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Systematics and Phytogeography |
Department of Horticulture, Auburn University, Auburn, Alabama 36849 USA
Received for publication January 27, 2004. Accepted for publication July 30, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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4 kb section of cpDNA. Variability within Citrullus was detected primarily at noncoding regions of high A + T content. Substitution rates varied from 0–0.48% for ndhF with A + T content of 68.4% to 0.39– 1.69% for the intergenic region of atpA with A + T content of 82.8%, mainly resulting in indels and transversions. Indels at several regions acted as valuable parsimony informative markers. Citrullus lanatus var. lanatus, the cultivated watermelon, and C. ecirrhosus and C. rehmii from Namibia, lacked molecular variability. The genus Citrullus is supported monophyletically and shows two main clades, one of which contains C. colocynthis. In the other clade, C. rehmii is sister to a clade containing C. ecirrhosus and C. lanatus. Two clades were recovered within C. lanatus, consisting of domesticated watermelon and wild citron, var. citroides. Five haplotypes within C. colocynthis were used to deduce colonization routes of the species. Biogeographic patterns point to separate colonization events into Africa and the Far East.
Key Words: Citrullus • colonization routes • Cucurbitaceae • nucleotide substitution rates • phylogeography • watermelon
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Studying the geographical distribution of genetic lineages can provide insights in different factors that shape the genetic diversity of species (Avise, 2000
).
Phylogeographic studies in plants have been dominated by investigations of the chloroplast (cp) genome (Schaal et al., 1998
). Since different portions of the chloroplast genome evolve at different rates, a wide range of possibilities exist for resolving relationships from the level of species and genus to family (Soltis et al., 1998
). In the complex mutational process of cpDNA (Clegg et al., 1994
), relative rates of synonymous substitution may be conserved over most chloroplast genes, while rates of nonsynonymous substitution are strongly influenced by locus-specific effects (Muse, 2000
). The study of codon bias has uncovered a substantial bias of genes favoring codons ending in A or T and strong dependence on adjacent nucleotide site composition in transition/transversion rates in noncoding regions of cpDNA. Insertion/deletion mutations (indels) appear to be context-dependent because their rate of occurrence is known to increase in specific regions of both coding and noncoding DNA (Morton and Clegg, 1995
). A number of factors contribute to this rate variation, including generation time; extinction; episodic changes of sequence divergence rate; and lineage specific rate variation (Soltis et al., 1998
). Although many factors influence nucleotide diversity, selection is likely one of the major contributors (Buckler et al., 2001
). Most studies of the effect of selection pressure on nucleotide diversity have focused on domesticated crops, comparing diversity between wild relatives and cultivars. During the selection of advantageous phenotypes, several crops appear to have passed through bottlenecks that substantially reduced diversity (Gepts, 2002
).
Despite the economic importance of watermelon, domestication events and phylogeographic relationships have only recently attracted scientific attention. The genus Citrullus Schrad. ex Eckl & Zeyh. contains four diploid (2n = 22) species: the domesticated watermelon, C. lanatus ((Thunb.) Matsum & Nakai) var. lanatus, grown in tropical and subtropical regions worldwide; the preserving melon or citron, C. lanatus var. citroides (Bailey) Mansf., grown in southern Africa; perennial C. colocynthis L. Schrad, the bitter apple grown for medicinal purposes from northern Africa to southwest Asia; perennial C. ecirrhosus Cogn. and annual C. rehmii De Winter, wild species endemic to desert regions of Namibia (Robinson and Decker-Walters, 1997
). The distribution of the wild taxa in southwest Africa point to Namibia as the center of domestication for watermelon. However, the available archaeological information does not support this. Watermelons were cultivated in the Nile Valley at least since the start of the second millennium BC when farming was not yet practiced in southwest Africa (Zohary and Hopf, 2000
). Seeds from C. colocynthis appear in several early Egyptian, Libyan, and near Eastern sites, and the species was probably used by humans prior to its domestication.
