A comparative study of cycad mucilages

The patterns of monosaccharide distribution of the mucilages of Cycadales are characteristic at the generic level. Arabinose, fucose, galactose, mannose, rhamnose and methylrhamnose were identified in the hydrolysed mucilage of Bowenia, Ceratozamia, Cycas, Dioon, Encephalartos, Lepidozamia, Macrozamia, Microcycas and Zamia. Stangeria contains no rhamnose and methylrhamnose, and Ceratozamia contains galactose only in traces. American genera may easily be distinguished from the others by means of their different monosaccharide composition. Lepidozamia appears to be well separated from Macrozamia.     

KARYOTYPIC DATA ON NORTH AND CENTRAL AMERICAN ZAMIACEAE (CYCADALES) AND THEIR PHYLOGENETIC IMPLICATIONS

Chromosome numbers and karyotypes of species from four American Zamiaceae (Cycadales) are reported. Zamia shows interspecific and intraspecific chromosome variation, whereas Microcycas, Ceratozamia, and Dioon have constant karyotypes within each genus. In Zamia, all karyotypes have the same number of submetacentric and acrocentric chromosomes, but they differ in the number of metacentric and telocentric chromosomes. Centric fission of metacentric chromosomes is proposed to explain the karyotypic variation in this genus. Zamia shows karyological relationships with Microcycas and Ceratozamia, whereas Dioon appears very distinct from the other American cycad genera. Affinity among Zamia, Ceratozamia, and Microcycas karyotypes and distinctiveness of Dioon karyotypes are supported by comparative analysis of phenotypic characters in the four genera.     

VARIATION IN LEAFLET MORPHOLOGY AMONG POPULATIONS OF CARIBBEAN CYCADS (ZAMIA)

Caribbean cycads (Zamia L.) are well known for variation in leaflet morphology. Here, I examine the variation in leaflet morphology among five populations of Zamia in Florida. Variables measured were leaflet length and width, leaflet length: width ratio, leaflet surface area, rachis and petiole lengths, number of leaflets per leaf, and total leaf area. In addition to comparisons among the Florida populations, these populations are compared with three previously studied populations in Puerto Rico (Newell, 1986). For the most part, the populations exhibited significant differences in all variables. In spite of the extensive morphological variation in this group of plants, leaflet morphology has long been important in Zamia taxonomy. The Florida populations would be considered by some to be either one, two, or three different species. The Florida and Puerto Rico populations together might be considered as few as one species or as many as five species. Based upon the data presented here, the five Florida populations appear to represent a single species; and the three Puerto Rico populations appear to represent two additional species. Further clarification of the taxonomy of Caribbean Zamia will require additional characters such as cone morphology and protein or DNA patterns.     

RFLP analysis for species separation in the generaBipolaris andCurvularia

Restriction fragment length polymorphism (RFLP) of the total DNA ofBipolaris andCurvularia species was analysed using arbitrarily chosen genomic clones of DNA fromCurvularia lunata andBipolaris maydis as probes. Clear differences among species in both genera, resulting in different banding positions, were obtained with some probe-enzyme combinations. Intraspecific polymorphism in banding positions with these probe-enzyme combinations was slight. These analyses allow discrimination between the species. DNA fingerprinting with intrageneric probes is a potentially useful tool for species separation and identification inBipolaris andCurvularia when coupled with another characteristic such as conidial morphology.Curvularia aeria comb. nov. was proposed forCurvularia lunata var.aeria on the basis of differences in RFLP banding patterns and differences in conidial morphology.     

Molecular systematic studies in cycads: Evidence fromtrnL Intron and ITS2 rDNA sequences

The results of a pilot DNA sequencing study of cycads conducted at the new molecular systematics laboratory at Fairchild Tropical Garden are presented and assessed with reference to previous phylogenetic analyses and classification schemes based on morphology and anatomy. Two DNA regions were sequenced and analyzed for variation, an intron in the trriL gene in the chloroplast genome (trriL intron) and the internal transcribed spacer region between the 5.8S and 26S ribosomal DNA subunits (ITS2). The trnL intron proved to be relatively conservative among cycad genera, while the ITS2 region contained higher levels of variation. Parsimony analysis of the sequences suggests a number of relationships, some of which were inferred by previous morphological studies, some of which are new. The sequences ofCycas are the most divergent among cycads, suggesting the longest isolation.Dioon is relatively isolated from the other genera and contains two major clades.Stangeria does not appear closely related toBowenia but does seem to have a weak affinity withZamia andMicrocycas. Lepidozamia is more closely related toEncephalartos than toMacrozamia. Sequence variation among the species ofCeratozamia is low.Microcycas andZamia are closely related.     

