Pyrrolizidine alkaloid profiles in Crotalaria species from Brazil: Chemotaxonomic significance

Gas chromatography–mass spectrometry analysis of seeds from 28 species of Crotalaria from Brazil (sections Calycinae, Crotalaria, Chrysocalycinae and Hedriocarpae) showed that pyrrolizidine alkaloids (PAs) are important as chemotaxonomic markers at the infrageneric level. The sections Calycinae and Crotalaria were characterized by 11-membered macrocyclic monocrotaline-type PAs. In the section Chrysocalycinae, a single species in the subsection Incanae, C. incana, showed integerrimine, a 12-membered macrocyclic senecionine-type, as main PA. The group close to the subsection Stipulosae (C. micans and C. maypurensis) showed distinctive PA patterns: C. micans presented mainly the 12-membered macrocyclic integerrimine, and C. maypurensis the unusual 7-hydroxy-1-methylene-8-pyrrolizidine. In the group close to the subsection Glaucae, the PAs with otonecine as the necine base were the main alkaloids, except in C. rufipila which showed an assamicadine-like PA (monocrotaline-type). The section Hedriocarpae showed main 12-membered macrocyclic senecionine-type PAs.     

Genetic exchange between two freshwater apple snails, Pomacea canaliculata and Pomacea maculata invading East and Southeast Asia

Two species of apple snails, Pomacea canaliculata and Pomacea maculata (formerly Pomacea insularum), have invaded many countries of East and Southeast Asia from their native range in South America. This study investigated the genetic structure of the two species invading these areas. Phylogenetic analysis based on sequences of the nuclear gene elongation factor 1-alpha (EF1α) detected two well-supported clades (Clade C and Clade M). Both P. canaliculata and P. maculata were represented in each clade. Some snails had both Clade C and Clade M EF1α sequences. These results suggest genetic exchange between snails of the two clades. A mating experiment between P. canaliculata with Clade C EF1α sequences and P. maculata with Clade M EF1α sequences resulted in viable F1 progeny under laboratory conditions. The genetic exchange was also inferred in some populations collected from Argentina, suggesting an existence of hybrid in the native range. Simple identification of EF1α types using a restriction enzyme, ApaLI, detected significant geographical structure of the EF1α variants in the invaded area. The divergent geographical structure could have resulted from either the founder effect or the bridgehead effect, although further genetic analysis is needed to clarify this. Average individual egg weight, which is an indicator of egg size, was higher in P. canaliculata than P. maculata in both field and laboratory reared samples, suggesting that some (probably most) P. canaliculata and P. maculata invading East and Southeast Asia still maintain species-specific populations.     

Phylogenetic relationships among members of the subfamily Sedoideae (Crassulaceae) inferred from the ITS region sequences of nuclear rDNA

Nucleotide sequences of the nuclear rDNA ITS regions were determined in 20 species of the subfamily Sedoideae (Crassulaceae). The phylogenetic relationships of these species with other members of the subfamily, occurring mainly in Southeast Asia, were analyzed. It was shown that the genus Orostachys was not monophyletic; its type subsection was significantly included into the clade of the genus Hylotelephium. Synapomorphic substitutions and indels, specific for the subsection Orostachys, were detected in ITS1. Sister relationships were established between clades Aziopsis and Phedimus, based on which they can be recognized as isolated genera.     

Chemosystematics of section Palaeonarthex of the Genus Ferula

Ferula species of the section Palaeonarthex contains various sesquiterpenoids. While C-15 terpenoid coumarins of the iresan type are the most characteristic constituents of species in the subsection Foetidissimae, sesquiterpene lactones and C-10 terpenoid coumarns are found in the subsection Tumidae and esters of the sesquiterpene alcohols in the subsection Jaeschkaenae. The biogenetic relationships of the Ferula sesquiterpenoids and their chemosystematic value are discussed.     

