The importance of species identity in the biocontrol process: identifying the subspecies of Acacia nilotica (Leguminosae: Mimosoideae) by genetic distance and the implications for biological control

Aims A molecular genetic distance study has been used in an initial survey to identify subspecies and genotypes of the weed Acacia nilotica in Australia, information needed to find suitable biocontrol agents. We use patterns of DNA sequence variation (in two DNA fragments) from each of the nine described subspecies of Acacia nilotica (L.) Delile (Leguminosae: Mimosoideae) that is to determine their genetic similarity, to verify if the Australian populations are A. nilotica ssp. indica (Benth.) Brenan, and to establish if any other subspecies are present in Australia. Location Australia and southern Africa through the Arabian peninsular to the Indo‐Pakistan subcontinent. Methods Representative specimens from the global distribution of the nine A. nilotica subspecies were sourced primarily from herbaria sheet specimens where available, and secondarily from field collections. These specimens together with related outgroups from Mimosoideae were genetically analysed using the DNA fragments trnL and internal transcribed spacer one (ITS1). We calculated a similarity index as set out in paup * using upgma (Unweighted Pair‐Group Method Arithmetic average) methods to cluster taxa to produce a genetic distance phenogram. Results Sequence results from ITS1 and trnL DNA fragments identified seven of the described subspecies of A. nilotica. Acacia nilotica ssp. cupressiformis (J. Stewart) Ali & Faruqi and A. nilotica ssp. adstringens (Schumach. & Thonn.) Roberty were not found to be genotypically distinct from A. nilotica ssp. indica and A. nilotica ssp. nilotica, respectively, based on the two DNA fragments. Subspecific ITS1 genotypes were geographically distributed similarly to previous reports that were based on morphology, with the exception that the hemispherica ITS1 genotype also occurred in Somalia. We confirmed that the Australian A. nilotica populations are mostly comprised of subspecies indica, but in addition, some individuals were found to be genetically identical to an unidentified Pakistan genotype not previously reported as occurring in Australia. Main conclusions Australian A. nilotica populations originated from India and Pakistan and we recommend further analysis to determine the complete genetic diversity profile and origins of the Australian populations. We highlight the importance of determining any hybridization between Australian populations of A. nilotica and native subgenus Acacia species. This study demonstrates the importance of genotyping weed species targeted for biocontrol and/or listed host specificity test species that may be easily misidentified. Biocontrol practitioners can justify genetic studies by considering the costs should a project fail through misidentification.     

RFLP analysis of the wild potato species,Solanum acaule Bitter (Solanum sect. Petota)

Summary Intraspecific variation of a wild potato species, Solanum acaule Bitt., was analyzed by RFLPs of genomic DNA. One hundred and five accessions were selected throughout the distribution area, including all subspecies, i.e., ssp. albicans (hexaploid), ssp. punae (tetraploid), ssp. acaule (tetraploid) and ssp. aemulans (tetraploid). Twenty-seven low-copy DNA clones (probes) were Southern hybridized with EcoRI, EcoRV, HindIII, and XbaI digests of total DNA of all accessions. In total, 238 RFLPs were detected from 94 enzyme x probe combinations. Among them, 49 RFLPs were specific to ssp. albicans, suggesting that the additional third genome is distinct from its two other genomes. RFLPs between and within subspecies were analyzed by principal component analysis. DNA similarities between subspecies coincided with a former taxonomic treatment in the sense that ssp. albicans is the most distantly related to ssp. acaule and ssp. aemulans is distantly related. Subspecies acaule and ssp. punae were indistinguishable. In addition, RFLPs could be used to distinguish groups within subspecies. Subspecies aemulans, confined to Argentina, was divided into two populations, one from the province of La Rioja and the other from the province of Jujuy. In ssp. acaule, some accessions from the southernmost distribution area were clearly distinguishable, while the others varied continuously, showing a geographical cline from Peru to Argentina.Reference to a specific brand or firm name does not constitute endorsement by the US Department of Agriculture over others of similar nature not mentioned     

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

A revision of the Nasa ranunculifolia group (Nasa ser. Grandiflorae pro parte,Loasaceae)

