Potential taxonomic use of random amplified polymorphic DNA (RAPD): a case study in Brassica

Summary The potential use of RAPDs for taxonomic studies were investigated using Brassica, Sinapis and Raphanus taxa. Principal coordinate analysis of 284 RAPD bands revealed the classical U triangle relationship between diploid and amphidiploid Brassica taxa. Raphanus sativus and S. alba were distinct from the Brassica taxa. It appears that at least ten primers with approximately 100 total bands are needed to adequately portray these relationships. Cultivars of cabbage and cauliflower were separated by RAPDs. Analysis of RAPDs from individual plants of B. carinata cv. dodola resulted in 69 RAPDs, with 91.7% monomorphic and 8.3% polymorphic bands. RAPDs appear to be useful for taxonomic studies at levels ranging from populations to species and perhaps genera.     

Examination of evolutionary relationships in the Cape fossorial skink species complex (Acontinae: Acontias meleagris meleagris) reveals the presence of five cryptic lineages

Variable morphological characters have obscured genealogical relationships in the Cape fossorial skink Acontias meleagris meleagris species complex. Currently the species complex contains four dubious operational taxonomic units (A. meleagris meleagris, A. m. orientalis, A. percivali tasmani and the morph lineicauda) with poorly defined species boundaries. In the present study we examine the evolutionary relationships within the species complex by sampling 24 skink populations from the known geographical distribution in the Western and Eastern Cape provinces of South Africa, representing a total of 119 specimens. We used partial sequence data derived from two mitochondrial DNA genes, 16S rRNA and COI, and one nuclear DNA gene, intron β‐fibrinogen (β‐fibint 7), to examine evolutionary relationships. Phylogenetic relationships were determined using both Maximum Parsimony (MP) and Bayesian inference (BI) from the combined mtDNA, nDNA and the total evidence data. Additionally we employed Maximum likelihood (ML) analyses on the total evidence data that comprised ～1.5 kb. Topologies derived from the combined mtDNA analyses were congruent with the total evidence analyses (mtDNA + nDNA) and retrieved five major clades with strong statistical support inferred from bootstrapping and posterior probabilities. The five clades were genealogically and geographically exclusive, diagnostic at both the mtDNA and nDNA level and characterized by pronounced sequence divergence, with no shared haplotypes between clades. Collectively these results suggest the presence of five putative cryptic operational taxonomic units within the A. meleagris meleagris species complex. Constraining the traditionally recognized taxa always retrieved a statistically worse topology suggesting that considerable taxonomic revision is required. Our results indicate that traditional morphological characters need to be reassessed to define the five novel lineages in the A. meleagris meleagris species complex. The phylogeographic pattern for the fossorial skinks we retrieved was novel compared to phylogeographic studies for codistributed above ground living taxa. These results suggest that the abiotic and biotic factors that impact subterranean taxa may differ from supraterranean taxa.     

Molecular diversity of species of the Elymus trachycaulus species complex and their relationships to non-North American taxa

Species of the E. trachycaulus complex species are known for their morphological variability, but little is known about their genetic basis. The delimitation of taxa within the complex has been controversial and difficult. E. trachycaulus is predominantly self-pollinating, and lacks clear morphological boundaries between it and E. alaskanus. Another controversial taxonomic issue of E. trachycaulus is the relationships of this complex species to non-North American E. caninus. The objectives of this study were to examine genetic diversity and the systematic relationships among the species of the E. trachycaulus complex and their relationships with E. caninus, E. alaskanus and E. mutabilis. Random amplified polymorphic DNA method was used to study 35 accessions of E. trachycaulus complex and other Elymus species. Higher genetic variation was detected within species of E. trachycaulus complex. Eurasian accessions are as variable as the North American ones. Both UPGMA and NJ analyses did not show clearly separation among species of the E. trachycaulus complex. No clear association between geographic origin and genetic grouping among these species was found. Eurasian E. trachycaulus probably originated from multiple North American populations.     