A molecular phylogeny of mosquitoes in the Anopheles barbirostris Subgroup reveals cryptic species: Implications for identification of disease vectors

The Barbirostris Subgroup of the genus Anopheles includes six mosquito species that are almost identical in adult morphology, but differ in their roles in the transmission of malaria and filariasis within Southeast Asia. The lack of robust, diagnostic morphological characters in adults has contributed to extensive misidentification of the species. Mosquitoes were collected from localities in Thailand and Indonesia, with an emphasis on specimens identified in the field as An. barbirostris and An. campestris. A 754 bp COI mitochondrial gene fragment was sequenced from 136 specimens and the rDNA ITS2 region (c.1600–1800 bp) from 51 specimens. Phylogenetic analyzes based on Bayesian methods, distance measures and Maximum-parsimony produced five clades (I–V) that are congruent between the nuclear and mitochondrial data sets. Based on adult female morphology, it is deduced that three of these clades, I–III, are members of the Barbirostris Complex whereas Clade V is An. campestris. The identity of Clade IV is as yet unknown. Using a haplotype network analysis, Clade III was found to have a star-like genealogy, suggesting population expansion. There were no shared haplotypes between clades. In Afrotropical anopheline mosquitoes, speciation has been linked to the expansion of human populations and the development of agriculture. We postulate that the radiation of species within the Barbirostris Subgroup in Southeast Asia may similarly be linked to human population expansion and the agrarian revolution. The development of a propensity for feeding on the blood of humans in some species of the Subgroup would have led to the transmission of malaria protozoa and filarial nematodes.     

Mechanical stress,fracture risk and beak evolution in Darwin's ground finches (Geospiza)

Darwin''s finches have radiated from a common ancestor into 14 descendent species, each specializing on distinct food resources and evolving divergent beak forms. Beak morphology in the ground finches (Geospiza) has been shown to evolve via natural selection in response to variation in food type, food availability and interspecific competition for food. From a mechanical perspective, however, beak size and shape are only indirectly related to birds'' abilities to crack seeds, and beak form is hypothesized to evolve mainly under selection for fracture avoidance. Here, we test the fracture-avoidance hypothesis using finite-element modelling. We find that across species, mechanical loading is similar and approaches reported values of bone strength, thus suggesting pervasive selection on fracture avoidance. Additionally, deep and wide beaks are better suited for dissipating stress than are more elongate beaks when scaled to common sizes and loadings. Our results illustrate that deep and wide beaks in ground finches enable reduction of areas with high stress and peak stress magnitudes, allowing birds to crack hard seeds while limiting the risk of beak failure. These results may explain strong selection on beak depth and width in natural populations of Darwin''s finches.     

The affinities of Chenopodium flabellifolium (Chenopodiaceae): Evidence from seed coat surface and flavonoid chemistry

Evidence from scanning electron microscopy of seed coat surfaces and leaf flavonoid chemistry has provided new insights into the relationships of the problematical Chenopodium flabellifolium from San Martín Island, Baja California. The seed surface of C. flabellifolium is basically smooth and thus is essentially the same as members of the subsection Lejosperma of section Chenopodium. This feature distinguishes the species from the taxon that has commonly been considered its nearest relative, namely C. neomexicanum of subsection Cellulata, which has an alveolate seed surface. Leaf flavonoid chemistry also serves to separate C. flabellifolium and C. neomexicanum. Chenopodium flabellifolium has also been viewed as conspecific with C. inamoenum (= C. hians or C. leptophyllum) of subsection Lejosperma. While the seed surface indicates that C. flabellifolium is best treated as a member of this subsection, other morphological as well as chemical data suggest that its closets affinities within the subsection lie with C. fremontii. The leaves of the two species are essentially of the same shape and collectively differ strikingly from those of C. hians and C. leptophyllum. Leaf flavonoid chemistry indicates that C. flabellifolium is closer to C. fremontii than to other taxa of subsection Lejosperma occurring in the western United States because both contain kaempferol 3-O-glycosides, which have not been detected elsewhere. The two species differ consistently in that the former has the pericarp attached to the seed whereas it is separable from the seed in the latter.     

Subfamilial relationships within Caryophyllaceae as inferred from 5' ndhF sequences

DNA sequences of the 5' end of the chloroplast ndhF gene for 15 species of Caryophyllaceae have been analyzed by parsimony and neighbor-joining analyses. Three major clades are identified, with little or no support for monophyly of traditionally recognized subfamilies. The first of the three major clades identified (Clade I) is constituted by part of the subfamily Paronychioideae. It includes members of the tribe Paronychieae and members of tribe Polycarpeae. The second (Clade II) contains members of the Paronychieae exclusively. Tribe Paronychieae is thus apparently polyphyletic and tribe Polycarpeae is at least paraphyletic. The third clade (Clade III) includes members of subfamilies Alsinoideae and Caryophylloideae along with the genus Spergularia. The genus Scleranthus is also part of Clade III, while Drymaria groups with the other genera of tribe Polycarpeae in Clade II. We conclude that morphological characters previously used to delimit subfamilial groupings in the Caryophyllaceae are apparently unreliable estimators of phylogeny.     

