Isozyme and seed protein phylogeny of the genusCitrullus (Cucurbitaceae)

Electrophoretic analysis of 26 enzyme coding genes was conducted on accessions of threeCitrullus species and the relatedPraecitrullus fistulosus andAcanthosicyos naudinianus. The isozyme phylogeny of the genusCitrullus and the related species was constructed based on pairwise measurements of the respective genetic distances between the species and races.P. fistulosus andA. naudinianus form two distinct outgroups toCitrullus which is characterized by two main clusters: The first includes twoC. colocynthis races and the second,C. lanatus andC. lanatus var.citroides, which are more closely related to each other than they are toC. ecirrhosus. The isozyme phylogeny is consistent with the variability in six seed protein bands and with the crossability relations among the examined species.     

Isozyme polymorphism and phylogenetic interpretations in the genus Cicer L.

Summary Allozyme variation among 50 accessions representing the cultivated chickpea (Cicer arietinum L.) and eight wild annual Cicer species was scored and used to assess genetic diversity and phylogeny. Sixteen enzyme systems revealed 22 putative and scorable loci of which 21 showed polymorphism. Variation was prevalent between species (D_st = 0.510) but not within species (H_s = 0.050). No variation for isozyme loci was detected in the cultivated chickpea accessions. Cicer reticulatum had the highest proportion of polymorphic loci (0.59) while the loci Adh-2 and Lap were the most polymorphic over all the species accessions. The phylogeny of annual Cicer species, as determined by allozyme data, generally corroborated those based on other characters in previous studies. Cicer arietinum, C. reticulatum and C. echinospermum formed one cluster, while C. pinnatifidum, C. bijugum and C. judaicum formed another cluster. Cicer chorassanicum was grouped with C. yamashitae, whereas C. cuneatum formed an independent group and showed the largest genetic distance from C. arietinum.     

Species relationships and genetic variation in the diploid wheats (Triticum,Aegilops) as revealed by starch gel electrophoresis

Twenty enzyme loci were examined in the diploid species ofTriticum andAegilops for allelic variation by starch gel electrophoresis. SectionSitopsis, including the five species,Ae. speltoides, Ae. lingissima, Ae. sharonensis, Ae. bicornis andAe. searsii form a close subgroup withAe. speltoides slightly removed from the others.T. monococcum s. lat., was found to be closest to the species of theSitopsis group.Ae. comosa, Ae. umbellulata andAe. uniaristata form a second subgroup withAe. caudata most closely related to these species.Ae. squarrosa appears almost equally related to all of the species, showing no special affinity for any one species group. Nineteen out of twenty loci examined were polymorphic with a mean of 6.7 alleles per locus. Species could be, for most loci, characterized by the presence of predominant alleles. A conspicious genetic characteristic ofTriticum-Aegilops is the sharing of these predominant alleles between species. Within species variation is characterized by a diffuse distribution of secondary alleles.     

Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).     

Genetic relationships among annual species of Cicer(Fabaceae) using isozyme variation

In order to determine the pattern of genetic diversity within and among the species of Cicer and to estimate interspecific genetic relationships, allelic variation was assayed for 23 isozyme loci in 63 accessions of 11 species of Cicer using starch gel electrophoresis. The total allozymic variation observed in the genus (H _t )was equal to 0.60. When partitioned (G st), 96% of this allelic diversity was found among rather than within species. The allelic diversity among species (D st)and allelic diversity within species (H s)were equal to 0.58 and 0.02, respectively. Cicer reticulatum and C. pinnatifidum had the highest proportion of polymorphic loci (17.39%) and the highest mean number of alleles per locus (1.22 and 1.17, respectively). UPGMA cluster analysis of Nei's unbiased genetic distance revealed four genetic groups. One includes C. reticulatum, C. arietinum and C. echino spermum where the first 2 species represent a putative derivative-progenitor pair. A second cluster contains C. bijugum, C. pinnatifidum and C. judaicum. Cicer yamashitae, C. chorassanicum, C. anatolicum and C. songoricum form a third group. Finally, C. cuneatum, which has a very distinct isozyme profile and peculiar morphological features, is the only member of a fourth species group. This species grouping agrees partially with those obtained from crossability and cytogenetic studies. The results suggest that the annual habit arose from perennial progenitors at least twice in the genus Cicer.     