Organization of a dispersed repeated DNA element in the Zamia genome

Occurrence and genomic organization of dispersed elements containing ZpS1 satellite repeats have been investigated in a wide representation of species of the old plant genus Zamia (Zamiaceae, Cycadales). In Z. paucijuga, the ZpS1 repeat is organized as long satellite DNA arrays and as short arrays inserted into AT-rich dispersed elements. A comparative study by Southern analysis shows that these unusual dispersed elements containing the ZpS1 repeat are present with different organizations in all investigated Zamia species. In some species these elements are present with a low copy number, while in other species secondary amplification events, involving specific sequence clusters, appear to have generated characteristic dispersed elements in a high copy number. Among Zamia species, several groups share similar restriction patterns, as the Zamia loddigesii complex and the Caribbean species suggesting a general correlation between organization and genomic representation of the dispersed repeated sequence and the pattern of phyletic relationships in the genus. However, the finding of different patterns also among closely related species suggests a complex history of amplifications and losses of these dispersed repetitive elements that cannot be always easily traced through the phylogenetic reconstruction of this ancient plant group.     

SYSTEMATIC STUDIES ON THE MEXICAN ZAMIACEAE. I. CHROMOSOME NUMBERS AND KARYOTYPES

Chromosome numbers and karyotypes are reported for seven species in three genera, of Mexican cycads. Karyotypic analysis of Cycads is made difficult by differential contraction, of their large chromosomes but idiograms are presented. Satellite variation is reported for three species of Ceratozamia. No satellites are reported for the three species of Zamia.     

Stomatal development in the cycad family Zamiaceae

Background and AimsThe gymnosperm order Cycadales is pivotal to our understanding of seed-plant phylogeny because of its phylogenetic placement close to the root node of extant spermatophytes and its combination of both derived and plesiomorphic character states. Although widely considered a ‘living fossil’ group, extant cycads display a high degree of morphological and anatomical variation. We investigate stomatal development in Zamiaceae to evaluate variation within the order and homologies between cycads and other seed plants.MethodsLeaflets of seven species across five genera representing all major clades of Zamiaceae were examined at various stages of development using light microscopy and confocal microscopy.Key ResultsAll genera examined have lateral subsidiary cells of perigenous origin that differ from other pavement cells in mature leaflets and could have a role in stomatal physiology. Early epidermal patterning in a ‘quartet’ arrangement occurs in Ceratozamia, Zamia and Stangeria. Distal encircling cells, which are sclerified at maturity, are present in all genera except Bowenia, which shows relatively rapid elongation and differentiation of the pavement cells during leaflet development.ConclusionsStomatal structure and development in Zamiaceae highlights some traits that are plesiomorphic in seed plants, including the presence of perigenous encircling subsidiary cells, and reveals a clear difference between the developmental trajectories of cycads and Bennettitales. Our study also shows an unexpected degree of variation among subclades in the family, potentially linked to differences in leaflet development and suggesting convergent evolution in cycads.     

Biflavonoids from the seed testa of cycadales

Biflavonoid patterns from ethanolic extracts of the brightly coloured testa of eight species of Cycadales have been determined. Species from the genus Macrozamia are characterized by the occurrence of cupressuflavone- and amentoflavone-based patterns, while the Cycas species contain only amentoflavone-based patterns. The other species from the genera Encephalartos, Lepidozamia, and Zamia contain a very minor or no detectable biflavonoid component. This is the first report of the occurrence of cupressuflavone in the Cycadales.     

Intrageneric Variability Between the Chloroplast Genomes of Trachelomonas grandis and Trachelomonas volvocina and Phylogenomic Analysis of Phototrophic Euglenoids