CpDNA studies in the genus Citrullus using PCR-RFLP analysis revealed 7 haplotypes with nucleotide diversity at five main regions. Three haplotypes were detected within C. lanatus, one haplotype being associated with the cultivated watermelon and two with wild citron types (Dane et al., 2004
). Most of the cpDNA variation within Citrullus is the result of large indels and nucleotide substitutions, primarily at noncoding regions. Sequencing analysis of informative regions should shed light on the domestication patterns of watermelon and the evolution of species in the genus. This molecular study was undertaken to investigate the phylogenetic relationships among Citrullus haplotypes in order to deduce colonization routes and estimate divergence times. Rates and patterns of nucleotide substitutions and indels in cpDNA regions of different A + T content were studied and their effect on phylogeny estimation discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Seeds from C. rehmii, a recently discovered species from the Namib desert in Namibia (De Winter, 1990
) were obtained from the NPGC or from the PI Station via B&T World Seeds. Praecitrullus fistulosus (Stocks) Pang., cultivated in India for its edible fruit, was chosen as outgroup. The species was once considered a Citrullus species, but has been given its own taxonomic category (Sujatha and Seshadri, 1989
). PCR-RFLP analysis had also indicated that Acanthosicyos naudinianus (Sond.) C. Jeffrey (tribe Beninsaceae as Citrullus and Praecitrullus) and Cucumeropsis mannii Naudin (tribe Melothrieae) are more distant (Dane, 2002
). DNA was extracted from seedlings or cotyledon tissues using the Qiagen plant DNA easy extraction kit (Qiagen, Valencia, California 91355 USA). Chloroplast genome sections were amplified using the following primer pairs: 803F (CTA TGG TAG CGG CGG GAW TTT TTC)-1955R (CGA TTA TAT GAC CAA TCA TAT A) for the ndhF 3' coding region; 1955F (TAT ATG ATT GGT CAT ATA ATC G)-607R (ACC AAG TTC AAT GTT AGC SAG ATT AGT C) for the ndhF 3' flanking region (Olmstead and Sweere, 1994
); ycf6F (CTT GGG CTG CTT TAA TGG) -psbMR (GTA AAT ATT CTT TAA TGG) for the intergenic region of ycf6-psbM; primer orf62P (CTT GCT TTC CAA TTG GCT GT)-trnGP (GCC AAG GAG AAG ATG CGG G) for the intergenic region ycf9-trnG; primer atpAF (AGG TTC AAA TCC TAT TGG ACG CA)-trnRR (TTT TGA AAG AAG CTA TTC ARG AAC) for the intergenic region of atpA-trnR, designed by Heinze (2002)
. The primers were chosen for sequence analysis upon the discovery of phylogenetically informative restriction enzyme site differences within Citrullus species (Dane, 2002
, 2004). Accessions from different areas of origin were used for sequencing to detect evolutionary patterns. Amplified fragments were purified using Qiaquick PCR Purification Kit (Qiagen) and sequence analysis was performed using an ABI 3100 sequencer. All nucleotide sequences were submitted to GenBank (see Table 1 for accession numbers). Sequences were aligned using Vector NTI® software (InforMaxTM, Frederick, Maryland 21704 USA), followed by manual adjustments. Single base indels were crosschecked to the original chromatograms for accuracy. Indels were scored as single binary (0 vs. 1 or ?) characters appended to the main matrix, according to Graham et al. (2000)
. All character states were specified as unordered and equally weighted. To test for congruence of the different cpDNA regions, partition homogeneity tests with heuristic searches were conducted in PAUP* with 100 replicates and tree-bisection-reconnection (TBR) branch swapping. Maximum parsimony trees were constructed with separate or combined data sets, with or without indel, as characters using PAUP* (Swofford, 2002
). Heuristic searches were performed with random taxon sequence additions, 500 replicates, and TBR branch swapping. A 50% majority rule consensus tree was constructed. Support for the clades was evaluated using 1000 bootstrap replicates.
