome-specific substitutions. Thus,
chloroplast and nuclear genes place G. gossypioides in incongruent positions within the larger Gossypium topology. This
incongruity can be explained by assuming that the chloroplast
topology accurately tracks the taxon evolution and that the
FAD2-1, the 5S ribosomal repeat, and the ribosomal 18S–26S
DNA repeats are mosaics that have recombined with their Agenome orthologs. Alternatively, one can propose that the
FAD2-1 and 5S ribosomal genes accurately track the taxon
evolution. This implies that G. gossypioides obtained the G.
raimondii-like chloroplast via later interspecific introgression
rather than by inheritance. At the moment the weight of evidence favors the latter hypothesis. The key may lie in a better
understanding of G. raimondii. The chloroplast DNA topology
indicates that G. gossypioides and G. raimondii were in physical contact at some point either via interspecific introgression
or via a recent common ancestor. Understanding how G. raimondii came to its present geographic isolation from its New
World congeners, particularly G. gossypioides, will be vital to
finally resolving the incongruence in topological placement of
G. gossypioides in chloroplast and nuclear topologies.
Gossypium mustelinum is basal among the five
allotetraploid species—The t