An expanded phylogeny of Cuphea (Lythraceae) and a North American monophyly

A phylogenetic analysis of the New World genus Cuphea was conducted employing sequences from the nuclear rDNA internal transcribed spacer (ITS) and chloroplast trnL-trnF spacer and rpl16 intron. The analysis expands the number of Cuphea species from 53 in an earlier ITS study to 70 and adds two chloroplast data sets in order to generate a more complete and robust phylogeny and to test a previous result that suggested the presence of a large North American clade. Results reaffirm the monophyly of Cuphea with Pleurophora as the sister genus and recover a basal divergence event that mirrors the two subgenera of the current classification. Phylogenies of the two chloroplast regions are largely unresolved beyond the initial dichotomy and some resolution at the terminal and subterminal nodes. Based on the ITS phylogeny, five major clades are recognized. Subgenus Cuphea (Clade 1), defined morphologically by the synapomorphic loss of bracteoles, is sister to the much larger subg. Bracteolatae (Clades 2–5). Clades 2–4, comprising the South American and Caribbean species, grade successively to Clade 5, an exclusively North American lineage of 29 species. Among the 12 sections included in the study, only section Trispermum, a subclade of Clade 4, is monophyletic. Section Pseudocircaea is nested within Clade 3, which is largely equivalent to section Euandra. The North American endemic clade includes four sections, of which none are recovered as monophyletic in this study.     

Cross-linking Chemistry of Squid Beak

In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance (¹H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.     

Phylogenetic perspective on the relationships and evolutionary history of the Acipenseriformes

The Acipenseriformes, as one of the earliest extant vertebrates, plays an important role in the evolution of fishes and even the whole vertebrates. Here we collected and analyzed all complete mitochondrial genomes of Acipenseriformes species. Phylogenetic analyses demonstrated that the polytomous branch included Acipenseridae and Polyodontidae formed five clades. The Polyodontidae clade and the Scaphirhynchus clade both were monophyletic group, whereas the Acipenser species and the Huso species both were polyphyletic group. The Bayesian divergence times showed that the origin time for Acipenseriformes was at 318.0 Mya, which was similar to the some previous results of 312.1 Mya, 346.9 Mya and 389.7 Mya. The result was in good consistent with the paleontological data available and the split time of the Pacific and Atlantic Oceans from the Jurassic to the Cretaceous (Laurasia splits in North America and Eurasia). The dN/dS ratios showed the evolutionary rates gradually slow down in five major Acipenseriformes clades from the Clade A (the Pacific sturgeons species) to Clade C (the genus Scaphirhynchus), which was related to the process of geographical formation.     

Species tree phylogeny,character evolution,and biogeography of the Patagonian genus <Emphasis Type="Italic">Anarthrophyllum</Emphasis> Benth. (Fabaceae)

Geologic events promoting the aridization of southern South America contributed to lineage divergences and species differentiation through geographic (allopatric divergence) and biotic and abiotic factors (ecological divergence). For the genus Anarthrophyllum, which is distributed in arid and semi-arid regions of Patagonia, we assessed how these factors affected species diversification and reconstructed its possible biogeographic history in South American arid environments. Sequences were obtained from two molecular markers: the ITS nuclear region and the trnS-trnG plastid region. Using Parsimony, Maximum likelihood and Bayesian inference individual gene trees were reconstructed, and a species tree was obtained using multi-species coalescent analysis. Divergence times among species were estimated using secondary calibrations. Flexible Bayesian models and stochastic character mapping were used to elucidate ancestral geographic distributions and the evolution of the floral and vegetative phenotypes in the genus. Gene trees and species tree analyses strongly support Anarthrophyllum as monophyletic; all analyses consistently retrieved three well-supported main clades: High Andean Clade, Patagonian Clade 1, and Patagonian Clade 2. Main diversification events occurred concomitant with the Andean uplift and steppe aridization; the Andean mountain range possibly acted as a species barrier for the High Andean Clade. Vegetative traits showed adaptations to harsh climates in some clades, while pollinator-related floral features were associated with independent diversification in bee- and bird-pollinated clades within both Patagonian Clades. In conclusion, evolutionary and biogeographic history of Anarthrophyllum resulted from the action of ecological, historical, and geographic factors that acted either alternatively or simultaneously.     