Allozyme divergence and evolution in the genusLens

The genusLens includes 5 taxonomic species:L. culinaris is cultivated andL. orientalis, L. odemensis, L. ervoides, andL. nigricans are wild. All the species are annual and almost exlusively selfers. The wild lentils are distributed over a large geographical area and form small disjunct populations which are composed of a small number of plants. 67Lens populations were assayed electrophoretically for 9 enzyme systems; 15 enzymic genes with 37 alleles were identified. The genetic distances (D) measured between the pairs of populations indicated a significantly greater similarity between populations belonging to the same taxonomic species. Assuming the populations represent a random sample of the variability in each of the species the genetic distances (D) between the 5 taxa were calculated. The shortest genetic distance was found betweenL. orientalis andL. culinaris. Another significant feature of the data is the apparent isolation ofL. nigricans from the other 4 species. The genetic distances between theLens species are compared to the patterns of crossability barriers between them.     

Allozyme investigations on the genetic differentiation between closely related pines —Pinus sylvestris, P. mugo, P. uncinata, andP. uliginosa (Pinaceae)

In eight natural European populations of four closely related taxa of pines (Pinus sylvestris, P. mugo, P. uncinata, andP. uliginosa) starch-gel electrophoreses revealed altogether 58 alleles at 15 loci from nine enzyme systems. With Nei's genetic distance (D) the largest mean genetic distance (D = 0.171) was found betweenP. sylvestris andP. mugo, a distance corresponding to that between other closely related pine species. Mean genetic distances between the other taxa were less than half that value and characteristic for subspecies or varieties rather than for species. On the basis of our results we suggest that both,P. uliginosa andP. uncinata, could be the result of the ancient hybridization betweenP. sylvestris andP. mugo.     

Isozyme studies in the genusCheirolophus (Asteraceae:Cardueae-Centaureinae) in the Iberian Peninsula, North Africa and the Canary Islands

The phylogenetic and phenetic analysis of 109 RAPD polymorphisms inS. nebrodensis, a perennial and self-incompatible endemic of four mountain ranges in Spain, andS. viscosus, a self-compatible annual widespread in Europe, as well asS. lividus, S. sylvaticus andS. vulgaris revealed a sister group relationship between the first two species. This result contrasts sharply with the earlier hypothesis based on isozyme variation thatS. viscosus originated from within a paraphyleticS. nebrodensis and that the two species represent a progenitor-derivative pair. After considering possible reasons for the sister group relationship found, including the possibility of rooting artefacts, it is concluded that neither the RAPD data nor the isozyme data allow to draw safe conclusions about the mode of speciation and therefore the relative age of the two species. As a consequence, the limited genetic variation ofS. viscosus in comparison toS. nebrodensis as revealed by both the RAPD and the isozyme data may reflect its population history, geographical distribution, reproductive ecology, or mode of dispersal just as well as its recent origin from a paraphyleticS. nebrodensis. The result of this study calls for a critical reexamination of other taxon pairs postulated to have a progenitor-derivative relationship on the basis of isozyme evidence.     

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.     

Isozyme polymorphism in some yellow- and blue-floweredVigna species complexes (Fabaceae, Phaseoleae)

An electrophoretic comparison of variation at 28 isozyme loci was performed for 58Vigna accessions belonging to theV. luteola, V. ambacensis, andV. racemosa groups of species. In all three groups, strong divergence is noted between results and actual nomenclature.     