Genetic and morphological variation in the Bulbophyllum exaltatum (Orchidaceae) complex occurring in the Brazilian “campos rupestres”: implications for taxonomy and biogeography

The Bulbophyllum exaltatum complex comprises 15 described taxa, and present a number of unresolved taxonomic questions, especially among populations found in the Brazilian campo rupestre vegetation. Allozymes were examined in 33 populations to determine the degree of genetic variability between them and their degree of differentiation to better define the taxa of this group. Additionally morphometric analyses were also performed on representatives of 24 populations. All of the populations examined demonstrated high levels of variability and none of the species formed distinct groups comprising all of the conspecific populations. However, the populations primarily grouped according to their regional occurrence, with a distinction between populations of the states of Minas Gerais and Bahia, which coincided with the geophysical disjunction of the mountain chains where they occur. It is probable that hybridization or incipient differentiation is contributing to the elevated genetic identity observed among the populations, generating a reticulated grouping pattern.     

Fungi from the roots of the common terrestrial orchid Gymnadenia conopsea

The fungal community associated with the terrestrial photosynthetic orchid Gymnadenia conopsea was characterized through PCR-amplification directly from root extracted DNA and cloning of the PCR products. Six populations in two geographically distinct regions in Germany were investigated. New ITS-primers amplifying a wide taxonomic range including Basidiomycetes and Ascomycetes revealed a high taxonomic and ecological diversity of fungal associates, including typical orchid mycorrhizas of the Tulasnellaceae and Ceratobasidiaceae as well as several ectomycorrhizal taxa of the Pezizales. The wide spectrum of potential mycorrhizal partners may contribute to this orchid's ability to colonize different habitat types with their characteristic microbial communities. The fungal community of G. conopsea showed a clear spatial structure. With 43 % shared taxa the species composition of the two regions showed only little overlap. Regardless of regions, populations were highly variable concerning taxon richness, varying between 5 and 14 taxa per population. The spatial structure and the continuous presence of mycorrhizal taxa on the one hand and the low specificity towards certain fungal taxa on the other hand suggest that the fungal community associated with G. conopsea is determined by multiple factors. In this context, germination as well as pronounced morphological and genetic differentiation within G. conopsea deserve attention as potential factors affecting the composition of the fungal community.     

Seed protein electrophoresis of some cultivated and wild species of <Emphasis Type="Italic">Chenopodium</Emphasis>

Seed protein profiles of 40 cultivated and wild taxa of Chenopodium have been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The relative similarity between various taxa, estimated by Jaccard’s similarity index and clustered in UPGMA dendrogram, is generally in accordance with taxonomic position, crossability relationships and other biochemical characters. Eight accessions of C. quinoa studied are clustered together and show genetic similarity with closely related C. bushianum and C. berlandieri subsp. nuttalliae. The taxa included under C. album complex are clustered in two groups which show that these taxa are a heterogenous assemblage and their taxonomic affinities need a reassessment. Other wild species studied are placed in the dendrogram more or less according to their taxonomic position.     

GENETIC POLYMORPHISM IN THE MORPHOLOGICALLY REDUCED DWARF MISTLETOES (ARCEUTHOBIUM,VISCACEAE): AN ELECTROPHORETIC STUDY

Arceuthobium is a well defined genus of obligate parasites of conifers. Assessment of taxonomic and phylogenetic relationships in this group is difficult owing to morphological reductions and complex host relationships. In the present study, genetic relationships within and among 19 taxa were examined using starch gel electrophoresis of triploid seed endosperm tissue. Allelic frequency data for eleven polymorphic loci were derived from analysis of 40 natural populations collected from the U.S. and Mexico. The genus showed remarkably high levels of genetic diversity: averaged across the 19 taxa, 66.7% of the loci were polymorphic with an average of 2.23 alleles per locus. This level of polymorphism is approximately double the average value reported for many dicotyledons and stands in contrast to the overall uniform morphology of these parasites. Unweighted pair group cluster analysis (UPGMA) of genetic similarity measures was conducted for all sampled populations and the results compared to a phenetic system of classification for the genus. Similarities between the two studies include the recognition of two subgenera based upon the segregation of the verticillately branched Arceuthobium americanum from the remainder of the taxa examined. Analysis of isozyme data supported a grouping of six taxa: A. vaginatum ssp. cryptopodum, A. vaginatum ssp. durangense, A. gillii, A. rubrum, A. divaricatum, and A. douglasii. The placement of the latter three taxa in other groups by phenetic criteria provides evidence for molecular divergence not seen using morphological features. The Campylopodum group of taxa comprised eleven members that were linked at similarity values of 80% or greater. Populations were not unambiguously grouped according to species as defined by the phenetic study or at similarity levels comparable to other well defined species in this study. This group is either not reproductively isolated or molecular differentiation is cryptic due to rapid adaptive radiation onto numerous host tree species.     

