Cytological analysis of ginseng carpel development

Panax ginseng Meyer, commonly known as ginseng, is considered one of the most important herbs with pharmaceutical values due to the presence of ginsenosides and is cultivated for its highly valued root for medicinal purposes. Recently, it has been recognized that ginseng fruit contains high contents of triterpene such as ginsenoside Re as pharmaceutical compounds. However, it is unclear how carpel, the female reproductive tissue of flowers, is formed during the three-year-old growth before fruit is formed in ginseng plants. Here, we report P. ginseng carpel development at the cytological level, starting from the initial stage of ovule development to seed development. The carpel of P. ginseng is composed of two free stigmas, two free styles, and one epigynous bilocular ovary containing one ovule in each locule. Based on our cytological study, we propose that the female reproductive development in P. ginseng can be classified into seven stages: early phase of ovule development, megasporogenesis, megagametogenesis, pre-fertilization, fertilization, post-fertilization, and seed development. We also describe the correlation of the female and male gametophyte development and compare morphological differences in carpel development between ginseng and other higher plants. One unique feature for ginseng seed development is that it takes 40 days for the embryo to develop to the early torpedo stage and that the embryo is small relative to the seed size, which could be a feature of taxonomic importance. This study will provide an integral tool for the study of the reproductive development and breeding of P. ginseng.     

Molecular characterisation of four echinostomes (Digenea: Echinostomatidae) from birds in New Zealand,with descriptions of <Emphasis Type="Italic">Echinostoma novaezealandense</Emphasis> n. sp. and <Emphasis Type="Italic">Echinoparyphium poulini</Emphasis> n. sp.

Morphological and molecular characterisation of echinostome specimens (Digenea: Echinostomatidae) recovered in one Anas platyrhynchos L. and one Cygnus atratus (Latham) (Anseriformes: Anatidae) from New Zealand revealed the presence of two known species, Echinostoma miyagawai Ishii, 1932 and Echinoparyphium ellisi (Johnston & Simpson, 1944) and two species new to science. Comparative morphological and phylogenetic analyses supported the distinct species status of Echinostoma novaezealandense n. sp. ex Branta canadensis (L.), A. platyrhynchos and C. atratus, and Echinoparyphium poulini n. sp. ex C. atratus. Echinostoma novaezealandense n. sp., a species of the “revolutum” species complex characterised by the possession of a head collar armed with 37 spines, keyed down to E. revolutum but was distinguished from the latter in having a much narrower body with almost parallel margins, longer oesophagus, wider cirrus-sac, larger seminal vesicle, much smaller ventral sucker, ovary, Mehlis’ gland and testes, more anteriorly located ovary and testes, and distinctly smaller eggs (81–87 × 42–53 vs 106–136 × 55–70 µm). This new species appears similar to Echinostoma acuticauda Nicoll, 1914 described in Australia but differs in having a longer forebody, more posteriorly located ovary and testes, and much smaller eggs (81–87 × 42–53 vs 112–126 × 63–75 µm). Echinoparyphium poulini n. sp. is differentiated from the four species of Echinoparyphium possessing 37 collar spines considered valid as follows: from E. chinensis Ku, Li & Chu, 1964 in having a much smaller body, four (vs five) angle spines and simple seminal vesicle (vs bipartite); from E. schulzi Matevosyan, 1951 in having a less robust body at a comparable body length, much smaller ventral sucker, ovary and testes, and longer but narrower eggs (87–109 × 50–59 vs 70–85 × 60–84 µm); and from the two smaller forms, E. serratum Howell, 1968 and E. aconiatum Dietz, 1909, in a number of additional metrical features correlated with body size and especially in the possession of much larger collar spines. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for representative isolates of the four species and analysed together with sequences for Echinostoma spp. and Echinoparyphium spp. available on GenBank. Phylogenetic analyses based on the mitochondrial nad1 gene revealed congruence between the molecular data and species identification/delineation based on morphology; this was corroborated by the 28S rDNA sequence data.     