Mating system and genetic differentiation inDasypyrum villosum (Poaceae) in Italy

Genetic differentiation at the morphological, isozyme, and DNA levels among sevenDasypyrum villosum (Poaceae, Triticeae, 2n = 14, VV genomes) populations from Italy was studied. A measure of the mating system was also obtained. Genetic diversity was mainly distributed within populations (90%) rather than among populations (10%), typical for most allogamous species. Interpopulation diversity, however, was greatest between the most geographically distant populations (about 750 km apart, 911 m altitude difference). The mating system was estimated to vary from 55 to 100% outcrossing. One population (I-16, Bomarzo) deviated from the others in its uniform early flowering habit and presence of isozyme alleles not found in other populations. It had genetic diversity similar to other populations for isozyme (Got-V2, Got-V3, andEst-VF) and ribosomal RNA (Nor-Vl) loci. This population is believed to be the product of a few migrant founder seeds and its unique characters point out that generalized population biology parameters of genetic diversity are not sufficient for describing species variation nor for developing conservation strategies.     

Effect of the breeding system on the genetic structure in threeCephalanthera spp. (Orchidaceae)

The genetic variation of seven enzymes for a total of nine loci was investigated in three species of terrestrial orchids of the genusCephalanthera:C. longifolia, C. rubra, andC. damasonium. These species are characterized by presenting different breeding types: outbreeding , outbreeding with facultative vegetative reproduction, and inbreeding, respectively. Electrophoretic evidence points to a difference in the behaviour of each of the three species which seems strictly related to the breeding type. On the basis of our resultsC. longifolia behaves as a normal outbreeder, whileC. rubra presents the influence of vegetative reproduction in some populations and not in others.C. damasonium shows a total lack of both among and within populations genetic variation, which is most probably due to the autogamic breeding type.     

Isoenzyme variation in the wild relatives ofVicia faba (Fabaceae)

Electrophoretic analysis of five enzyme systems, LAP, PGI, SKDH, SOD and 6-PGDH, among 102Vicia accessions representingV. bithynica and seven species of theV. narbonensis complex, namelyV. eristalioides, V. kalakhensis, V. johannis, V. galilaea, V. serratifolia, V. narbonensis andV. hyaeniscyamus, has been performed. The recorded variation was tentatively assigned to 41 allelic genes at eight loci; intraspecific variation was observed in all species except forV. eristalioides. The results obtained were compared with the corresponding data reported earlier forV. faba. Hierarchical grouping of the investigated taxa, includingV. faba, was based onNei's genetic identities calculated from the allozyme frequency data.Vicia faba andV. bithynica were shown to be most distantly related to one another and to the remaining species investigated.Vicia serratifolia appeared to be a peripheral member of theV. narbonensis complex. The results are discussed with reference to genetic diversity and taxonomic relationships of the species under study.     

Allozyme variability and phylogenetic relationships in the cultivated potato (Solanum tuberosum) and related species