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), since MEGA version 2.1 (Kumar et al., 2001
) was found to underestimate divergence because of elimination of parsimony informative indels. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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4.0 kb, representing 2.6% of the maternally inherited cucurbit genome of 155 kb (Havey et al., 1998
). Variant sites within Citrullus at the sequenced regions are characterized in Table 2. Sequences from the 17 accessions and the outgroup were easily aligned, and gaps were detected mainly in alignments with the outgroup. A low number (42) of phylogenetically informative characters were identified within Citrullus, varying from five for the 3' coding region of ndhF to 14 for the ycf6-psbM intergenic region. This resulted in a total of 1.1% variant sites. Between the outgroup P. fistulosus and Citrullus a high number of additional substitutions (77) and indels (16) were detected (Table 2). The C. rehmii accessions obtained by the PI Station in Griffin, GA from B&T World Seeds are misclassified. PI 432717 shows complete sequence homology to C. lanatus var. citroides; PI 632755 has complete sequence homology to C. colocynthis accessions; while accessions NAM 1303 and NAM 1887 obtained from the National Plant Resource Centre in Namibia constitute the unique Citrullus rehmii species described by De Winter (1990)
and will be discussed as such.
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). At the 3' flanking region of ndhF only six parsimony informative sites were detected within Citrullus, while an additional 20 substitutions and two indels, of 9 and 15 base pairs (bp), separate all Citrullus accessions from P. fistulosus. Among the Citrullus substitutions, five were detected in C. lanatus var. lanatus, three of which also occurred in C. lanatus var. citroides, one also in C. ecirrhosus, and one unique substitution in C. rehmii. C. rehmii showed complete homology with P. fistulosus at six of the variant Citrullus sites, C. colocynthis at five of the variant Citrullus sites. Sequence analysis of the 3' flanking region of Cucumeropsis mannii PI 532721 (GenBank accession number AY527172) was conducted to consider ingroup status. The 50% bootstrap majority tree constructed using only ndhF3' sequence data, and with P. fistulosus and C. mannii as outgroups, recovered Citrullus as a monophyletic clade (93% bootstrap value).
Nucleotide variation at ycf6-psbM in Citrullus
Sequence variation at the ycf6-psbM region was more informative. The A + T content of this region is high (70.4%, Table 2) with nucleotide substitutions at 16 different sites within Citrullus. While several unique substitutions were detected in all species, intraspecific variability was detected only in C. colocynthis. Between Citrullus and the outgroup a total of three indels and 14 additional single base substitutions were observed.
Nucleotide variation at ycf9-trnG in Citrullus
Intrageneric divergence at the ycf9-trnG region was similarly very low with 0.35% variant sites (Table 2). The A + T content of the region varies from 71.1% for C. lanatus var. lanatus to 74.8% for C. colocynthis. One transversion separates C. ecirrhosus from the other Citrullus species; another distinguishes C. lanatus and C. ecirrhosus. However, evolutionary splits between the different species were easily detected through the occurrence of overlapping indels with high A + T content (90%) ranging in size from 5 to 132 bp (Fig. 1). Since other cucurbit species (Cucumis melo L., C. sativus L., Cucumeropsis mannii) show PCR fragments of similar size (700–750 bp) as C. colocynthis, large deletions at this region must have occurred in C. ecirrhosus (127 bp) and C. lanatus (132 bp). The deletions were scored as three characters: 1) 1 (presence) versus 0 (absence) of a 118 bp section; 2) 1 versus ? (missing data) of two indels; and 3) 1 versus ? of a 5 bp indel (Fig. 1) according to Graham et al. (2000)
. Six substitutions and one indel are present between the 132 bp section in C. colocynthis and P. fistulosus. The large indel is nested in a (AT)4 microsatellite region and contains several (AT)2 and (AT)3 tandem repeats. At another section of the ycf9-trnG region, an 18 bp insertion completely identical to a 17 bp adjacent flanking region with A + T content of 94% is present in C. lanatus, C. ecirrhosus and C. rehmii. Part of this insertion contains a 5 bp deletion in C. colocynthis. These indels were scored as 2 characters: 1) 1 (presence) versus 0 (absence) of the 18 bp insertion, 2) 1 versus ? (missing data) of a 5 bp deletion. An additional 6 bp duplication of a flanking region and 6 more variant sites were detected between Citrullus and P. fistulosus.