<Emphasis Type="Italic">Parapharyngodon hugoi</Emphasis> n. sp., a new nematode (Oxyuroidea: Pharyngodonidae) of the tree frog <Emphasis Type="Italic">Trachycephalus typhonius</Emphasis> (Linnaeus) from the Brazilian Pantanal,including a key to the congeners from amphibians of the American continent

A new nematode Parapharyngodon hugoi n. sp. (Oxyuroidea: Pharyngodonidae) is described parasitising the large intestine of the tree frog Trachycephalus typhonius (Linnaeus) (Anura: Hylidae) from the wetlands of Pantanal, State of Mato Grosso do Sul, Brazil. The new species exhibits a unique structure of the posterior cloacal lip in males, which is supported by a rigid V-shaped structure. Parapharyngodon hylidae parasitic in hylid frogs, including T. typhonius, from Mexico, is the most similar congener to P. hugoi n. sp. but is distinguished from the new species by the presence of a gubernaculum (vs absence), by the lateral alae in males ending far anterior to cloacal opening (vs near to it) and because in gravid females the ovaries encircle the oesophageal corpus. Additionally, the new species differs from its congeners as well as from species of Thelandros Wedl, 1862, a very closely related genus, by the combination of features such as spicule length, number of caudal papillae, morphology of the anterior cloacal lip, which is echinate, and position of ovaries. The geographical distribution of hosts seems to play an important role in the speciation process of Parapharyngodon spp.; however, due the lack of molecular data this issue along with the validity of both Thelandros and Parapharyngodon are still questions to be solved in the future, after improvement of the genetic database. A key to the species of Parapharyngodon parasitic in amphibians from the American continent is provided.     

Dynamics of Malaria Mosquito Species Composition in Siberian Populations Detected by Restriction Analysis

The peculiarities of the dynamics of malaria mosquito species proportions were studied in natural populations. Twenty-one collections from five larval habitats of the Anopheles maculipennis complex malaria mosquitoes were taken in the vicinity of Novosibirsk (Russia). It was detected that the Anopheles messeae and An. daciae are dominant species in the collected samples. Three An. beklemishevi individuals were also detected. The dynamics of the species proportions within the reproduction seasons for a number of years and also the differences between localities in the species composition were studied. It was revealed that the An. daciae proportion is maximal in July and falls in August. The species proportions can change sharply from year to year. For example, the An. messeae species prevailed in localities on the left bank of Novosibirsk Reservoir in 2013 and 2014 with the frequency of 54–68%, while An. daciae began to prevail on this territory in 2016 with the frequency of 73–85%. Our data on the proportions of species in different reservoirs were compared with data of other authors for the collections of malaria mosquitoes of the Anopheles maculipennis complex in Russia (Tomsk oblast) and in Germany. A high correspondence of models provided by these authors for the territory that we studied was demonstrated. Thus, the ecological peculiarities of the An. messeae and An. daciae species are highly stable even in geographically and climatically distant localities. An increase in the portion of relatively anthropophilic An. daciae in the middle of summer can be a risk factor relative to malaria transmission in this period of time.     

Effect of habitat structure on reproduction and prey capture of a rare carnivorous plant, <Emphasis Type="Italic">Pinguicula lutea</Emphasis>

Habitat degradation is one of the greatest threats to biodiversity worldwide and the main contributor to the decline of many carnivorous plant species. For carnivorous plants in the southeastern United States, including many Pinguicula species (butterwort, Lentibulariaceae), degradation via altered fire regime has been implicated in their decline. Despite this decline, limited empirical research has been conducted examining the influence of habitat structural changes (through natural succession or human management) on reproduction and prey capture by carnivorous plants. The objectives of our study were to compare reproduction and prey capture for Pinguicula lutea (yellow butterwort) in habitats with different vegetation structures in the Florida Panhandle, where differences were largely due to management history. Pinguicula lutea is a self-compatible carnivorous plant that inhabits fire-dependent longleaf pine savannas of the southeastern United States and is threatened in the state of Florida. In 2014 and 2015, 13 sites were identified occupying three different habitat structures: maintained (intermittently mowed), grassy (dominated by Aristida stricta var. beyrichiana), and woody (encroachment by Hypericum and Ilex). Reproductive output was determined by assessing fruit set and ovule fertilization rate at each site. Additionally, prey availability and prey capture were assessed at each habitat site. In general, there were no differences in either measure of reproduction across habitat structure types. There were differences in prey abundance of Collembola, Diptera, and total arthropods both in terms of availability and capture. Total arthropod availability and prey capture were lowest in grassy sites compared to maintained habitat sites and woody habitat sites. Microclimatic conditions associated with each habitat structure and leaf morphology or physiology could explain the observed arthropod abundance and prey capture patterns. This study is the first ecological assessment of plant–insect interactions for Pinguicula species of the southeastern US and highlights the importance of habitat quality and management for this understudied group of carnivorous plants.     

