Insights into the Witheringia solanacea (Solanaceae) Complex in Costa Rica. II. Insect Visitors and Pollination Biology of W. asterotricha and W. meiantha1

Isolating mechanisms are important in maintaining the taxonomic integrity of closely related sympatric taxa. A previous study found strong post‐zygotic isolating barriers between two species, Witheringia asterotricha and W. meiantha, of the W. solanacea (Solanaceae) species complex in Costa Rica. This study examines the presence of pre‐zygotic barriers between the two species at La Selva Biological Station in Costa Rica. Both species offer pollen and nectar as floral rewards and are visited primarily by solitary or semi‐social bees, some of which sonicate (“buzz”) the anthers to discharge pollen. No evidence was found for phenological differences in flowering time between W. asterotricha and W meiantha, but pre‐zygotic factors, such as ethological isolation and possibly fine‐scale ecological or geographic barriers, may be responsible for restricting gene flow between the two species.     

CYTOLOGICAL AND INTERFERTILITY RELATIONSHIPS OF ARACHIS SECTION ARACHIS

Twenty-nine recently introduced diploid (2n = 2x = 20) accessions of section Arachis plus an A. correntina (Burk) Krap. et Greg. nom. nud. control were hybridized to the diploid A-genome species A. duranensis Krap. et Greg. nom. nud. (ace. 7988), the diploid B-genome species A. batizocoi Krap. et Greg. (acc. 9484), and with two subspecies of the A-B genome (2n = 4x = 40) A. hypogaea cultivars NC 4 and Argentine. Most attempted crosses were successful and the resulting plants were vigorous. However, A. batizocoi × accession 30008 hybrids died as seedlings and A. batizocoi × accession 30017 produced only dwarf plants. The 710 diploid F₁s from A. batizocoi were generally sterile, while those from A. duranensis had fertility ranges from 5% to 84%. Meiotic chromosome relationships in diploid crosses were cytologically evaluated in 185 plants plus tester accessions. Most taxa in section Arachis have an A genome, only A. batizocoi accessions have a B genome, a D genome is represented by accessions 30091 and 30099, and two other genomic groups, represented by accessions 30011 and 30033, may be present in the section. Most cytological differentiation was found among species originally collected in southern and eastern Bolivia. On the other hand, species collected at the extremes of the distribution of section Arachis species (northern Argentina to north-central Brazil) were cytologically very similar. Evidence is presented for speciation in Arachis being associated with both genetic differentiation and with translocated chromosomes. All taxa in the section except the D-genome species are believed to be cross-compatible with A. hypogaea, so germplasm introgression from most Arachis species should be possible.     

Ribosomal DNA repeat unit polymorphism in 49 Vicia species

DNA restriction endonuclease fragment analysis was used to obtain new information on the genomic organization of Vicia ribosomal DNA (rDNA), more particularly among V. faba and its close relatives and the taxa within three (Narbonensis, Villosa, Sativa) species' complexes. Total genomic DNA of 90 accessions representing 49 Vicia species was restricted with 11 enzymes, and the restriction fragments were probed with three ribosomal clones. Twenty-eight repeat unit length classes were identified. The number of length classes (1–2) per accession did not correspond to the number of nucleolar organizing regions (NORs). The number of rRNA genes was independent of the 2C nuclear DNA amount present in the taxon. Each of the 90 accessions had 2 (rarely 1)-4 DraI sites. Those taxa with the same number of DraI sites generally could be distinguished from each other by different configurations. Probing of the DNA samples digested with tetranucleotide recognition restriction endonucleases emphasized differences between divergent spacer regions and enabled relative homologies between the coding regions to be established. Overall, rDNA restriction site variation among the species showed a good correlation with taxonomic classification. The rDNA analysis indicated evolutionary relatedness of the various taxa within the Narbonensis species complex. rDNA diversity within two other species complexes (Villosa, Sativa), on the other hand, was more extensive than expected. With few exceptions, data on the two complexes give evidence of taxon-specific divergences not seen with other approaches. The restriction site variability and repeat length heterogeneity in the rDNA repeat exhibited startling differences between V.faba and its close wild relatives included in the Narbonensis species complex. This analysis provides new evidence that none of the species within the complex can be considered to be putative allies of broad bean.     