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Maximum parsimony trees were constructed using separate and combined data analysis with and without indel characters. Partition homogeneity tests with heuristic searches implemented in PAUP* showed congruent results using the five databases, and parsimony analyses of the different data sets showed similar topologies. The use of DNA sequence data from ycf6-psbM and atpA-trnR resulted in more informative topologies than sequence data analysis from the ndhF region at the inter- and intraspecific level. The bootstrap 50% majority-rule consensus tree using combined data analyses (tree length = 148, CI = 0.960, RI = 0.962; Fig. 2) shows two main clades. One clade contains C. colocynthis and the other the rest of the species. In the latter clade, C. rehmii is sister to a clade containing C. ecirrhosus and C. lanatus, which are sister to one another. Two well-supported clades were recovered within C. lanatus containing the domesticated watermelon (var. lanatus) and the wild citron (var. citroides) (Fig. 2). Trees constructed with sequence data alone (omitting presence/ absence indel characters) failed to detect intraspecific topologies, but were otherwise similar.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) were initially chosen for sequence analysis because of the presence of indels, but provided even lower numbers of informative sites and were not analyzed further. Similarly, mitochondrial DNA sequencing was conducted using nad1B and nad1C (Demesure et al., 1995
), but was abandoned because of lack of variant sites among Citrullus species.
Within C. lanatus variability was detected at 8 sites only (0.2% variant sites), separating the cultivated var. lanatus from wild var. citroides. No variability was detected between cultivated accessions (var. lanatus) with different morphological characteristics collected from varying geographic locations. One of these is the Egusi watermelon (PI 494529), which provides grain for inhabitants of the southern coast of West Africa. Two haplotypes were detected among citron type watermelon (var. citroides) at the atpA-trnR region, which can now be used to study its biogeographic patterns. Intraspecific variability was not detected in C. rehmii (NAM 1303 and NAM 1887) or C. ecirrhosus. Lack of intraspecific variability hinders understanding of domestication events in the genus. It was surprising to discover that sequencing of variable cpDNA sites only increased the number of haplotypes within the genus from seven (Dane et al., 2004
) to 10, and that the additional haplotypes were detected within C. colocynthis only.
Variability within Citrullus was detected at noncoding regions of primarily high A + T content (Table 2). The substitutions rate varied from 0–0.48% for ndhF with A + T content of 68.4% to 0.39–1.69% for the atpA-trnR region with A + T content of 82.8%, mainly resulting in transversions and large indels. Indels in noncoding cpDNA regions can act as valuable markers of deep evolutionary splits (Hilu and Alice, 1999
), a consequence of their low rate of occurrence as compared to nucleotide substitutions (Kelchner, 2000
). Graham et al. (2000)
detected an inverse relationship between natural log-transformed frequency of indels and their natural log-transformed length. Large indels separating C. colocynthis (and P. fistulosus) from the other Citrullus species at several cpDNA regions (trnS-trnfMM, ndhA, trnC-trnD; Dane, 2002
) are rare. The informative indels within Citrullus varied in length from a 132 bp deletion with A + T content of 90.1% (at ycf9-trnG); to a 23 bp perfect repeat insertion in some C. lanatus var. citroides accessions (at atpA-trnR); and a 7 bp perfect repeat insertion (at ycf6-psbM) among C. colocynthis accessions. The large indel (Fig. 1) is clearly the result of slip strand mismatching since the flanking regions contain simple repeat sequences (TA). A 127 bp deletion is phylogenetically very informative, since it is shared by C. ecirrhosus and C. lanatus and indicates a close relationship between these species. Indels are more common in the outgroup taxon (Table 2), but the frequency of occurrence is not related to indel length as reported by Graham et al. (2000)
. Indels range in size from 1 to 15 bp and several are perfect repeat insertions or deletions, with tandem repeat sequences contiguous with and homologous to a flanking region. The highest number of indels was detected at atpA-trnR, while no indels were detected at the ndhF coding region (Table 2). The frequent occurrence of indel events in noncoding plastid DNA sequences is well known (Clegg et al., 1994
). This distribution follows a general pattern with a mix of different simple repeat sequences, and large indels associated with more complex sequence structure as described by Hahn (2002)
.