Gene frequencies at 13 isozyme loci were determined in three South American taxa of cultivated potatoes [the diploid group (gp.) Stenotomum, the diploid subgroups (subgp.) Goniocalyx, and the tetraploid gp. Andigena ofS. tuberosum], in the diploid weed speciesS. sparsipilum, and in most of the main cultivars now raised in the Northern Hemisphere (the tetraploid gp. Tuberosum ofS. tuberosum). High levels of genetic variability (mean number of alleles per locus, percentage of polymorphic loci, and mean heterozygosity) were detected, being higher in tetraploid potatoes. An equilibrium among the evolutionary factors which increase genetic variability and artificial selection for maximum yield would explain the high uniformity of heterozygosity values we observed in both Andigena (0.36 ± 0.02) and Tuberosum (0.38 ± 0.01) cultivars.—The low value of genetic distance (D = 0.044) between Stenotomum and Goniocalyx does not support the status of species forS. goniocalyx.—In most isozyme loci, the electromorphs of gp. Andigena were a combination of those found in both gp. Stenotomum andS. sparsipilum, suggesting an amphidiploid origin of gp. Andigena from that two diploid taxa. The presence in Andigena of unique electromorphs, which were lacking in both gp. Stenotomum andS. sparsipilum, suggests that other diploid species could be also implied in the origin of tetraploid Andean potatoes. Furthermore, since Andigena were more related to Stenotomum (D = 0.052) than toS. sparsipilum (D = 0.241), the autopolyploidization of Stenotomum individuals and the subsequent hybridization with gp. Andigena may also have occurred. Thus, our study suggests a multiple origin (amphidiploidy, autoploidy, and hybridization at tetraploid level) of gp. Andigena.—Most of the electromorphs of gp. Tuberosum were also found in gp. Andigena; both the direct derivation of that group from the Andean tetraploid potatoes and the repeated introgression provided by breeding programmes could explain this result. However, the allele c of Pgm-B, present in 30 out of 76 Tuberosum cultivars from Northern Hemisphere as well as in 3 Chilean Tuberosum cultivars, lacks in the 258 Andigena genotypes sampled, suggesting that Chilean germplasm could have taken part in the origin of at least the 39% of the potato cultivars from Europe and North America analyzed here.—The distanceWagner procedure provides an estimate of a 30% of heterogeneity in the evolutionary divergence shown by different groups of cultivated potatoes. Diploid groups show a higher (22.5%) evolutionary rate than tetraploids, which can be attributed to both tetrasomic inheritance and facultative autofecundation that exists in Andigena and Tuberosum groups. Thus, artificial selection acting since 10000 years has not resulted in a higher rate of molecular evolution at the isozyme level in the tetraploids.     

Protein variation,taxonomy and differentiation in five species of marmosets (genusCallithrix Erxleben, 1777)

The electrophoretic patterns of 15 protein systems codified for 20 genetic loci were investigated using horizontal electrophoresis. A total of 150 blood samples, from five species of the genusCallithrix were analyzed. Polymorphic variation was observed in 10 out 20 loci analyzed. The genotypic distributions are in Hardy-Weinberg equilibrium. The average heterozygosity (H) varied from 1% to 5%, similar to those observed for other Neotropical primates. The genetic distance coefficients revealed a phylogenetic separation of these species into two groups: (1) “argentata” (C. humeralifer andC. emiliae); (2) “jacchus” (C. jacchus, C. penicillata, andC. geoffroyi). This arrangement is according to the taxonomic arrangement proposed byHershkovitz (1977),de Vivo (1988), andMittermeier et al. (1988). The results in each group are compatible with the subspecies values recorded for the Platyrrhini. These values showed that:C. humeralifer andC. emiliae are subspecies ofC. argentata;C. jacchus, C. penicillata, andC. geoffroyi are subspecies ofC. jacchus. These results also suggest thatC. j. geoffroyi is the “jacchus” group taxon, most similar genetically to the “argentata” group.     

Patterns of genetic variation of the genusCapsicum (Solanaceae) in Mexico

The evolutionary relationships of 186 accessions ofCapsicum from Mexico were studied through enzyme electrophoresis. A total of 76 alleles representing 20 genetic loci coding for nine enzyme systems were observed and the allelic variations of enzymes were studied for geographical distribution. Allele frequencies were used to estimate the apportionment of gene diversity within and between populations and to construct a dendrogram based on a similarity matrix containingNei genetic distances. — The gene diversity estimates suggest that the structure ofCapsicum populations in Mexico consists of predominantly homozygous genotypes presumably due to a self-pollinated breeding system and population bottlenecks. Significant genetic differentiation was found mainly between populations of differing geographical regions.—Based on the results of this study, three species of domesticatedCapsicum can be identified in Mexico,C. annuum var.annuum, C. chinense, andC. pubescens. Semidomesticated and wild forms include two species,C. frutescens andC. annuum var.glabriusculum. A sharp geographical division results between the latter species;C. frutescens was collected exclusively in the southeastern states of Oaxaca, Chiapas, and Tabasco; whereas wild and semidomesticated forms from the rest of the country areC. annuum. Based upon the similarity of enzyme genotypes of semidomesticated and wild forms, the primary center of domestication of cultivatedC. annuum was estimated to be the region comprising the states of Tamaulipas, Nuevo Leon, San Luis Potosi, Veracruz, and Hidalgo in eastern Mexico. A possible second center of domestication is suggested to be localized in the state of Nayarit, western Mexico.     