CHLOROPLAST DNA VARIATION AND THE PHYLOGENY OF HORDEUM (POACEAE)

Restriction site variation in the chloroplast genome (cpDNA) was surveyed among 37 taxa or cytotypes (40 accessions) of the genus Hordeum. Seventeen restriction enzymes were employed, and a total of 491 restriction sites were assayed. Of these, 120 were variable among the taxa, including 70 synapomorphies. The level of sequence divergence (p) among species of Hordeum varied from 0.0 to 0.017, indicating that Hordeum possesses an about-average level of cpDNA diversity as compared to most other genera of flowering plants for which data are available. Wagner and polymorphism parsimony phytogenies were constructed from the restriction site data. These analyses divided the genus into several distinct groups; 1) American taxa; 2) diploid H. marinum; 3) Asian taxa; 4) H. vulgare-H. bulbosum; and 5) the H. murinum complex. Bootstrap-based confidence limits provided statistical support for the monophylesis of the latter three groups. The cpDNA data showed remarkably good congruence with previously published isoenzymatic, molecular, cytological, and crossing data.     

Conservation unit status inferred for plants by combining interspecific crosses and AFLP

Hybridization and introgression are common in plants and lead to morphological similarity between species and taxonomic confusion. This gene flow with closely related species can complicate efforts to determine whether an endangered taxon is evolutionarily distinctive and should be identified as a separate conservation unit. Potentilla delphinensis is a rare and threatened endemic species of the Southern French Alps. Two common related taxa (P. grandiflora and P. thuringiaca) are morphologically similar and occur in the same geographical locations. Thus, whether P. delphinensis represents a reliable conservation unit remained unclear. Our evaluation procedure based on a combination of molecular biology and interspecific crosses was used to define taxa within these plants. Plants were sampled from a total of 23 single and mixed localities for the three supposed taxa and were genotyped with 68 polymorphic Amplified Fragment Length Polymorphism (AFLP) loci. Fourty-one seedlings from interspecific crosses were obtained and genotyped. Amplified Fragment Length Polymorphism markers identified four genetically distinct units (P. delphinensis, P. grandiflora and two distinct groups of P. thuringiaca). All individuals of P. delphinensis formed a homogeneous and distinct taxon. This taxon was most probably an old allopolyploid from P. grandiflora and the related group of P. thuringiaca. Interspecific crosses gave low seed set and low germination rate. Furthermore, assignment test indicated that seedlings obtained from interspecific crosses were essentially apomictic rather than hybrids. These results suggest that a reproductive barrier exists between the different taxa. In conclusion, all results supported P. delphinensis as a true biological species and justified its conservation unit status. A surprising outcome of this work was the evidence of a potential new cryptic species. This study demonstrated the need to combine a molecular marker-based approach and pollination experiments for an accurate evaluation of plant taxa.     

Genetic characterization of Paspalum notatum accessions by AFLP markers

Paspalum notatum Flügge is a warm-season forage grass with sexual diploid and apomictic tetraploid races. Genetic improvement was achieved in out-breeding diploids. The acquisition of artificial sexual tetraploids has raised the possibility of performing crosses and plant improvement at the tetraploid level. The objective of our study was to obtain a genetic and cytoembryological characterization of a germplasm collection of P. notatum, including 31 accessions from seven countries of America and 11 experimentally obtained genotypes. Morphology of mature gametophytes was observed to assess the mode of reproduction of the accessions. A total of 1342 AFLP fragments were generated across the 42 genotypes and from two reference taxa: P. urvillei and P. procurrens. AFLP data were converted into a binary matrix and similarity relationships were established. The genetic distance among all the accessions showed a maximum value of 0.36. In addition, eleven AFLP fragments were observed exclusively in apomictic plants, which could be linked to genomic regions implicated in the control of apospory.     

A reexamination of infraspecific taxa of a wild potato,Solanum microdontum (Solanum sect.Petota:Solanaceae)

Current taxonomic interpretations ofSolanum microdontum Bitter partition the species into two or three infraspecific taxa, variously recognized as subspecies or varieties. The present study reexamines these taxa using morphological data from four individuals each of 69 accessions from most of the range of the species, planted in a common field plot. Our results show that the character states used to recognize infraspecific taxa inS. microdontum often vary within accessions and have no correlation with geography. We conclude that past hypotheses have used typological concepts and that infraspecific taxa are not warranted. This study questions other hypotheses of infraspecific taxa in sect.Petota.     