Rates of nucleotide substitution in Citrullus
The numbers of nucleotide substitutions per site with respect to P. fistulosus ranged from 1.66% to 6.67%, the substitution rate within Citrullus from 0.0% to 1.69%, and the intraspecific rate from 0.0% to 0.25%. Low substitution rates were observed at most regions with the exception of the atpA-trnR site. Comparison of plastid DNA evolution in other taxa indicated that noncoding regions are generally more useful than coding regions. Because of lack of codon-based constraints, sequences of noncoding DNA are known to show less transition:transversion (Ts : Tv) bias than coding plastid DNA (Asmussen and Chase, 2001
; Hahn, 2002
). Ts : Tv ratios at variant sites within Citrullus range from 0.2 for atpA-trnR to 0.7 for ycf6-psbM, while only transversions were observed at the 3' flanking region of ndhF and ycf9-trnG. Similarly Ts : Tv ratios of nucleotide substitutions separating Citrullus from Praecitrullus at the studied regions are variable (ranging from 0.1 to 1.3) but on average (0.43) approach the expected value of 0.5. Almost all transversions are in regions where one or two neighboring bases are A and T, supporting findings of Morton and Clegg (1995)
and Small et al. (1998)
that positions flanked by A and T are more likely to undergo transversions. A 5' pyrimidine in both strands resulted in substitution rates two to three times higher than substitution rates at sites flanked by a 5' purine. This was observed within Citrullus and between Citrullus and the outgroup, which indicates that similar evolutionary processes occur across cucurbit genera.
The substitution rate in Citrullus is low in comparison to substitution rates in other species (Parducci and Szmidt, 1999
; Mohanty et al., 2001
; Manos and Stanford, 2001
; Davis et al., 2002
; Yang et al., 2002
; Ingvarsson et al., 2003
). This is especially true at the 3' region of ndhF (Table 2), although ndhF is useful for inferring relationships among closely related species (Davis et al., 2002
). In Cercis, values of 0.0–2.2% between ingroup taxa and 5.5–6.4% for sequence divergence with the outgroup were observed (Davis et al., 2002
). Similarly higher values were detected at other noncoding cpDNA regions, such as trnD-trnT in Brassica (3.1–3.5%; Yang et al., 2002
), atpB-rbcL in Pleurozium species (4.44%; Chiang and Schaal, 2000
). However, Xu et al. (2000)
using sequence variation at nine non-coding regions in Glycine and Soja similarly found low substitution rates ranging from 0.5 to 2.5%. Recent studies have suggested dramatic variation in rates of molecular evolution among land plant lineages (Soltis et al., 2002
). It is clear that the interplay among divergence times and rates is complex. In angiosperms the rate of morphological evolution correlates with the rate of neutral molecular substitutions (Barraclough and Savolainen, 2001
). In this genus with only four species, two perennial (C. colocynthis, C. ecirrhosus) and two annual (C. lanatus, C. rehmii), no relationship is apparent between morphology and molecular evolution. C. lanatus var. lanatus, known for variability in fruit and seed morphology, lacks molecular evolution at cpDNA regions. This might be related to domestication and subsequent dispersal events of this cultivated species all over the world. Dispersal from the center of domestication must have involved limited amounts of seed, thus reducing the genetic diversity of the species. However, the bitter apple (C. colocynthis) cultivated for medicinal purposes shows substitutions at several sites. Since the annual species show a lower level of sequence divergence than the perennial species, the genus lacks a generation time effect on molecular evolution. Other molecular marker studies in the genus similarly have encountered low levels of genetic diversity, and this has hindered the development of watermelon linkage maps (Hawkins et al., 2001
).