On the Occlusal Fit of Tribosphenic Molars: Are We Underestimating Species Diversity in the Mesozoic?

The complex occlusal fits between tribosphenic teeth are a rich source of information for taxonomic, phylogenetic, and evolutionary analysis. The degree of fit between upper and lower cheek teeth has been used to refer specimens to species-level taxa, but statistical data on occlusal fit in relation to taxonomic identity have been lacking. We used landmarks on upper and lower first molars of 20 bat populations representing 16 species to assess the degree of occlusal fit (1) between teeth from the same individual; (2) between teeth from different individuals belonging to the same populations; and (3) between teeth belonging to different populations. We found that the fit of teeth belonging to different populations was significantly worse than between those of the same population and that the degree of misfit increased linearly with time since common ancestry, albeit with substantial variance. We used our comparisons to assess the species-level diversity within Batodon, the smallest known placental mammal from the Cretaceous. Our data suggest, with caveats, that instead of belonging to a single species, the specimens assigned to Batodon represent at least two species as different as those belonging to different genera or families of living bats.     

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.     

Isozyme differentiation in the endemic genusArgyranthemum (Asteraceae: Anthemideae) in the Macaronesian Islands

The genusArgyranthemum (Asteraceae: Anthemideae) comprises 38 taxa and is restricted to the archipelagos of the Canaries, Selvagens, Madeira, and Desertas in the Macaronesian biogeographic region. An electrophoretic study, including 17 enzyme loci and at least one population of each of the described taxa, was carried out. High identity (low distance) values between taxa (mean of 0.893) were obtained despite the old age of the islands, their close proximity to the African continent, and the fact thatArgyranthemum is the most species-rich and variable genus in Macaronesia. These results suggest that the genus is monophyletic and that it has evolved very rapidly in these islands. There is little correspondence between taxonomy and neighbor-joining analysis based on Roger's genetic distances, but in several instances populations from the same islands cluster together despite being from different species or even different sections. It is suggested that repeated genetic bottlenecks associated with the founding of new populations during radiation of the genus resulted in lineage sorting of ancestral allozyme polymorphisms. Because every population has a high average identity with all other populations, lineage sorting could result in populations of different taxa being slightly more similar than populations of the same taxon. Gene flow between different species on the same island could account for some populations clustering by island of origin rather than taxonomic disposition. Average allozyme diversity within populations (0.098) is 50% higher than the mean total diversity for species endemic to oceanic islands.     

A phylogenetic analysis of Pisum based on morphological characters, and allozyme and RAPD markers

Cladistic analyses of 17 wild and cultivated pea taxa were performed using morphological characters, and allozyme and RAPD (random amplified polymorphic DNA) markers. Both branch-and-bound and bootstrap searches produced cladograms that confirmed the close relationships among the wild species and cultivars of Pisum proposed by a variety of systematic studies. Intraspecific rankings were supported for northern P. humile, southern P. humile, P. elatius and P. sativum, which together comprise a single-species complex. P. fulvum, while clearly the most divergent of the pea taxa, could also be assigned to the same species complex without violating the hierarchial logic of the cladogram. Its inclusion or exclusion depends on whether the level of interfertility it displays with other pea taxa or its overall morphological and chromosomal distinction are emphasized. As suggested by previous studies, northern P. humile was the most likely sister taxon to cultivated P. sativum; although, rigorous phylogenetic evaluation revealed a close genealogical affinity among P. elatius, northern P. humile and P. sativum. Despite their limited number, the 16 morphological characters and allozyme markers used precisely organized the pea taxa into established taxonomic groupings, perhaps in part reflecting the role morphology has played historically in pea classification. The RAPD data also generally supported these same groupings and provided additional information regarding the relationships among the taxa. Given that RAPDs are relatively quick and easy to use, are refractory to many environmental influences, can be generated in large numbers, and can complement traditional characters that may be limited in availability, they provide a valuable new resource for phylogenetic studies.     