The monophyletic Nasa ranunculifolia group (Nasa ser. Grandiflorae pro parte) is revised on the basis of extensive field studies and a revision of copious herbarium material. All species of this group are from the high Andes of Peru and Ecuador (mostly 3000–4300 m). The overall degree of morphological divergence is much lower than in other groups of Nasa and many of the various local ‘races’ are best recognized as ecogeographically isolated subspecies. Thus, six species and 13 subspecies are recognized on the basis of differences in leaf and floral morphology, growth habit, life history and distribution. Nasa ranunculifolia, N. macrantha, N. cymbopetala and N. macrorrhiza are united as subspecies under N. ranunculifolia. An additional five subspecies of N. ranunculifolia are newly described (ssp. pamparomasii, ssp. guzmangoensis, ssp. bolivarensis, ssp. patazensis and ssp. huanucoensis). Nasa rugosa is subdivided into four subspecies, three of them new (ssp. llaqtacochaensis, ssp. gracilipes and ssp. pygmaea). Nasa tulipadiaboli sp. nov. and N. basilica sp. nov. are described as new species. Eight taxa (seven subspecies and one species) are endemic to the Amotape–Huancabamba Zone, and seven of them (six subspecies and one species) are new to science. Only a single species ranges into the Amotape–Huancabamba Zone from the south, underscoring the high levels of endemism (and taxonomic novelty) in this area. Illustrations, distribution maps, a key and diagnoses are given for all taxa recognized. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167 , 47–93.     

Genetic variability in Plantago species in relation to their ecology

Summary Genetic variation in leaf and inflorescence morphology and in generative development within the species Plantago major has been analysed by means of crosses between members of two different subspecies. The variable characters chosen are supposed to be important for determining the ecological differences between the subspecies and other ecotypes. The analyses of F₂'s indicated that a substantial number of loci controlling the above mentioned characters are situated near the Pgm-1 locus, forming a gene complex. This gene complex can exist in at least three different forms in ssp. pleiosperma, ssp. major lawn type and ssp. major roadside type, respectively. In addition, some important factors for ecotypic differentiation are situated in the neighbourhood of the Got-1 locus and in a linkage group containing three other allozyme loci. These linkages between allozyme loci and fitness-affecting loci can explain the restriction of some enzyme alleles to a particular subspecies.Grassland Species Research Group Publication No. 50     

Phylogeography of silver‐studded blue,Plebejus subsolanus (Lepidoptera: Lycaenidae), in the central mountainous regions of Japan

The silver‐studded blue, Plebejus subsolanus, is widely distributed in the Russian Altai mountains, northeastern China, the Korean Peninsula, and the Japanese archipelago. In Japan, the species is distributed across wide elevation ranges from the lowlands of Hokkaido to the subalpine zone of Honshu. Current subspecies classification in Japan is as follows: ssp. iburiensis, occurring in lowland grasslands in Hokkaido; ssp. yaginus in lower mountain grasslands in Honshu; and ssp. yarigadakeanus in higher mountain grasslands in Honshu. The habitat of this species has been markedly reduced due to recent habitat destruction and land‐use changes. Here, we undertook phylogeographic analyses of two subspecies, ssp. yaginus and yarigadakeanus in the central mountainous regions of Japan, based on two mitochondrial gene sequences, in order to collect information for establishing effective conservation strategies. From 57 samples from the four mountain ranges, we obtained a haplotype network comprised of 12 haplotypes. Because of the haplotype network topology, the geographic distribution of haplotypes and the correspondence of haplotype divergence to subspecies taxonomy, we provisionally divided the haplotypes into three haplogroups: YR1 and YR2, which comprised ssp. yarigadakeanus, and YG, which comprised ssp. yaginus. Mitochondrial DNA genetic differentiation generally agreed with morphological subspecies classification. The haplotype network suggested that ssp. yarigadakeanus populations had multiple origins, and the subspecies character of “bright blue of the male's wings” was assumed to have evolved independently in each subalpine meadow. We found that P. subsolanus was genetically differentiated depending upon the elevation at each mountain region, suggesting that each haplogroup should be a conservation unit.     

