Phylogeny of the cycads based on multiple single-copy nuclear genes: congruence of concatenated parsimony,likelihood and species tree inference methods

Background and aims

Despite a recent new classification, a stable phylogeny for the cycads has been elusive, particularly regarding resolution of Bowenia, Stangeria and Dioon. In this study, five single-copy nuclear genes (SCNGs) are applied to the phylogeny of the order Cycadales. The specific aim is to evaluate several gene tree–species tree reconciliation approaches for developing an accurate phylogeny of the order, to contrast them with concatenated parsimony analysis and to resolve the erstwhile problematic phylogenetic position of these three genera.

Methods

DNA sequences of five SCNGs were obtained for 20 cycad species representing all ten genera of Cycadales. These were analysed with parsimony, maximum likelihood (ML) and three Bayesian methods of gene tree–species tree reconciliation, using Cycas as the outgroup. A calibrated date estimation was developed with Bayesian methods, and biogeographic analysis was also conducted.

Key Results

Concatenated parsimony, ML and three species tree inference methods resolve exactly the same tree topology with high support at most nodes. Dioon and Bowenia are the first and second branches of Cycadales after Cycas, respectively, followed by an encephalartoid clade (Macrozamia–Lepidozamia–Encephalartos), which is sister to a zamioid clade, of which Ceratozamia is the first branch, and in which Stangeria is sister to Microcycas and Zamia.

Conclusions

A single, well-supported phylogenetic hypothesis of the generic relationships of the Cycadales is presented. However, massive extinction events inferred from the fossil record that eliminated broader ancestral distributions within Zamiaceae compromise accurate optimization of ancestral biogeographical areas for that hypothesis. While major lineages of Cycadales are ancient, crown ages of all modern genera are no older than 12 million years, supporting a recent hypothesis of mostly Miocene radiations. This phylogeny can contribute to an accurate infrafamilial classification of Zamiaceae.     

Serine proteinase inhibitors in seeds of Cycas siamensis and other gymnosperms

Seeds of 32 species selected from two of the four major groups of gymnosperms, the ancient Cycadales and the economically important Coniferales, were analysed for inhibitors (I) of the serine proteinases trypsin (T), chymotrypsin (C), subtilisin (S) and elastase (E) using isoelectric focusing (IEF) combined with gelatin replicas. Subtilisin inhibitors were detected in 17 species, being particularly active in the Cycadales. Several species of the genera Cephalotaxus, Pseudotsuga and Cycas contained inhibitors active against elastase while strong CSTIs and CSIs were also present in Cycas pectinata and C. siamensis. No inhibitors were detected in seeds of Chamaecyparis, Thuja, Abies, Larix, Picea and Pinus spp. Serine proteinase inhibitors were purified from seeds of C. siamensis by affinity chromatography using trypsin and chymotrypsin, IEF and SDS-PAGE. Several CSTI components with M_r ranging from 4000 to 18,000 were partially sequenced using Edman degradation and mass spectrometry. Most of the sequences were similar to a hypothetical protein encoded by an mRNA from sporophylls of C. rumphii which in turn was similar to Kunitz-type proteinase inhibitors from flowering plants. Analysis of expressed sequence tag (EST) databases confirmed the presence of mRNAs encoding Kunitz-type inhibitors in the Cycadales and Coniferales and also demonstrated their presence in a third major group of gymnosperms, the Ginkgoales. This is the first report of Kunitz-type serine proteinase inhibitors from plants other than Angiosperms.     

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.     

Données nouvelles sur la paléoflore de Colombie

From samples collected during two missions in Columbia and from samples stored at the University of Medellin, are successively described three fossile floras: a poor and fragmentary Devonian flora from Floresta, a rich Upper Jurassic flora from the «Formación Valle Alto in the Central Cordillera composed of Ferns, Bennettitales, Cycadales and Caytoniales with the genus: Gleichenites, Cladophlebis, Sphenopteris, Pachypteris, Sagenopteris, Nilssoniopteris, Otozamites, Anomozamites, Zamites, Ctenozamites, Ptilophyllum and Desmiophyllum, a lower Cretaceous flora from the «Quebrada campanas and San Felix area in the Central Cordillera composed of Ferns, Bennettitales, Cycadales, Ginkgoales, Caytoniales and Coniferales with Coniopteris, Piazopteris, Klukia, Sphaenobaiera, Sagenopteris, Nilssoniopteris, Zamites, Podozamites, Cupressinocladus, Cyparissidium, and Elatocladus. These three floras are of euramerian type.     