A biosystematic study of theOryza meyeriana complex (Poaceae)

To determine the taxonomic status and to clarify the biosystematic relationships of wild rice species, i.e.,Oryza granulata andO. meyeriana in theO. meyeriana complex (Poaceae), a series of intraspecific and interspecific hybridization experiments, genomic analyses through meiotic pairing, and morphological studies were conducted. Hybridization results demonstrate comparatively high crossability with about 34–39% of seed set, and very limited reproductive isolation betweenO. granulata andO. meyeriana. Chromosome pairing in the parental species and their F₁ hybrids indicate very high genomic affinity between the two taxa. Morphological analyses of the parental accessions and their intra- and interspecific hybrids show a large variation among the samples included, but no appreciable grouping was observed. The traditionally used character, i.e., spikelet length, does not contribute significantly to the grouping of the samples included. Based on the facts that the two taxa have negligible reproductive barriers and share almost identical genomes, the authors believe thatO. granulata andO. meyeriana do not warrant separate taxonomic status, and recommend that they be combined asOryza granulata.     

Inter- and intraspecific morphometric variability in Juniperus L. seeds (Cupressaceae)

In this study, a statistical classifier for Mediterranean taxa of Juniperus, based on 98 seed morphometric parameters, was tested at interspecific, specific and intraspecific levels. Ripe cones of 10 taxa were collected in different regions of the Mediterranean Basin to compare and discuss their taxonomic treatments according to two different sources. High percentages of correct identification were reached for both taxonomic treatments at the specific and intraspecific level and from the comparison among taxa of the J. oxycedrus, J. communis and J. phoenicea complexes. Moreover, ripe cones of J. macrocarpa were collected from four Sardinian populations, in two seasons, and from plant and soil, in order to analyse inter-population, seasonal and source variability in seed morphology. This statistical classifier discriminated J. macrocarpa seeds collected in spring more accurately than those collected in autumn, but it failed to distinguish between the seeds collected from plants and soil, or between those collected from different populations of the same geographic region.     

Genetic differentiation of metallicolous and non-metallicolous Armeria maritima (Mill.) Willd. taxa (Plumbaginaceae) in Central Europe

This study investigates the genetic differentiation within the Central European Armeria maritima (Mill.) Willd. complex with special reference to the metallicolous populations using AFLP markers. Our sampling comprised all metallicolous (ssp. halleri, hornburgensis, bottendorfensis, eifeliaca, calaminaria), and non-metallicolous taxa (ssp. maritima, elongata, alpina). Geographical and genetic distances between populations were moderately positively correlated. Genetic variability of metallicolous and non-metallicolous populations was not significantly different. Lowland populations were clearly differentiated from the alpine populations. Within the lowland group metallicolous and non-metallicolous populations were not genetically differentiated. All lowland populations show a regional differentiation and close relationships to ssp. elongata. Thus, the metallicolous taxa should not be maintained as subspecies. Likewise, their treatment as varieties of a ssp. halleri s.l. is critical because this taxon cannot be consistently characterized throughout its geographical range and may be an artefact itself. If a taxonomical recognition should be considered necessary it is advisable to treat the microendemics as varieties of ssp. elongata.     

Taxonomical study ofAster leiophyllus complex (Compositae) in Kanto district,Japan, with special reference to ploidy level

TheAster leiophyllus complex is complicated taxonomically and includes many related taxa representing polyploid series. It has been reported that this group is diversified throughout the Kanto district, central Japan, and there are three taxa endemic to the Kanto district. However, as neither morphological variation range nor distribution pattern is sufficiently investigated, taxonomic confusion sometimes occurs. In this study, morphological variation was examined in relation to the ploidy level, and taxonomic consideration was given. As a result, four morphologically recognized taxa occur in the Kanto district;A. leiophyllus (2x, 6x),A. leiophyllus var.sawadanus (2x, 3x, 4x),A. semiamplexicaulis (2x), andA. sugimotoi (4x, 5x). The distribution ranges ofA. leiophyllus var.sawadanus, A. semiamplexicaulis andA. sugimotoi are small, whileA. leiophyllus is distributed over most of the Kanto district. Morphological and cytological observations indicate that one of the reasons of the taxonomic confusion is the presence of presumed hybridization betweenA. leiophyllus and other taxa.     