Allozyme diversity and the evolution ofCrepidiastrum (Compositae) on the Bonin (Ogasawara) Islands

The genusCrepidiastrum is distributed in East Asia and includes 7 species. In the Bonin Islands, three species ofCrepidiastrum occur, and all of them are endemic to the islands. For detecting the origin and speciation of these endemic species, electrophoretic studies have been done in three endemic species of the Bonin Islands as well as in the remaining four species ofCrepidiastrum, andYoungia denticulata which is considered to be closely related toCrepidiastrum. A total of 386 individuals were sampled from 14 populations. As a result, 17 loci of 10 enzyme systems were resolved and gene frequencies for each population were calculated. The genetic variability was low in island species, as reported in some oceanic island plants. Four groups were recognized in the dendrogram generated by the UPGMA method. The Bonin endemics were clustered together, suggesting a monophyletic origin.C. ameristophyllum andC. linguaefolium were found to be genetically very similar, and this may suggest recent and rapid speciation within the islands.     

Allozyme diversity and the evolution ofSymplocos (Symplocaceae) on the Bonin (Ogasawara) Islands

To study the origin and speciation of plants in oceanic islands, electrophoretic analyses have been done on three endemic species ofSymplocos in the Bonin Islands as well as on three other species;S. kuroki, S. nakaharae andS. tanakae which are considered to be closely related to the Bonin endemics. There occur three species:S. kawakamii, S. pergracilis andS. boninensis in Bonin. The genusSymplocos is one which is considered to be diversified in the Bonin Islands. Seven enzyme systems presumed to be encoded by 18 loci were examined. The genetic diversity was low in the island species, as reported in some oceanic island plants of Hawaii and the Bonin Islands. The three endemics share high genetic identities and they clustered together in the tree drawn by the UPGMA method, suggesting that they are a monophyletic group, that is, they result from a single introduction.     

Allozyme diversity and systematics inAnnonaceae—a pilot project

Five species ofAnnona and one species fromArtabotrys, Cananga, Polyalthia, andRollinia were investigated in regard to 11 different allozyme loci. Preliminary studies on small population samples ofAnnona suggest genetic uniformity in three species and variability within and between populations in two other species. The allotetraploid origin ofA. glabra is clearly shown by its hybrid enzyme bands. The genetic distance between fiveAnnona species partly corresponds with their morphological relationships; onlyA. muricata appears more separated than is suggested by morphology. A comparison of the five genera demonstrates close relationship betweenAnnona andRollinia. Two enzyme loci are identical within all taxa investigated and possibly may serve as a genetic marker for the family.     

Intergeneric relationships ofLolium,Festuca, andVulpia (Poaceae) and their phylogeny

Morphological and seed protein analyses of 26 species of the generaLolium, Festuca andVulpia confirmed their close systematic affinities. Six inflorescence characters readily differentiatedFestuca fromLolium. Protein similarities betweenFestuca of sect.Bovinae and cross-pollinated species ofLolium, coupled with cytogenetic and crossability data, substantiate that they should be united into one genus.Vulpia had phenetic similarities with sect.Scariosae, Montanae andOvinae ofFestuca. Lolium, Festuca, andVulpia are most likely derived from a common ancestral form which was close toFestuca pratensis andLolium perenne.