The latest studies of chloroplast genomes of phototrophic euglenoids yielded different results according to intrageneric variability such as cluster arrangement or diversity of introns. Although the genera Euglena and Monomorphina in those studies show high syntenic arrangements at the intrageneric level, the two investigated Eutreptiella species comprise low synteny. Furthermore Trachelomonas volvocina show low synteny to the chloroplast genomes of the sister genera Monomorphina aenigmatica, M. parapyrum, Cryptoglena skujae, Euglenaria anabaena, Strombomonas acuminata, all of which were highly syntenic. Consequently, this study aims at the analysis of the cpGenome of Trachelomonas grandis and a comparative examination of T. volvocina to investigate whether the cpGenomes are of such resemblance as could be expected for a genus within the Euglenaceae. Although these analyses resulted in almost identical gene content to other Euglenaceae, the chloroplast genome showed significant novelties: In the rRNA operon, we detected group II introns, not yet found in any other cpGenome of Euglenaceae and a substantially heterogeneous cluster arrangement in the genus Trachelomonas. The phylogenomic analysis with 84 genes of 19 phototrophic euglenoids and 18 cpGenome sequences from Chlorophyta and Streptophyta resulted in a well‐supported cpGenome phylogeny, which is in accordance to former phylogenetic analyses.     

Systematic significance of achene morphology in Soroseris,Syncalathium and Parasyncalathium (Asteraceae: Cichorieae)

The systematic significance of the morphological structure of achenes in the Himalayan–Tibetan Plateau endemic genera Soroseris, Syncalathium and Parasyncalathium is described and discussed. The achene surface sculpturing of 15 samples representing 13 species of the three genera was investigated using scanning electronic microscopy (SEM) to evaluate inter‐ and intrageneric relationships of the three genera. Characters such as cell arrangement, shape of the epidermis, type of ornamentation of the outer cell wall and degree of wax development are described. The results show that characters such as the shape of the epidermis and the type of ornamentation of the outer cell wall are distinct between the three genera and useful for species‐level classification. Parasyncalathium souliei differs from Syncalathium, in which it has traditionally been placed, in the short, stout beak of the achene and especially in the obscure outline of the epidermal cells and their long acuminate, steeple‐like, end walls. Combined with karyological and molecular data, the differences in achene morphology and sculpturing further support our recognition of Parasyncalathium as distinct from Syncalathium. The achene characters are not only useful for assessing relationships, but are also useful for delimiting species. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 476–486.     

Conserved genetic regions across angiosperms as tools to develop single‐copy nuclear markers in gymnosperms: an example using cycads

Several individuals of the Caribbean Zamia clade and other cycad genera were used to identify single‐copy nuclear genes for phylogeographic and phylogenetic studies in Cycadales. Two strategies were employed to select target loci: (i) a tblastX search of Arabidopsis conserved ortholog sequence (COS) set and (ii) a tblastX search of Arabidopsis‐Populus‐Vitis‐Oryza Shared Single‐Copy genes (APVO SSC) against the EST Zamia databases in GenBank. From the first strategy, 30 loci were selected, and from the second, 16 loci. In both cases, the matching GenBank accessions of Zamia were used as a query for retrieving highly similar sequences from Cycas, Picea, Pinus species or Ginkgo biloba. After retrieving and aligning all the sequences in each locus, intron predictions were completed to assist in primer design. PCR was carried out in three rounds to detect paralogous loci. A total of 29 loci were successfully amplified as a single band of which 20 were likely single‐copy loci. These loci showed different diversity and divergence levels. A preliminary screening allowed us to select 8 promising loci (40S, ATG2, BG, GroES, GTP, LiSH, PEX4 and TR) for the Zamia pumila complex and 4 loci (COS26, GroES, GTP and HTS) for all other cycad genera.     

Macroecology of Aquatic Insects: A Quantitative Analysis of Taxonomic Richness and Composition in the Andes Mountains of Northern Ecuador

To determine the spatial dynamics of Neotropical lotic insect species, specimens were collected from 41 streams on the eastern and western flanks of the Andes Mountains in Ecuador. We examined the manner in which taxonomic richness and composition differed with elevation, latitude, and versant. Statistical analyses were limited to 5 families (Ephemeroptera: Baetidae, Leptohyphidae, Oligoneuriidae; Heteroptera: Naucoridae; Coleoptera: Elmidae), comprising 32 genera and 85 species, for which identifications or morphospecies assignments were reliable. Assessment of taxonomic diversity was based on the richness of genera and species at each site. In addition, each site was characterized by species richness within each of 4 families with more than 10 species. The effects of versant and transect on composition and structure were family‐specific. Mean site differences between versants in elmid richness at generic and specific levels depended on transect. Only baetid richness was affected by versant and transect in a consistent manner. Variation among sites in composition based on all genera and species was captured using multidimensional scaling (MDS). Subsequent MANOVAs based on scores from MDS axes revealed that differences between versants were much stronger in the southern transect, although transects differed from each other at specific and generic levels. A Jaccard's similarity matrix was computed for each family to reflect the spatial organization of taxonomic composition. Mesogeographic patterns of species composition for each of the four families were correlated (Mantel analysis) at both the regional level and at the level of the entire study area. At the regional level, the only pair of families to exhibit correlated patterns of species composition was elmids and naucorids in the southwestern region. The pattern of species composition for each family was correlated with the patterns for one or more other families at the level of the entire study area. Thus, spatial dynamics of species composition was similar for the families examined, suggesting that the Andes exert a consistent influence on species distributions within families, regardless of ordinal affiliation. At a local scale, however, the way in which taxonomic composition changed with latitude and versant was family‐specific. Mayflies, the most vagile of the taxa studied, had the highest percentage of species overlap between versants. Of three genera of Naucoridae collected, species of Ambrysus, of probable Mexican origin, were found only on the eastern versant, corroborating other evidence that the genus is recent in South America. Moreover, dispersion by Ambrysus across the Andes Mountains may not have occurred, as it has for Cryphocricos and Limnocoris, which are of probable South American origin.     