A chemical perspective on the evolution of variation in Eucalyptus globulus

It is becoming increasingly easy to generate genotypic data but much harder to gather an equivalent amount of phenotypic information, particularly for chemical traits. In this study of Eucalyptus globulus ssp. globulus, we measured about 60 chemical leaf traits of trees growing in a common garden to address the following questions: (1) how much variation is there between geographic regions, populations within regions and within populations? (2) How do chemical traits vary over the species’ geographic range? (3) If so, does E. globulus ssp. globulus exhibit distinct chemotypes – plants that are morphologically similar but which differ chemically? (4) Are the affinities between E. globulus ssp. globulus and closely related subspecies apparent in the chemical variation? Variation among trees within populations contributed most variation in leaf chemistry followed by variation between geographic regions. For many traits, variation among populations within proposed races and variation among proposed races within geographic regions explained little of the total variation. There was a cline in the concentration of secondary chemicals with the lowest concentrations in Tasmanian populations and the highest in those from eastern Victoria, with intermediate concentrations in populations from Bass Strait Islands. We identified three chemotypes, characterised by specific terpenes and formylated phloroglucinol compounds. The frequency of occurrence of these chemotypes showed a geographic pattern also, with “chemotype 1” predominating in Tasmania, while “chemotypes 2 and 3” occurred at highest frequency in eastern Victoria. We suggest that the chemotypes reflect introgression between E. globulus ssp. globulus and the three closely related subspecies – E. globulus ssp. bicostata, E. globulus ssp. maidenii and E. globulus ssp. pseudoglobulus. Although the formation of land-bridges with fluctuating sea levels has no doubt shaped the evolutionary history of all four subspecies, we propose that the migratory swift parrot (Lathamus discolor), an important pollinator and a species closely associated with E. globulus, has helped shape the evolution of the four tree subspecies.     

Analysis of the genetic diversity and relationships among and within species of Hippophae (Elaeagnaceae) based on RAPD markers

RAPD markers were used to assess the genetic diversity and inter- and intra-specific relationships of the genus Hippophae L. and to study the correlation between genetic distances and geographic distances among populations of H. rhamnoides ssp. sinensis. The results analyzed by the percentage of polymorphic loci and Shannon information index indicated that a high level of genetic diversity existed both among and within species of the genus Hippophae. In the UPGMA dendrogram, the species or subspecies were clustered into two main groups but not strictly grouped according to sect. Hippophae and sect. Gyantsensis Lian. The multiple regression analysis and Mantel test both indicated a significant correlation between genetic distance and altitude distance among populations of H. rhamnoides ssp. sinensis, and the cluster analysis suggested that the genetic variation among populations of H. rhamnoides ssp. sinensis was linked to their monophyletic origin. Moreover, some degree of genetic differentiation was found among samples collected at different times.     

Deep sequencing of amplicons reveals widespread intraspecific hybridization and multiple origins of polyploidy in big sagebrush (Artemisia tridentata; Asteraceae)

• Premise of the study: Hybridization has played an important role in the evolution and ecological adaptation of diploid and polyploid plants. Artemisia tridentata (Asteraceae) tetraploids are extremely widespread and of great ecological importance. These tetraploids are often taxonomically identified as A. tridentata subsp. wyomingensis or as autotetraploids of diploid subspecies tridentata and vaseyana. Few details are available as to how these tetraploids are formed or how they are related to diploid subspecies. • Methods: We used amplicon sequencing to assess phylogenetic relationships among three recognized subspecies: tridentata, vaseyana, and wyomingensis. DNA sequence data from putative genes were pyrosequenced and assembled from 329 samples. Nucleotide diversity and putative haplotypes were estimated from the high-read coverage. Phylogenies were constructed from Bayesian coalescence and neighbor-net network analyses. • Key results: Analyses support distinct diploid subspecies of tridentata and vaseyana in spite of known hybridization in ecotones. Nucleotide diversity estimates of populations compared to the total diversity indicate the relationships are predominately driven by a small proportion of the amplicons. Tetraploids, including subspecies wyomingensis, are polyphyletic occurring within and between diploid subspecies groups. • Conclusions: Artemisia tridentata is a species comprising phylogenetically distinct diploid progenitors and a tetraploid complex with varying degrees of phylogenetic and morphological affinities to the diploid subspecies. These analyses suggest tetraploids are formed locally or regionally from diploid tridentata and vaseyana populations via autotetraploidy, followed by introgression between tetraploid groups. Understanding the phylogenetic vs. ecological relationships of A. tridentata subspecies will have bearing on how to restore these desert ecosystems.     