The evolution of the reproductive system of Urodasys (Gastrotricha: Macrodasyida)

Macrodasyidan gastrotrichs are hermaphrodites with complex reproductive organs that function in sperm transfer and receipt, but homology among the organs of members of different clades remains undetermined, as does a broader understanding of evolutionary trends in the reproductive biology of macrodasyidans. In this study, we investigate the evolution of reproduction in Urodasys, a clade of 15 macrodasyidan species that shows variability in reproductive mode (hermaphroditic and parthenogenetic) and sexual anatomy. We use partial 18S rDNA sequence data from 30 specimens representing five described species, sequence data from one undescribed species in GenBank, and sequence data from a potentially new species found at Capron Shoal, Florida, to gain insight into the phylogeny of the clade and clarify evolutionary trends in reproductive modality. Based on a total of 33 specimens of seven potential species, we found that members of Urodasys can be separated into three clades reflective of different reproductive modalities: Clade I, species with paired male and female gonads but without accessory sexual organs; Clade II, species with a single left testis, paired ova, and accessory organs including a sclerotic stylet; and Clade III, parthenogenetic species without testes or accessory organs. In addition, we find that the potentially new species from Florida can form spermatophores, a condition shared with another species in Clade I. Herein, we describe this novel spermatophore‐bearing species and discuss the significance of spermatophore formation in the genus.     

PHYLOGENY OF PHOTOSYNTHETIC EUGLENOPHYTES BASED ON COMBINED CHLOROPLAST AND CYTOPLASMIC SSU RDNA SEQUENCE ANALYSIS1

Eighteen new 16S rDNA and 16 new 18S rDNA sequences from 24 strains, representing 23 species of photoautotrophic euglenoids, were obtained in nearly their entire length. Maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses were performed on separate data (39 sequences of 16S rDNA and 58 sequences of 18S rDNA), as well as on combined data sets (37 sequences). All methods of sequence analysis gave similar results in those cases in which the clades received substantial support. However, the combined data set produced several additional well‐supported clades, not encountered before in the analyses of green euglenoids. There are three main well‐defined clades (A, B/C/D, and G) on trees from the combined data set. Clade G diverges first, while clades A and B/C/D form sister groups. Clade A consists of Euglena species sensu stricto and is divided into three sub‐clades (A1, A2, and A3). Clade A3 (composed of E. deses and E. mutabilis) branches off first; then, two sister clades emerge: A1 (composed of E. viridis‐like species) and A2 (consisting of E. agilis and E. gracilis species). Clade B/C/D consists of the Strombomonas, Trachelomonas, Cryptoglena, Monomorphina, and Colacium genera. Clade G comprises Phacus and Lepocinclis, as well as the Discoglena species of Euglena, with Discoglena branching off first, and then Phacus and Lepocinclis emerging as sister groups.     

Age of spent Octopus vulgaris and stress mark analysis using beaks of wild individuals

Age estimation of the cephalopod Octopus vulgaris by using beaks has improved in recent years, but maximum age and longevity in the wild have not been confirmed due to the low availability of senescent wild octopuses. In this study, a beak analysis of lateral wall surfaces (LWS) from 20 spent specimens confirmed the 1-year lifecycle of the species in Central East Atlantic waters. Stress marks (checks) were clearly located in the daily increment sequence of rostrum sagittal sections (RSS). The highest daily variations in sea surface temperature (ΔT) that occurred during the last months of their lifetimes coincided with the locations of the marks on the beak, enabling confirmation of O. vulgaris beaks as life recorders for the first time. It also supports the daily deposition of RSS beak increments in the wild. Individuals were grouped into two main zones, at 20ºN and 18ºN, respectively. Both groups showed different thermal check patterns, in accordance with the oceanographic differences. Two other checks (not coinciding with high values of ΔT) were observed in RSS at averages of 15 and 28 days before death, respectively, which were interpreted as responding to senescent-related events.     