Recognition of a species-poor, geographically restricted but morphologically diverse Cape lineage of diving beetles (Coleoptera: Dytiscidae: Hyphydrini)

Aim To establish the phylogeny and geographical origin of the genera of the diving beetle tribe Hyphydrini in order to investigate the origin of differences in geographical range size, intrageneric species‐richness and morphological disparity. In particular, we tested the hypothesis that the geographically restricted, species‐poor and morphologically deviating genera found in the Cape Region of South Africa are a paraphyletic pool of ‘primitive’ Hyphydrini, from which the morphologically more uniform, species‐rich and geographically widespread genera have originated. Location Worldwide, with special reference to the Cape Region of South Africa. Methods We constructed a genus‐level molecular phylogeny of 10 of the 14 known genera of Hyphydrini, including the five endemic to the Cape Region, using sequences from four gene fragments (two mitochondrial, rrnL and cox1; and two nuclear, 18S rRNA and histone 3, c. 2200 bp). Phylogenies were built with Bayesian methods, and linearized using penalized likelihood. Morphological disparity was characterized by correspondence analysis of a data matrix of 21 binary characters. We compare morphological disparity among groups using distance to the global and local centroids and the total range of morphospace occupied. Geographical range was estimated using the number of 6° longitude × 8° latitude Universal Transverse Mercator squares known to contain any species of each genus. Results Hyphydrini is made up of four well supported clades of similar relative genetic divergence: (1) Hyphydrus (Old World plus Australasia, 133 species), (2) the five endemic genera of the Cape Region, sister to Hovahydrus (Madagascar) (10 species), (3) Desmopachria (America, 92 species), and (4) two Oriental genera (Microdytes and Allopachria, 68 species). The morphological disparity within the Cape Region lineage has apparently increased with time, with the two genera closest to the global centroid paraphyletic and basal with respect to the three more recent, morphologically deviating genera. Differences in the number of species between each of the four lineages were not significant. The correlation between the number of species in each lineage and geographical range extent was highly significant, with the low species number of the Cape Region (six) well within the 95% confidence interval of the regression. Main conclusions Contrary to expectations, the species‐poor, morphologically deviating endemic genera of the Cape Region are not a ‘primitive’ relictual pool from which the widespread, species‐rich and morphologically uniform genera have originated. The morphological disparity within the Cape lineage has increased with time, and the apparent lack of species‐level diversification disappears when species–area relationships are considered. A major unanswered question is why one of the four main lineages of Hyphydrini has remained restricted to a very reduced area (the Cape Region), but despite this evolved the highest degree of morphological diversity seen in the tribe.     

Electrophoretic protein patterns of Geotrichum and its teleomorphs

Total patterns of water-soluble proteins of 35 strains (7 species) of Galactomyces and Dipodascus strains with their respective Geotrichum anamorphs are compared. Quantitative differences among a number of species are found with iso-electric focusing; bands that characterize species are selected. Qualitative differences are found between the teleomorph genera.     