Collapse of species boundaries in the wild potatoSolanum brevicaule complex (Solanaceae, S. sect.Petota): Molecular data

TheSolanum brevicaule complex is a group of morphologically very similar wild and cultivated potato taxa (Solanum sect.Petota). This study uses single to low-copy nuclear RFLPs and RAPDs to investigate their species boundaries and relationships. Cladistic analyses of both data sets are largely concordant with each other and with a recently published phenetic analyses of the same accessions using morphology. All three data sets separate members of the complex into populations from Peru and immediately adjacent northwestern Bolivia, including most cultivated species accessions, and populations from northwestern Bolivia to Argentina. The molecular results suggest that the complex is paraphyletic as currently circumscribed. Many species of theS. brevicaule complex should be relegated to synonymy.     

Crossing relationships and chromosome numbers of Solanum section Solanum in Uganda

The ability of taxa to cross/hybridize is useful information for plant systematists and breeders. Crossability reflects reproductive isolation and the biological species concept stresses the need for reproductive isolation between species to maintain morphological distincness. For plant breeders knowledge on crossing ability facilitate selection of taxa for character improvement breeding. In this study, the crossing relationships and chromosome numbers within and among Ugandan species of Solanum sect. Solanum is studied by making 800 crosses involving 246 combinations. Less than half of these combinations were successful, producing F1 offspring. All studied accessions are self‐compatible and most accessions crossed readily with accessions of their own species. Interspecific crossings failed either to yield seeds, yielded F1 seeds that did not germinate, or resulted in F1s that did not have stainable pollen – implying a crossing barrier; or stainable pollen, but with chromosome numbers that indicated reproduction by apomixis. The results support the taxonomic treatment of Solanum based on classical, numerical and partly molecular evidences. The material studied represents eight Ugandan taxa: S. americanum, a diploid (2n = 2x = 24); five tetraploids (2n = 4x = 48) S. florulentum, S. memphiticum, S. tarderemotum, S. villosum ssp. villosum and S. villosum ssp. miniatum; and two hexaploids (2n = 6x = 72) S. scabrum subsp. scabrum and S. scabrum subsp. laevis. In addition to confirming the ploidy levels of the Ugandan accessions, the ploidy levels of S. florulentum, S. memphiticum and S. tarderemotum are reported for the first time. Non‐Ugandan material of Solanum sarrachoides was found to be diploid. Knowledge of the crossing behaviour and ploidy levels in Solanum will facilitate breeding for character improvement in these important species that are used commonly as food and/or medicine in eastern Africa.     

Cytotaxonomic observations in tropical Vaccinieae (Ericaceae)

Chromosome numbers are reported for 36 accessions representing 31 species from nine genera of the tribe Vaccinieae, family Ericaceae. The plants are tropical and come from Southeast Asia and Central and South America. The taxonomy of the tribe is outlined in these regions. Genera are often poorly defined and taxa were chosen to reflect the range of variation of the Vaccinieae. Most Southeast Asian Vaccinium species were diploid (2n= 24) as were those of Agapetes subgenus Agapetes (apart from the Himalayan A. flava), Agapetes scortechinii and Costera endertii. All other accessions were found to be polyploid. The correlation between polyploidy, geographical distribution and the possession of an ‘anatomical complex’ of the leaf and stem in Vaccinieae of New Guinea and the neotropics is discussed.     

Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L.

Restriction fragment length polymorphisms (RFLPs) were studied in fourteen accessions of CCDD genome allotetraploid wild rice species (Oryza latifolia, O. alta and O. grandiglumis). Fourteen nuclear RFLP markers previously mapped in AA genome-cultivated rice were used as probes. A phylogenetic tree, constructed by parsimony analysis based on RFLPs, grouped the accessions according to their geographic origin from Central or South America. Oryza alta, O. grandiglumis and one accession of O. latifolia grouped together as a subgroup, and our results suggested that the three taxa should be considered as populations of a single complex species. Duplicate loci, representing the two constituent genomes of the allotetraploid, were observed for most RFLP markers. By comparing RFLPs from the allotetraploids with those from a CC genome diploid wild species (O. officinalis), it was possible to detect RFLPs specific for both the CC and DD genomes of the allotetraploid. In inter-accession F2 populations, independent segregation of RFLP markers for CC and DD genomes was observed.     

Wild and cultivated barleys show similar affinities for mineral nitrogen

Summary The kinetics of net ammonium influx were very similar among several cultivars of barley (Hordeum vulgare) and several accessions of the wild taxa H.v. spontaneum and H. jubatum. For net nitrate influx, variation was greater among accessions than among species; accessions from warmer climates had faster rates than those from colder climates. These data indicate that domestication of barley has not reduced its affinity for mineral nitrogen.     