Spatio-temporal expression profile of matrix metalloproteinase (Mmp) modulators Reck and Sparc during the rat ovarian dynamics

Background

Matrix metalloproteinases (Mmps) and their tissue inhibitors (Timps) are widely recognized as crucial factors for extracellular matrix remodeling in the ovary and are involved in follicular growth, ovulation, luteinization, and luteolysis during the estrous cycle. Recently, several genes have been associated to the modulation of Mmps activity, including Basigin (Bsg), which induces the expression of Mmps in rat ovaries; Sparc, a TGF-β modulator that is related to increased expression of Mmps in cancer; and Reck, which is associated with Mmps inhibition. However, the expression pattern of Mmp modulators in ovary dynamics is still largely uncharacterized.

Methods

To characterize the expression pattern of Mmps network members in ovary dynamics, we analyzed the spatio-temporal expression pattern of Reck and Sparc, as well as of Mmp2, Mmp9 and Mmp14 proteins, by immunohistochemistry (IHC), in pre-pubertal rat ovaries obtained from an artificial cycle induced by eCG/hCG, in the different phases of the hormone-induced estrous cycle. We also determined the gene expression profiles of Mmps (2, 9, 13 14), Timps (1, 2, 3), Sparc, Bsg, and Reck to complement this panel.

Results

IHC analysis revealed that Mmp protein expression peaks at the early stages of folliculogenesis and ovulation, decreases during ovulation-luteogenesis transition and luteogenesis, increasing again during corpus luteum maintenance and luteolysis. The protein expression patterns of these metalloproteinases and Sparc were inverse relative to the pattern displayed by Reck. We observed that the gene expression peaks of Mmps inhibitors Reck and Timp2 were closely paraleled by Mmp2 and Mmp9 suppression. The opposite was also true: increased Mmp2 and Mmp9 expression was concomitant to reduced Reck and Timp2 levels.

Conclusion

Therefore, our results generate a spatio-temporal expression profile panel of Mmps and their regulators, suggesting that Reck and Sparc seem to play a role during ovarian dynamics: Reck as a possible inhibitor and Sparc as an inducer of Mmps.

     

Diversity within the <Emphasis Type="Italic">Hygrophorus agathosmus</Emphasis> group (Basidiomycota,Agaricales) in Northern Europe

The North European species of the Hygrophorus agathosmus group in subsection Tephroleuci were studied. Three new species are identified based on morphology, ecology and sequence data. Two species are associated with Pinus spp. One of these is described here as H. suaveolens, while the other one is only known from one locality in the Nordic countries and seems to have a more South European distribution range. A closely related sister species to H. agathosmus is identified based on ITS sequence data, H. cf. agathosmus. It is confirmed to have an intercontinental distribution range and to be associated with Picea spp. probably on more acidic to neutral soil, whereas H. agathosmus s.s. has a more limited North-East European distribution range and occurs in older and rich Picea abies forests. A neotype for H. agathosmus is here selected from South Sweden. Hygrophorus agathosmus f. albus and H. agathosmus f. aureofloccosus are confirmed as forms. No genetic differences in the ITS region between specimens with grey cap colour and the two forms were observed. A key to the species in Northern Europe is provided.     

Revision of the systematics of algae in the order Laminariales (Phaeophyta) from the Far-Eastern Seas of Russia on the basis of molecular-phylogenetic data

An overview of the literature changes in the systematics of algae in the order Laminariales (Phaeophyta) based on molecular phylogentic data is given. In a recent taxonomic revision by Lane et al., [45], the number and status of the families traditionally included in the order have been revised. One family was transferred to the order Tilopteridales; a new family, the Costariaceae, was described; and the genus Laminaria was split into 2 genera, Laminaria and a newly resurrected genus Saccharina. These innovations have necessitated a systematic revision of the Far Eastern species of the Laminariales. Our genetic studies indicate that 2 species of Laminaria and 12 intraspecific taxa (1 subspecies and 11 forms) from the Russian Pacific coasts should be transferred to the genus Saccharina. The following new nomenclatural combinations are proposed: Saccharina bongardiana, comb. nov. (including 4 forms: f. bifurcata, f. subsessilis, f. subsimplex, f. taeniata) and Saccharina gurjanovae, comb. nov. (including f. lanciformis). In addition, new nomenclature combinations are proposed for intraspecific taxa of the Laminaria species (L. angustata, L. cichorioides, L. japonica) that have already been transferred to the genus Saccharina [45]. These include S. angustata subsp. sibirica, comb. nov., 4 new combinations for the forms of S. cichorioides (f. coriacea, f. sacchalinensis, f. sikotanensis, and f. sinuicola), and 2 new combinations for the forms of S. japonica (f. diabolica, and f. longipes). The taxonomic status of the rest of the members of the Laminariales known from the seas of the Russian Far East is discussed. Laminariaceous algae in this area represent 6 of the 8 known families currently included in the Laminariales (Chordaceae, Pseudochordaceae, Alariaceae, Arthrothamnaceae, Laminariaceae, and Costariaceae).     