Biodiversity of Convolvulaceous species that contain ergot alkaloids,indole diterpene alkaloids,and swainsonine

Convolvulaceous species have been reported to contain several bioactive principles thought to be toxic to livestock including the calystegines, swainsonine, ergot alkaloids, and indole diterpene alkaloids. Swainsonine, ergot alkaloids, and indole diterpene alkaloids are produced by seed transmitted fungal symbionts associated with their respective plant host, while the calystegines are produced by the plant. To date, Ipomoea asarifolia and Ipomoea muelleri represent the only Ipomoea species and members of the Convolvulaceae known to contain indole diterpene alkaloids, however several other Convolvulaceous species are reported to contain ergot alkaloids. To further explore the biodiversity of species that may contain indole diterpenes, we analyzed several Convolvulaceous species (n = 30) for indole diterpene alkaloids, representing four genera, Argyreia, Ipomoea, Stictocardia, and Turbina, that had been previously reported to contain ergot alkaloids. These species were also verified to contain ergot alkaloids and subsequently analyzed for swainsonine. Ergot alkaloids were detected in 18 species representing all four genera screened, indole diterpenes were detected in two Argyreia species and eight Ipomoea species of the 18 that contained ergot alkaloids, and swainsonine was detected in two Ipomoea species. The data suggest a strong association exists between the relationship of the Periglandula species associated with each host and the occurrence of the ergot alkaloids and/or the indole diterpenes reported here. Likewise there appears to be an association between the occurrence of the respective bioactive principle and the genetic relatedness of the respective host plant species.     

Biflavones of Dioon

Seven biflavones, amentoflavone, bilobetin, sequoiaflavone, ginkgetin, sciadopitysin, 7,4′,7′,4?-tetra-O-methylamentoflavone, and diooflavone (amentoflavone hexamethyl ether), were identified from extracts of the cycad genus Dioon. The biflavones were identified by direct comparison with authentic samples using m.m.p., co-chromatography in 3 solvents, and NMR studies of the acetates. This is the first time amentoflavone hexamethyl ether has been identified as a natural product. After surveying numerous species of the Cycadales, no evidence could be obtained for the occurrence of biflavone glycosides or of biflavones based upon any other nucleus than apigenin.     

Santonian Angiosperm-dominated leaf-assemblage from Piolenc (Vaucluse,Sud-Est de la France)

Lower Santonian sandy to coaly-clayey beds, collected near the village of Piolenc (Vaucluse) contain amber bearing insects and extremely well-preserved leaf cuticle compressions of Corystospermales, Cycadales, Bennettitales, Gymnosperms and mostly Angiosperms. The sedimentological and taphonomic studies indicate that the clays bearing the fossil cuticles were deposited along the Alpine Sea shore into supratidal ponds or marshes around which the plants studied herein grew. Moreover, the ‘nearest living relative’ and ‘foliar physiognomy’ methods, and the sedimentological and palaeontological studies agree with the occurrence of a warm and humid, tropical climate, including a dry season. To cite this article: B. Gomez et al., C. R. Palevol 2 (2003).     

Xanthones of Swertia bimaculata

The whole plant of Swertia bimaculata Hf. & T. has been shown to contain four tetra-and five penta-oxygenated xanthones, three of which are previously unreported in nature. The xanthones are broadly based on 1,3,5- and 1,3,7-oxygenated systems with added oxygen functions at C₂, C₄ and/or C₈ positions and represent a number of methoxylated patterns. In addition, three xanthones have been found to be present in a bound form, the sugar moiety containing glucose and glucuronic acid. This is the first demonstration of the occurrence of xanthones and xanthone disaccharides in a Swertia species which are common to both Swertia and Frasera species. The results are thus of considerable phylogenetic significance.     

Correlations between flavonoid chemistry,anatomy and geography in the restionaceae

Comparisons of the flavonoid patterns in stems and inflorescences between Australasian and South African members of the Restionaceae indicate significant differences with geography. Nine of 14 Australasian species contain gossypetin or a related 8-hydroxyflavonoid and proanthocyanidins are uncommon. By contrast, the 33 South African taxa studied contain common flavonols, flavones and glycoflavones, while proanthocyanidins are present in 29. Two anatomically related South African genera, Chondropetalum and Elegia, contain, in addition, myricetin 3-galactoside, together with the 3-galactosides of the myricetin methyl ethers, larycitrin and syringetin. These results confirm the conclusions derived from anatomy that members of Hypolaena, Leptocarpus and Restio, genera represented in both Australia and South Africa, have the distinctive flavonoids characteristic of their geographic origin rather than of their systematic position. The family as a whole is different in flavonoid pattern from other monocotyledonous families with which it is sometimes associated.     