Use of chloroplast DNA polymorphisms for the phylogenetic study of seven Phaseolus taxa including P. vulgaris and P. coccineus

The genetic variability of seven Phaseolus taxa has been evaluated on the basis of molecular data and the results have used to clarify the phyletic relationships between several taxa of the P. coccineus L. complex. Chloroplast DNA (cpDNA) from 33 populations was digested with six restriction endonucleases, revealing some polymorphisms that made it possible to divide most of the taxa into two main groups: the subspecies of P. coccineus on the one hand, and P. vulgaris L., P. polyanthus Greenman and P. costaricensis (Freytag and Debouck) on the other hand. P. polyanthus is closer to P. vulgaris than the other taxa of the second group and should be considered as a separate species. The position of the wild species P. costaricensis is intermediate between P. coccineus and P. polyanthus. P. glabellus shows sufficient polymorphisms at the cpDNA level to be recognized as a separate species, as previously suggested from total seed-protein electrophoretic studies. These results favour the hypothesis of a common phylogeny for P. vulgaris, P. polyanthus, P. costaricensis and P. coccineus from a single wild ancestor. Although cpDNA is generally known to be uniform at the intraspecific level, some additional polymorphisms were also detected within P. vulgaris, P. polyanthus and P. coccineus. Further studies are required to understand the significance of the latter.     

Molecular phylogeny of three groups of acacias (Acacia subgenus Phyllodineae) in arid Australia based on the internal and external transcribed spacer regions of nrDNA

The phylogeny of three groups of arid Australian acacias ‐ the Acacia victoriae, A. murrayana and A. pyrifolia groups ‐ was constructed based on parsimony analysis of sequence data from the internal and external transcribed spacers (ITS and ETS) of the nuclear ribosomal DNA. Forty ingroup taxa were sequenced, including multiple accessions for some taxa and two species (A platycarpa and A. longispinea) that had been identified in other analyses as relatives of these acacias. Acacia anthochaera was used as the functional outgroup.

The ITS and ETS regions proved to be sufficiently variable to resolve relationships at both the specific and intra‐specific level. Two main clades were resolved. One clade confirmed the monophyly of the Acacia murrayana group, and relationships of species were strongly supported. All taxa in this clade have a similar pattern of seedling leaf development. In the second clade, the A. pyrifolia group is nested within the A. victoriae group and all taxa have spinose stipules. Acacia platycarpa and A. longispinea are related to this clade. Phyllode nerve number (uninerved or plurinerved) proved to be homoplasious.

Acacia victoriae is a widespread and very variable species. The molecular data identified two major groups: a group of populations occurring across northern Australia and a group of populations from the Western, Central and Eastern deserts. Further analysis of population variation is required to assess the taxonomic status of various forms in this species complex.

The geographic distributions of sister taxa suggest predominantly allopatric speciation. The degree of molecular divergence and position of the clades within subgenus Phyllodineae suggest that the lineages are not of recent origin, but have a history that relates to increased aridity in the Australian Eremean region during the Cenozoic.     

Is Oryza malampuzhaensis Krish. et Chand. (Poaceae) a valid species? Evidence from morphological and molecular analyses

Phylogenetic relationship between O. malampuzhaensis Krish. et Chand. (2n = 4x = 48; Poaceae, Oryzeae), a South Indian endemic wild rice with a disputed taxonomic identity, and eight other species belonging to the O. officinalis complex of the genus Oryza was examined using 62 morphological characters and 445 random amplified polymorphic DNA (RAPD) markers. Multivariate and cluster analyses using both the data sets clearly separated all accessions of O. malampuzhaensis into a distinct group. Genetic distances between O. malampuzhaensis and other species in O. officinalis complex were comparable with the distance between any other two taxa with species rank in this complex. Case-by-case taxonomic treatment of O. malampuzhaensis in relation to other species examined is presented. A taxonomic key for the discrimination of O. malampuzhaensis from other species in the O. officinalis complex has been constructed. Based on the present results, we strongly argue to restore the species rank to O. malampuzhaensis, as originally proposed by Krishnaswamy and Chandrasekharan (1958).     

Relationships between length and weight of freshwater macroinvertebrates in Japan

Relationships between weight (W; dry weight) and length (L; head capsule width, total body length or head carapace length) were examined in 31 Japanese freshwater macroinvertebrate taxa, using the form W = aL ^b . The relationships were expressed as data of the lowest taxonomic level and data of higher taxonomic levels. The length–weight relationships obtained in this study were similar to those obtained in North America and Europe at the lowest taxonomic level, whereas they could be different from those obtained in North America and Europe at the higher taxonomic levels. We suggest that researchers should make their own regressions for a target taxon or use the regression for the same taxon as possible lower taxonomic level in the local area.