Phylogenetic reconstruction
Intraspecific cpDNA variation has permitted the elucidation of evolutionary processes in many plant species and analyses of phylogeography (Soltis et al., 1992
; Dumolin-Lapegue et al., 1997
). Even though the amount of intraspecific information is generally low, domestication routes, long-distance gene flow patterns and progenitor-derived relationships have been determined in several species. C. colocynthis, a desert perennial species, has been cultivated for its bitter medicinal fruit since biblical times. On the island of Cyprus, the raising of colocynth has been a source of revenue since the fourteenth century (Yaniv et al., 1999
). Substitutions detected in the accessions point to separate colonization routes onto the African continent, one route leading into Northern Africa along the Mediterranean region to Chad, another into Ethiopia. Accessions from North Africa share a unique haplotype, distinguishable by one 7 bp insertion (at ycf6-psbM), while the accession from Afghanistan has a unique haplotype which shares two unique transitions with the haplotype from Ethiopia (Fig. 3). C. colocynthis haplotype from Ethiopia has three unique substitutions, and the highest level of intraspecific divergence with the haplotype from Pakistan (0.25%), which has four unique substitutions. A total of five different haplotypes can thus be detected within C. colocynthis: haplotype I in countries around the Mediteranean from Morocco to Cyprus, haplotype II in PI 549161 from Chad, haplotype III in the accession (PI 537277) from Pakistan, haplotype IV (PI 269365) in Afghanistan, and haplotype V (PI 195967) in Ethiopia. The biogeographic patterns of the species point to divergence from the Middle East, with one path leading into Afghanistan (haplotype IV), another into Ethiopia (haplotype V), another into Pakistan (haplotype III), and a fourth route from the Mediterranean area into Chad (I & II).
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Divergence date of Citrullus
Any attempt to apply dates to the radiation of a genus like Citrullus must be considered highly speculative because of the influence of human colonization. Gaut (1998)
and Xiang et al. (2000)
estimated an absolute synonymous substitution rate (Rs) of 1.24 x 10–9 substitutions/site/year using several chloroplast loci. Assuming Tdivergence = K/ 2 R (Sanderson, 1998
) and similar substitution rates at noncoding cpDNA regions, it can be estimated that C. lanatus diverged at least 0.8–0.9 million years ago (mya) from C. ecirrhosus (K = 0.0020–0.0023), 2.0–2.1 mya from C.rehmii (K = 0.0050–0.0053), 2.3–3.0 mya from C. colocynthis (K = 0.0058–0.0075), and 11.0–11.7 mya from Praecitrullus fistulosis (K = 0.0274–0.291) from India. The mid-Tertiary was a time of climate changes across Africa, which by the late Oligocene saw the development of seasonally dry climate and the spread of grassland and savanna at the expense of forest (Coetzee, 1993
). Continued cooling of the African climate during the late Oligocene and into the Miocene (24– 25.5 mya) resulted in increasingly dry habitats. The Pliocene (5.5 mya) saw the final establishment of the Sahara and Namib deserts (Goldblatt et al., 2002
). C. colocynthis is characterized by exceptional soil binding and drought resistance capacities and might have radiated early across continents, while other drought resistant Citrullus species are indigenous to the Namib desert. The present geographic distribution pattern might reflect patterns of survival more directly than patterns of origin. While many plant phylogenetic studies have relied on sequences of cpDNA, especially in studies of domestication routes, long-distance gene flow patterns and progenitor-derived relationships, the slow rate of sequence divergence in species like Citrullus have limited the utility of such analyses. Forthcoming studies with single-copy nuclear gene sequences (Sang, 2002
) will cast more light on the radiation of Citrullus against a background of aridification.
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FOOTNOTES |
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2 Author for correspondence (e-mail: danefen{at}auburn.edu
)
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