Distribution and Expression of Elicitin Genes in the Interspecific Hybrid Oomycete Phytophthora alni

Phytophthora alni subsp. alni, P. alni subsp. multiformis, and P. alni subsp. uniformis are responsible for alder disease in Europe. Class I and II elicitin gene patterns of P. alni subsp. alni, P. alni subsp. multiformis, P. alni subsp. uniformis, and the phylogenetically close species P. cambivora and P. fragariae were studied through mRNA sequencing and 3′ untranslated region (3′UTR)-specific PCRs and sequencing. The occurrence of multiple 3′UTR sequences in association with identical elicitin-encoding sequences in P. alni subsp. alni indicated duplication/recombination events. The mRNA pattern displayed by P. alni subsp. alni demonstrated that elicitin genes from all the parental genomes are actually expressed in this allopolyploid taxon. The complementary elicitin patterns resolved confirmed the possible involvement of P. alni subsp. multiformis and P. alni subsp. uniformis in the genesis of the hybrid species P. alni subsp. alni. The occurrence of multiple and common elicitin gene sequences throughout P. cambivora, P. fragariae, and P. alni sensu lato, not observed in other Phytophthora species, suggests that duplication of these genes occurred before the radiation of these species.     

Hamamelose and its derivatives as chemotaxonomic markers in the genus Primula

About 550 species of vascular plants as well as a few mosses were checked for the occurrence of hamamelose, the corresponding alcohol hamamelitol and a galactoside of the latter, clusianose. Hamamelose was found in most of the species investigated. It provides therefore no useful character for chemotaxonomic purposes. Hamamelitol, however, could only be detected in the genus Primula and was therefore more useful. Small amounts of hamamelitol were found in the leaves of species from the subgenera Auganthus, Aleuritia and Auricula but not from Primula and Sphondylia. Clusianose and large amounts of hamamelitol are produced by species of 4 subsections of the section Auricula, but not by species of the subsections Auricula and Erythrodrosum. A taxonomic separation of the two latter from the 4 former subsections is suggested. The taxonomic position of Primula allionii should be reconsidered, since neither clusianose nor hamamelitol could be demonstrated in this species, whereas the other species of the subsection Rhopsidium do contain these carbohydrates.     

Temporal Patterns of Larval Fish Occurrence in a Large Subtropical River

Knowledge of temporal patterns of larval fish occurrence is limited in south China, despite its ecological importance. This research examines the annual and seasonal patterns of fish larval presence in the large subtropical Pearl River. Data is based on samples collected every two days, from 2006 to 2013. In total, 45 taxa representing 13 families and eight orders were sampled. The dominant larval family was Cyprinidae, accounting for 27 taxa. Squaliobarbus curriculus was the most abundant species, followed by Megalobrama terminalis, Xenocypris davidi, Cirrhinus molitorella, Hemiculter leuscisculus and Squalidus argentatus. Fish larvae abundances varied significantly throughout the seasons (multivariate analyses: Cluster, SIMPROF and ANOSIM). The greatest numbers occurred between May and September, peaking from June through August, which corresponds to the reproductive season. In this study, redundancy analysis was used to describe the relationship between fish larval abundance and associated environmental factors. Mean water temperature, river discharge, atmospheric pressure, maximum temperature and precipitation play important roles in larval occurrence patterns. According to seasonal variations, fish larvae occurrence is mainly affected by water temperature. It was also noted that the occurrence of Salanx reevesii and Cyprinus carpio larvae is associated with higher dissolved oxygen (DO) concentrations, higher atmospheric pressure and lower water temperatures which occur in the spring. On the other hand, M. terminalis, X. davidi, and C. molitorella are associated with high precipitation, high river discharge, low atmospheric pressure and low DO concentrations which featured during the summer months. S. curriculus also peaks in the summer and is associated with peak water temperatures and minimum NH₃–N concentrations. Rhinogobius giurinus occur when higher atmospheric pressure, lower precipitation and lower river discharges occur in the autumn. Dominant fish species stagger their spawning period to avoid intraspecific competition for food resources during early life stages; a coexistence strategy to some extent. This research outlines the environmental requirements for successful spawning for different fish species. Understanding processes such as those outlined in this research paper is the basis of conservation of fish community diversity which is a critical resource to a successful sustainable fishery in the Pearl River.     