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.     

Hybrid crosses and the genetic basis of interspecific divergence in lifespan in Pristionchus nematodes

Characterizing the genetic basis of among‐species variation in lifespan is a major goal of evolutionary gerontology research, but the very feature that defines separate species – the inability to interbreed – makes achieving this goal impractical, if not impossible, for most taxa. Pristionchus nematodes provide an intriguing system for tackling this problem, as female lifespan varies among species that can be crossed to form viable (although infertile) hybrids. By conducting reciprocal crosses among three species – two dioecious (long‐lived Pristionchus exspectatus and short‐lived Pristionchus arcanus) and one androdioecious (short‐lived Pristionchus pacificus) – we found that female lifespan was long for all hybrids, consistent with the hypothesis that the relatively short lifespans seen for P. pacificus hermaphrodites and P. arcanus females are caused by independent, recessive alleles that are masked in hybrid genomes. Cross‐direction had a small effect on survivorship for crosses involving P. exspectatus, indicating that nuclear–mitochondrial interactions may also influence Pristionchus longevity. Our findings suggest that long lifespan in P. exspectatus reflects the realization of an ancestral potential for extended longevity in the P. pacificus species complex. This work demonstrates the utility of interspecific hybrids for ageing research and provides a foundation for future work on the genetic architecture of interspecific lifespan variation.     

Allozyme divergence among species ofWolffia (Lemnaceae)

The genusWolffia was surveyed electrophoretically at 14 allozyme loci. A total of 133 clones representing 10 of the 11 recognized species was examined. Genetic identities among most pairs of species are zero, with non-zero values ranging from 0.14 to 0.40.Wolffia angusta and the newly describedW. neglecta show the highest similarity, and the former species has an identity of 0.14 withW. australiana. The next highest similarity (0.34) occurs betweenW. globosa of Southeast Asia andW. cylindracea of southern Africa, which until recently, had generally been viewed as members of the same species. Other species showing some common alleles are members of a complex involvingW. arrhiza, W. columbiana, W. cylindracea, andW. globosa. WithinW. arrhiza, plants from South Africa and Europe are easily distinguished electrophoretically because each contains unique alleles at two loci. Strains from other parts of Africa vary at these loci and are not totally distinct from either the plants from South Africa or from Europe. Species ofWolffia are much more divergent at allozyme loci than the majority of congeners of flowering plants. This suggests that the species are quite old and that the difficulties in distinguishing taxa morphologically are the result of reduction rather than lack of divergence due to recent speciation. Because of the lack of shared alleles between the majority of species pairs inWolffia, enzyme electrophoresis provides limited resolution of species relationships in the genus.     

Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding approach

Sixty‐five accessions of the species‐rich freshwater red algal order Batrachospermales were characterized through DNA sequencing of two regions: the mitochondrial cox1 gene (664 bp), which is proposed as the DNA barcode for red algae, and the UPA (universal plastid amplicon) marker (370 bp), which has been recently identified as a universally amplifying region of the plastid genome. upgma phenograms of both markers were consistent in their species‐level relationships, although levels of sequence divergence were very different. Intraspecific variation of morphologically identified accessions for the cox1 gene ranged from 0 to 67 bp (divergences were highest for the two taxa with the greatest number of accessions; Batrachospermum helminthosum and Batrachospermum macrosporum); while in contrast, the more conserved universal plastid amplicon exhibited much lower intraspecific variation (generally 0–3 bp). Comparisons to previously published mitochondrial cox2–3 spacer sequences for B. helminthosum indicated that the cox1 gene and cox2–3 spacer were characterized by similar levels of sequence divergence, and phylogeographic patterns based on these two markers were consistent. The two taxa represented by the largest numbers of specimens (B. helminthosum and B. macrosporum) have cox1 intraspecific divergence values that are substantially higher than previously reported, but no morphological differences can be discerned at this time among the intraspecific groups revealed in the analyses. DNA barcode data, which are based on a short fragment of an organellar genome, need to be interpreted in conjunction with other taxonomic characters, and additional batrachospermalean taxa need to be analyzed in detail to be able to draw generalities regarding intraspecific variation in this order. Nevertheless, these analyses reveal a number of batrachospermalean taxa worthy of more detailed DNA barcode study, and it is predicted that such research will have a substantial effect on the taxonomy of species within the Batrachospermales in the future.     