Estimates of gene flow in forest trees

Qualitative and quantitative estimates of gene flow were obtained for fourteen gymnosperm and seven angiosperm forest tree species. High levels of gene flow were prevalent among gymnosperms while these levels varied from high to low among angiosperms. In both groups, species with greater pollen dispersal abilities appear to maintain high levels of gene flow. A detailed analysis of population structure in relation to gene flow was carried out on a gymnosperm species (Pinus rigida) and two angiosperm subspecies (Eucalyptus caesia ssp. caesia and ssp. magna). The results suggested that populations of many species may be concatenated systems bound by gene flow, and the overall levels of gene flow may be influenced by either single or clusters of populations. Different levels of gene flow was found between two closely related species of E. caesia growing under similar ecological conditions, suggesting a plausible link between pollinator behaviour and pollen flow.     

Chromosome evolution in the Gibasis linearis alliance (Commelinaceae)

Two related species of Gibasis, G. venustula (x = 6) and G. speciosa (x = 5) have been shown to be differentiated by a Robertsonian fusion. Meiotic analysis of the F₁ hybrids has revealed further chromosome differentiation of the parent species, involving several interchanges and inversions. These rearrangements tend to be concentrated in the Robertsonian group, and give rise to unusual meiotic configurations. The sets of the two species are nonetheless highly homologous as shown by high chiasma frequency and as much as 65% stainable pollen in the F₁. Models are proposed suggesting the possible evolutionary pathways of this karyotypic differentiation. The results are discussed in terms of chromosome evolution and its consequences for these species.     

Differences in nitrate uptake of species from habitats rich or poor in nitrogen when grown at low nitrate concentrations,using a new growth technique

Summary A method is described for culturing plants at extremely low nutrient concentrations. Using a Braun infusion pump, a fixed amount of nitrate or ammonium was supplied continuously to plants growing in a culture vessel at a rate limiting the uptake of the plants. At a very low nitrogen concentration an equilibrium was established where uptake rate of the plants is equal to the rate of supply by the infusion pump. The nitrogen concentrations reached appeared to be in the order of 1 μM. The method compared the nitrate uptake byHypochaeris radicata L.ssp.radicata, H. radicata ssp.ericetorum Van Soest andUrtica dioica L. and ammonium uptake byH. radicata ssp.radicata andH. radicata ssp.ericetorum. Plants were cultivated in monocultures or in mixed cultures (two species per culture vessel). For the mixed cultures competition for nitrate (or ammonium) between the species was maintained for long periods. The capacities of the uptake systems of two subspecies ofH. radicata from places different in nitrogen supply and pH were adapted equally well to both low nitrate and low ammonium concentrations. Apparently factors other than nitrogen uptake play a part in the distribution of the subspecies. The capacity of the uptake system ofU. dioica, a nitrophilous species, was lower than that ofH. radicata ssp.radicata, a species from places poorer in nitrogen. This difference is related to the different distribution of the two species in the field. The present results are compared with those of previous experiments where K_m and V_max were measured and the significance of both parameters is discussed.     

MITOCHONDRIAL DNA HOMOGENEITY IN THE PHENOTYPICALLY DIVERSE REDPOLL FINCH COMPLEX (AVES: CARDUELINAE: CARDUELIS FLAMMEA-HORNEMANNI)

Breeding redpoll finches (Aves: Carduelinae) show extensive plumage and size variability and, in many cases, a plumage polymorphism that is not related to age or sex. This has been ascribed to extreme phenotypic variation within a single taxon or to moderate variability within distinct taxa coupled with hybridization. The predominant view favors the recognition of two largely sympatric species: Carduelis flammea, comprised of four well-marked subspecies—flammea, cabaret, islandica, and rostrata; and C. hornemanni, comprised of two subspecies—hornemanni and exilipes. We studied representative samples of these putative subspecies (except islandica) for variation in mitochondrial DNA (mtDNA). Using 20 informative restriction enzymes that recognized 124 sites (642 base pairs [bp] of sequence or ≈ 3.7% of the molecule), we identified 17 RFLP haplotypes in the 31 individuals surveyed. The haplotypes formed a simple phylogenetic network with most clones diverging by a single site difference from a common haplotype found in almost half of the individuals. Within populations and taxa, levels of mtDNA diversity were similar to those observed in other avian species. The pattern of mtDNA divergence among populations was statistically unrelated to their geographic or traditional taxonomic relationships, and the estimated distance between the two traditionally recognized species was very small relative to those typically observed among avian sister species.     