Quantifying Shark Distribution Patterns and Species-Habitat Associations: Implications of Marine Park Zoning

Quantifying shark distribution patterns and species-specific habitat associations in response to geographic and environmental drivers is critical to assessing risk of exposure to fishing, habitat degradation, and the effects of climate change. The present study examined shark distribution patterns, species-habitat associations, and marine reserve use with baited remote underwater video stations (BRUVS) along the entire Great Barrier Reef Marine Park (GBRMP) over a ten year period. Overall, 21 species of sharks from five families and two orders were recorded. Grey reef Carcharhinus amblyrhynchos, silvertip C. albimarginatus, tiger Galeocerdo cuvier, and sliteye Loxodon macrorhinus sharks were the most abundant species (>64% of shark abundances). Multivariate regression trees showed that hard coral cover produced the primary split separating shark assemblages. Four indicator species had consistently higher abundances and contributed to explaining most of the differences in shark assemblages: C. amblyrhynchos, C. albimarginatus, G. cuvier, and whitetip reef Triaenodon obesus sharks. Relative distance along the GBRMP had the greatest influence on shark occurrence and species richness, which increased at both ends of the sampling range (southern and northern sites) relative to intermediate latitudes. Hard coral cover and distance across the shelf were also important predictors of shark distribution. The relative abundance of sharks was significantly higher in non-fished sites, highlighting the conservation value and benefits of the GBRMP zoning. However, our results also showed that hard coral cover had a large effect on the abundance of reef-associated shark species, indicating that coral reef health may be important for the success of marine protected areas. Therefore, understanding shark distribution patterns, species-habitat associations, and the drivers responsible for those patterns is essential for developing sound management and conservation approaches.     

Glowing Seashells: Diversity of Fossilized Coloration Patterns on Coral Reef-Associated Cone Snail (Gastropoda: Conidae) Shells from the Neogene of the Dominican Republic

The biology of modern Conidae (cone snails)—which includes the hyperdiverse genus Conus—has been intensively studied, but the fossil record of the clade remains poorly understood, particularly within an evolutionary framework. Here, ultraviolet light is used to reveal and characterize the original shell coloration patterns of 28 species of cone snails from three Neogene coral reef-associated deposits from the Cibao Valley, northern Dominican Republic. These fossils come from the upper Miocene Cercado Fm. and lower Pliocene Gurabo Fm., and range in age from about 6.6-4.8 Ma. Comparison of the revealed coloration patterns with those of extant species allow the taxa to be assigned to three genera of cone snails (Profundiconus, Conasprella, and Conus) and at least nine subgenera. Thirteen members of these phylogenetically diverse reef faunas are described as new species. These include: Profundiconus? hennigi, Conasprella (Ximeniconus) ageri, Conus anningae, Conus lyelli, Conus (Atlanticonus?) franklinae, Conus (Stephanoconus) gouldi, Conus (Stephanoconus) bellacoensis, Conus (Ductoconus) cashi, Conus (Dauciconus) garrisoni, Conus (Dauciconus?) zambaensis, Conus (Spuriconus?) kaesleri, Conus (Spuriconus?) lombardii, and Conus (Lautoconus?) carlottae. Each of the three reef deposits contain a minimum of 14–16 cone snail species, levels of diversity that are similar to modern Indo-Pacific reef systems. Finally, most of the 28 species can be assigned to modern clades and thus have important implications for understanding the biogeographic and temporal histories of these clades in tropical America.     

Pyrrolizidine alkaloids in the antipodean genus Brachyglottis (Asteraceae)

The phylogeny of the genus Brachyglottis suggests that its constituent species should contain pyrrolizidine alkaloids. Consistent with this hypothesis, and the established occurrence of such alkaloids in Brachyglottis repanda, Brachyglottis kirkii, and Brachyglottis hectori, an investigation of Brachyglottis adamsii revealed the presence of senecionine and retrorsine; Brachyglottis huntii was found to contain senkirkine and retrorsine; 7-O-angelylheliotridine was the predominant alkaloid in Brachyglottis perdicioides, and the same alkaloid together with senecionine, senkirkine and intergerrimine was present in the Brachyglottis hectori × B. perdicioides “Alfred Atkinson” horticultural hybrid; Brachyglottis sciadophila contained clivorine and neopetasitenine (acetylfukinotoxin); the latter alkaloid was also present in B. kirkii together with the previously reported senkirkine and senkirkine 12-acetate.     