Allozyme variation among the spontaneous species of Sorghum section Sorghum (Poaceae)

Summary A survey of allozyme variation among the spontaneous taxa of Sorghum section Sorghum was undertaken. Eight plants each of 90 accessions representing the diploid S. bicolor (ssp. arundinaceum and drummondii) and the tetraploids S. almum and S. halepense were analyzed for 17 enzyme systems encoded by 30 loci. Low levels of variation were found within and among accessions, although there was more variation than is typical of inbreeding species. We found an average of 3.2 alleles per locus in ssp. arundinaceum, with a mean expected heterozygosity for the accessions of 0.034 and total panmictic heterozygosity of 0.154. An analysis of the apportionment of genetic variation among accessions of ssp. arundinaceum indicated that 26% of the variation occurs within accessions and 74% among accessions. Cultivated sorghum contains far less allozymic variation than ssp. arundinaceum, its presumed progenitor. This is consistent with the prediction that cultivated sorghum experienced a loss of genetic variation during domestication. For the most part, cultivated sorghum contains a subset of the allozymes found in ssp. arundinaceum. Principal component analysis revealed continuous variation among the accessions and geographic regions, with accessions failing to segregate into discrete clusters. However, accessions of race virgatum of ssp. arundinaceum occupied one end of the continuum and were, in that sense, distinguished from the other accessions. Similarly, most accessions of S. halepense and S. almum occupied the central portion of the continuum. The allozymic data presented here are consistent with the hypothesized origin of S. halepense via autopolyploidy or segmental allopolyploidy.     

Variation of isozyme patterns among Arachis species

The genus Arachis contains a large number of species and undescribed taxa with patterns of genetic variation that are little understood. The objectives of this investigation were to estimate genetic diversity among species of Arachis by utilizing electrophoretic techniques and to establish the potential for use of isozymes as markers for germplasm introgression. One-hundred-and-thirteen accessions representing six of the seven sections of the genus were analyzed for isozyme variation of 17 enzymes. Section Rhizomatosae species were not included because they produce very few seeds. Seeds were macerated and the crude extract was used for starch-gel electrophoretic analyses. Although the cultivated species has few polymorphic isozymes, the diploid species are highly variable and two-to-six bands were observed for each isozyme among accessions. Because of the large number of isozyme differences between A. hypogaea and A. batizocoi (the presumed donor of the B genome), this species can no longer be considered as a progenitor of the cultivated peanut. Seed-to-seed polymorphisms within many accessions were also observed which indicate that germplasm should be maintained as bulk seed lots, representative of many individuals, or as lines from individual plants from original field collections. The area of greatest interspecific genetic diversity was in Mato Grosso, Brazil; however, the probability of finding unique alleles from those observed in A. hypogaea was greatest in north, north-central, south and southeast Brazil. The large number of polymorphic loci should be useful as genetic markers for interspecific hybridization studies.     

Rapid recent radiation of S-RNase lineages in Witheringia solanacea (Solanaceae)

Strong frequency-dependent selection as found in the self-incompatibility loci of flowering plants maintains allelic lineages for extremely long time scales, such that allelic genealogies can shed insight into long-term demographic patterns of species. Effective mutation rate, as well as demographic change such as population bottlenecks, can influence genealogical structure. In addition, loss of functionality at the self-incompatibility locus is likely to affect radiation rates. Partial sequences for 21 S-RNase alleles of the mid-elevation tropical species Witheringia solanacea were obtained in order to compare their substitution rates and genealogy with those of Witheringia maculata and two species in the closely related genus Physalis. Sequences for W. solanacea fell into the three clades within the Solanaceae already identified for the genus. Terminal branch lengths for W. solanacea, scaled to the total depth of its phylogeny, were intermediate between the unusually short terminal branches of W. maculata and those of the two Physalis species. In contrast to the Physalis species, where interspecific dN/dS for closely related alleles exceeded 1.0 to the same degree as did intraspecific dN/dS, in Witheringia only intraspecific comparisons showed an excess of nonsynonymous substitutions, suggesting postspeciation radiation of alleles. Alleles associated with lowered S-RNase production and self-compatibility showed extremely short terminal branches. In summary, it appears that rapid recent diversification of alleles characterizes the Witheringia lineages. In some cases, this rapid diversification can be attributed to relaxed constraints due to breakdown of self-incompatibility.