Genetic structure and differentiation of Plantago major reveals a pair of sympatric sister species

Seeds of the widespread weed Plantago major were collected from 10 European countries, as well as Trinidad and North America. The seed collections were from populations of two taxa which are ecologically rather than geographically separated and formally recognized as the subspecies Plantago major ssp. major and P.m. ssp. intermedia (also called P.m. ssp. pleiosperma). Eight polymorphic allozyme loci and 73 random-primed DNA fragments were scored, as well as 11 morphological characters. Complete concordance between morphological traits and genetic data provides evidence that these two taxa, although very similar, are distinct species. They are both widespread, they are broadly sympatric and capable of interbreeding. However, slight morphological and ecological differences coincide with genetic clustering of populations from widely separated locations. In addition, P. major and P. intermedia differ in their population structure: P. intermedia has greater genetic diversity among populations and less genetic variance within populations than P. major. We suggest that differences between the two species in their levels of selfing may explain the distinctive genetic structure of each species. We hypothesize a link between selfing rate and lifespan of the two taxa. P. major is characterized by lower genetic variation among populations, a higher rate of outcrossing, longer lifespan and production of fewer seeds per seed capsule. P. intermedia is more highly structured with much differentiation among populations, a higher rate of inbreeding and it often grows as an annual.     

Races of Phytophthora megasperma f.sp. glycinea (Phytophthora Rot) Affecting Soybeans in North Alabama

Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin (Pmg) was isolated for the first time from the northern counties of Alabama (MORGAN and MADISON). TWO predominant Pmg races, R₂ and R₁₁, were identified by the reactions of standard soybean differential cultivars. Other soil-borne pathogens isolated from the soil and suspected Pmg-affected root samples in Morgan, Adadison, and Limestone counties were Pytbium spp., Fusarium ssp., Sclerotium rolfsii, and Rbizoctonia solani. Further study is in progress to obtain a proper assessment of Pmg races in Alabama.     

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.     

Patterns of allozyme variation in western Eurasian Fagus

Fagus sylvatica L. is one of the most widespread broad‐leaved trees in the temperate forests of the northern hemisphere. In addition to two subspecies, F. sylvatica ssp. sylvatica in Europe and F. sylvatica ssp. orientalis in south‐western Asia, two further taxa were described: F. moesiaca (Maly) Czeczott in the south‐western Balkans and F. taurica Popl. in Crimea. The opinions about the number and ranks of taxa within this complex are highly controversial. To assess the degree of genetic differentiation among them, and to reveal geographical patterns of genetic diversity and their relationships to history and biogeography of beech populations, genetic variation at 12 allozyme loci was studied in 279 populations in western Eurasia. A Bayesian analysis of population structure revealed the existence of two clusters, which fairly well coincided with F. sylvatica ssp. sylvatica and F. sylvatica ssp. orientalis, whereby the populations from the south‐western Balkans and Crimea contained a mixture of these two gene pools. On the other hand, a neighbour‐joining tree based on pairwise F_ST failed to separate the subspecies into well‐defined distinct clades. Populations of F. sylvatica ssp. orientalis proved to be incomparably more differentiated than ssp. sylvatica (F_ST = 0.157 and 0.032, respectively). Asian populations also showed higher levels of allelic richness both on population and taxon levels than the European ones (the number of alleles after rarefaction was 3.40 and 4.27 in F. sylvatica ssp. sylvatica and ssp. orientalis, respectively). This indicates that the gene pool of F. sylvatica ssp. orientalis has not been depleted by reduced population sizes during the Pleistocene glaciations, as is the case of F. sylvatica ssp. sylvatica. Genetic similarities between isolated regional populations are explained by shared ancestral polymorphisms and/or range overlaps with subsequent hybridization in the past. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 154 , 165–174.