Flavonoid chemistry of Chenopodium fremontii. Infraspecific variation and systematic implications at the interspecific level

Four flavonoid geographical races based on twenty flavonol 3-O-glycosides were found to exist in Chenopodium fremontii with those populations from the northern part of the range (northern Colorado, Wyoming. and western Nebraska) producing 7-methyl ethers and 3-O-galactosides and glucosides. Plants from Arizona. southern Colorado and New Mexico lack 7-methyl ethers and contain 3-O-rhamnogalactosides and rhamnoglucosides (rutinosides). California populations are chemically similar to those from Arizona, southern Colorado and New Mexico but contain arabinosides while lacking rutinosides. No morphological features could be correlated with the chemical races. Chenopodium fremontii can be distinguished chemically from other closely related diploid species of the western U.S., all of which exhibit simpler flavonoid patterns. It is suggested that the simpler chemical patterns for the latter species (which include C. atrovirens, C. desiccatum, C. hians. C. incanum, C. leptophyllum, and C. pratericola) are a derived condition relative to C. fremontii.     

Production of BMAA and DAB by diatoms (Phaeodactylum tricornutum,Chaetoceros sp., Chaetoceros calcitrans and,Thalassiosira pseudonana) and bacteria isolated from a diatom culture

Microalgae have previously been reported to contain β-N-methylamino-l-alanine (BMAA), and the global presence of these primary producers has been associated with the widespread occurrence of BMAA in marine organisms. It has been repeatedly shown that filter-feeding bivalves accumulate phytoplankton species and their toxins. In this study, the concentrations of total soluble BMAA and DAB as a function of growth phase were observed for four non-axenic diatom species (i.e. Phaeodactylum tricornutum, Chaetoceros sp., Chaetoceros calcitrans and Thalassiosira pseudonana). These strains had previously been shown to contain BMAA using a highly selective HILIC-MS/MS method. BMAA cell quota appeared to be species-specific, however, highest BMAA concentrations were always obtained during the stationary growth phase, for all four species, suggesting that BMAA is a secondary metabolite. While DAB was detected in a bacterial culture isolated from a culture of P. tricornutum, the presence or absence of a bacterial population did not influence production of BMAA and DAB by P. tricornutum, i.e. no significant difference was noted for BMAA and DAB production between axenic and non-axenic cultures. The presence of DAB in bacteria had previously been shown, and raised the question as to whether DAB observed in many species of microalgae may arise from the non-axenic culture conditions or from the microalgae themselves.     

Alkaloid profiles of Dermatophyllum arizonicum,Dermatophyllum gypsophilum,Dermatophyllum secundiflorum,Styphnolobium affine,and Styphnolobium japonicum previously classified as Sophora species

Sophora arizonica, Sophora gypsophila, Sophora secundiflora, Sophora affinis, and Sophora japonica were recently reclassified as Dermatophyllum arizonicum, Dermatophyllum gypsophilum, Dermatophyllum secundiflorum, Styphnolobium affine, and Styphnolobium japonicum, respectively. Some legumes of the sub family Papilionoideae including Sophora species are reported to contain a variety of quinolizidine alkaloids. The quinolizidine alkaloid profiles of D. arizonicum, D. gypsophilum, D. secundiflorum, S. affine, and S. japonicum were investigated qualitatively and quantitatively using field collections and herbarium specimens throughout their range of geographical distribution for the native species. This is the first report of the alkaloid profiles of D. arizonicum and D. gypsophilum. Alkaloid profiles of the other species were compared to previous reports. The Dermatophyllum species contain quinolizidine alkaloids, and the teratogen anagyrine (11), while the Styphnolobium species do not contain quinolizidine alkaloids. The chemotaxonomic data are consistent with the reclassification of each species.     

A chemotaxonomic survey of the tribe psoraleeae in africa

A study of the leaf flavonoids, furanocoumarins and essential oils of 51 species (three genera) of the tribe Psoraleeae (Fabaceae) has provided chemical data to support the recent subdivision of the large worldwide genus Psoralea into a number of segregate genera. The flavonoid patterns in most species were very similar, consisting largely of proanthocyanidins and complex mixtures of flavone O- and C-glucosides. Psoralea repens, the only maritime species to be examined, is unique in containing only rutin. Three isoflavones (daidzein, formononetin and genistein) were of widespread occurrence. The genera Psoralea and Otholobium, which could not be separated on flavonoid data, were clearly demarcated by their essential oils. Furanocoumarin patterns were useful for the characterization of species.