Japanese species of the Longibrachiatum Clade of Trichoderma

The Longibrachiatum Clade of the genus Trichoderma in Japan was examined, among which two new species and three new records are herewith reported. The new species, T. tsugarense and T. kunigamense were isolated from a bed log (cultivation of Lentinula edodes) and volcanic ash soil, respectively. These species are distinguished from closely related species by growth and morphological characteristics and in phylogenetic analysis. Additional species new to Japan were T. ghanense, T. parareesei and T. sinense. The significance of their distribution is discussed. Most species of the Longibrachiatum Clade are tropical rather than temperate in distribution. Their in vitro optimum growth tends to be >35 °C but the optimum temperature for some Japanese species was lower. Some species are endophytes of temperate plant species, some of which are endemic in Japan.     

Numerical analysis of morphology of natural hybrids between Carex hostiana and the members of Carex flava agg. (Cyperaceae)

Uni‐variate and multi‐variate statistical methods, based on data taken from dried specimens, were used to determine the morphological variance of Carex hostiana 3 Carex flava agg. hybrids and to establish their parents among members of the C. flava complex. The following hybrids were found: C. demissa 3 hostiana [C. 3 fulva], C. hostiana 3 lepidocarpa [C. ×leutzii] and C. flava 3 hostiana [C. 3 xanthocarpa]. The least variable traits, namely beak length, utricle length, ratio of beak length to the overall utricle length, female spike width, and width of the lowest bract, proved to be the most useful in delimiting the hybrids. Carex flava 3 hostiana specimens usually have long utricles and beaks, wide male and female spikes, as well as wide bracts and leaf blades. Carex hostiana 3 lepidocarpa specimens are characterized by relatively short beaks (with low ratio of beak length to the overall utricle length) and narrow bracts. Common features of C. demissa 3 hostiana specimens, on the other hand, are male spikes with long peduncles, usually longly parted female spikes and a long beak compared to the overall utricle length.     

Insight into genetic relation and diversity of cultivated and semi-domesticated under-utilized Crotalaria species gained using start codon targeted (SCoT) markers

To augment conventional crop improvement approaches in cultivated sunnhemp (Crotalaria juncea L.) and other under-utilized Crotalaria species, genetic diversity of 94 genotypes from seven Crotalaria species was studied using 20 Start Codon Targeted (SCoT) markers. High allele number (1.32), polymorphism information content (0.37) and resolving power (6.59) established SCoT as a reliable marker system for genetic analysis in Crotalaria. All the species except Crotalaria retusa L. exhibited high number of SCoT amplicons. Analysis of molecular variance revealed significant variability between (24.0%) the species as well as within species (76.0%). A cluster analysis identified distinct groups corresponding to the seven species and also identified sub-groups within the species. The sunnhemp cultivars were distant from the landraces, suggesting the need of population improvement using distantly related genotypes. Species relationship identified Crotalaria pallida Aiton to be a close relative of C. juncea. The results of principal coordinate analysis were comparable to that of cluster analysis, revealing high genetic variability in sunnhemp and other semi-domesticated Crotalaria species. The study further suggests some measure for conservation of genetic resources and genetic improvement of these species based on the results of diversity analysis.     

The systematic value of flower structure in Crotalaria and related genera of the tribe Crotalarieae (Fabaceae)

Flowers in the tribe Crotalarieae of the family Fabaceae are generally adapted to bee pollination mechanisms. Molecular systematics have recently provided a major step towards a profound insight into generic relationships, thereby creating the opportunity to re-evaluate the taxonomic and functional significance of flower structure in the tribe, with emphasis on the large genus Crotalaria. A representative sample of flowers from 211 species was dissected to record morphological character states. These data were supplemented from the literature to allow for generalizations for the tribe as a whole. Six structural–functional flower types were identified: (1) pump; (2) gullet; (3) hugging; (4) saddle; (5) tunnel and (6) brush (saddle and tunnel types described here for the first time). Crotalaria uniquely has the brush type, characterized by a rostrate keel, highly dimorphic anthers, stylar trichomes and elaborate callosities on the standard petal. Remarkably, Crotalaria and Bolusia are the only genera of the tribe Crotalarieae with callosities present in all of the species. In other genera, callosities are generally absent or infrequent. Trends towards specialization of pollination syndromes are apparent as assemblages of apomorphic states that co-occur in what we refer to here as “specialized flowers”; individual characters are labile or non-homologous (e.g. callosities) and diagnostically less valuable. Unique combinations of flower characters are often useful to support current generic concepts in Crotalarieae.