Vertical oviposition activity of mosquitoes in the Atlantic Forest of Brazil with emphasis on the sylvan vector,Haemagogus leucocelaenus (Diptera: Culicidae)

This study aimed to assess the vertical patterns of oviposition and temporal changes in the distribution of mosquito species in an area of the Atlantic Forest in Rio de Janeiro State, Brazil, and in particular, the behavior and oviposition of potential yellow fever virus vectors. Mosquito samples were collected from the Ecological Reserve Guapiaçu (REGUA, Brazil), which includes a somewhat disturbed forest, with a large diversity of plants and animals. In all, 5,458 specimens (ten species from seven genera) were collected. Haemagogus leucocelaenus was the most frequently captured species, representing 73% of the specimens collected. Species richness and diversity were the highest in the samples collected from the ground‐level ovitraps and decreased with height. Species composition also differed significantly among heights. The largest species differences were detected between ovitraps set at the ground level and those set at 7 m and 9 m; Hg. leucocelaenus, Limatus durhamii, and Limatus paraensis contributed most to these differences. Sampling month and climatic variables had significant effects on species richness and diversity. Species diversity and richness decreased with height, suggesting that the conditions for mosquito breeding are more favorable closer to the ground. Species composition also showed vertical differences.     

A character‐based approach in the Mexican cycads supports diverse multigene combinations for DNA barcoding

A DNA barcoding study was conducted to determine the optimal combination of loci needed for successful species‐level molecular identification in three extant cycad genera—Ceratozamia, Dioon, and Zamia—that occur in Mexico. Based on conclusions of a previous multigene study in representative species of all genera in the Cycadales, we tested the DNA barcoding performance of seven chloroplast coding (matK, rpoB, rpoC1, and rbcL) and non‐coding (atpF/H, psbK/I, and trnH‐psbA) regions, plus sequences of the nuclear internal transcribed spacer. We analysed data under the assumptions of the “character attributes organization system” (CAOS), a character‐based approach in which species are identified through the presence of ‘DNA diagnostics’. In Ceratozamia, four chloroplast regions and one nuclear region were needed to achieve > 70% unique species identification. In contrast, the two‐gene combination atpF/H + psbK/I and the four‐gene combination atpF/H + psbK/I + rpoC1 + ITS2 were needed to reach 79% and 75% unique species identification in Dioon and Zamia, respectively. The combinations atpF/H + psbK/I and atpF/H + psbK/I + rpoC1 + ITS2 include loci previously considered by the international DNA barcoding community. However, none of the three combinations of potential DNA barcoding loci found to be optimal with a character‐based approach in the Mexican cycads coincides with the ‘core barcode’ of chloroplast markers (matK + rbcL) recently proposed for universal use in the plant kingdom.     

A molecular framework for the phylogeny of the Pseudocerotidae (Platyhelminthes,Polycladida)

Systematic relationships within the cotylean family Pseudocerotidae were examined using nucleotide sequences of the D3 expansion segment of the 28S rDNA gene. A previously suggested separation of Pseudoceros and Pseudobiceros based on the number of male reproductive systems was confirmed. Regardless of the algorithm employed, Pseudoceros always formed a monophyletic clade. Pseudobiceros appeared to be paraphyletic; however, a constrained maximum parsimony tree was not significantly longer (2 steps, = 0.05). Additionally, the genera Maiazoon, Phrikoceros and Tytthosoceros were validated as taxonomic entities, and their relationships to other genera within the family were determined. Molecular data also supported species separations based on colour patterns. An intraspecific genetic distance of 1.14% was found for Pseudoceros bifurcus, whereas the intrageneric distance was 3.58%. Genetic distances among genera varied, with the closest distance being 2.048% between Pseudobiceros and Maiazoon, and the largest distance (8.345%) between Pseudoceros and Tytthosoceros.     

Seed-coat microsculpturing and its systematic application in Isatis (Brassicaceae) and allied genera in Iran

In order to examine the systematic application of seed-coat microsculpturing in Isatis, seed surfaces of 23 species (41 populations) in four genera of tribe Isatideae were examined using scanning electron microscopy (SEM). Eight types of basic ornamentation patterns were recognized among the studied specimens. Of these, the reticulate–areolate type was the most common and was found in the genera Isatis, Pachypterygium, Samerari and Tauscheria and 15 species (e.g., I. cappodocica, I. kotschyana and I. tinctoria). The reticulate type, the second most frequent, occurred in 7 species while other types each were represented by only one or 2 species. Although different populations of a given species show similar seed-surface sculpturing in most cases, in some polymorphic species like I. cappadocica and I. kotschyana these patterns were variable among populations. To some extent the variation corresponds to infraspecific taxa for some species, but the differences are not significant enough to be useful in the delimitation of the subspecies recognized by previous workers. Moreover, seed-coat characters do not support the separation of genera Isatis, Pachypterygium, Sameraria and Tauscheria.