Species radiation in the Alps: multiple range shifts caused diversification in Ringlet butterflies in the European high mountains

The distributions of European high mountain species are often characterised by small and geographically isolated populations and, in many cases, have highly complex biogeographic histories. The butterfly genus Erebia represents one of the best examples for small-scale diversification in the European high mountain systems and therefore to understand speciation processes and associated range dynamics of high mountain species. In this study, we analysed 17 polymorphic allozyme loci of 1731 individuals from 49 populations representing four species, one of which has three subspecies: Erebia nivalis; Erebia tyndarus; Erebia ottomana; and Erebia cassioides cassioides, Erebia cassioides arvernensis, and Erebia cassioides neleus. Samples were collected in the high mountain systems of Europe (i.e. Pyrenees, Massif Central, Alps, Apennines, Carpathians, Balkan high mountains). Genetic analyses supported all previously accepted species. However, the genetic differentiation within E. cassioides sensu lato into three geographically delimited groups is justifying species rank: E. arvernensis distributed in the Pyrenees, Massif Central and western Alps; E. cassioides sensu stricto in the eastern Alps and Apennines; and E. neleus in the Balkan mountains and the south-western Carpathians. While the differentiation between western Alps and Massif Central as well as eastern Alps and Apennines was low, the Pyrenees as well as the south-western Carpathians were significantly differentiated from the other regions within the respective taxon. In general, the differentiation among the populations of E. neleus was stronger than between populations of the other taxa. Within E. cassioides, we found a west-east gradient of genetic similarity over the eastern Alps. Based on the obtained genetic structures, we are able to delineate glacial refugia and interglacial range modifications. Based on the genetic structures and genetic diversity patterns, we conclude that, triggered by the glacial-interglacial cycles, repeated range modifications have taken place with subsequent differentiation and speciation in the region of the Alps and Balkans. Colonisations to Pyrenees (E. arvernensis pseudomurina, E. arvernensis pseudocarmenta), Massif Central (E. ottomana tardenota, E. a. arvernensis) and Apennines (E. cassioides majellana) appear to be recent and most probably not older than the last interglacial period.     

Chromosomal distribution of H3K4me2, H3K9me2 and 5-methylcytosine: variations associated with polyploidy and hybridization in <Emphasis Type="Italic">Brachiaria</Emphasis> (Poaceae)

Key message

Assessment of chromosomal distribution of modified histones and 5-methylcytosine shown that there are diversification of chromosomal types among species of Brachiaria and its interspecific hybrids.

Abstract

Histone post-translational modifications and DNA methylation are epigenetic processes that are involved in structural and functional organization of the genome. This study compared the chromosomal distribution of modified histones and 5-methylcytosine (5-mCyt) in species and interspecific hybrids of Brachiaria with different ploidy levels and reproduction modes. The relation between H3K9me2 and 5-mCyt was observed in the nucleolus organizer region, centromeric central domain and pericentromeric region. H3K4me2 was detected in euchromatic domains, mainly in the terminal chromosomal regions. Comparison of chromosomal distribution among species and hybrids showed greater variation of chromosomal types for the H3K9me2 in B. decumbens (tetraploid and apomictic species) and the 963 hybrid, while, for the H3K4me2, the variation was higher in B. brizantha and B. decumbens (tetraploid and apomictic species) and 963 hybrid. The chromosome distribution of 5-mCyt was similar between B. brizantha and B. decumbens, which differ from the distribution observed in B. ruziziensis (diploid and sexual species). Significant alterations in DNA methylation were observed in the artificially tetraploidized B. ruziziensis and in the interspecific hybrids, possibly as result of hybridization and polyploidization processes. The monitoring of histone modifications and DNA methylation allowed categorizing nuclear and chromosomal distribution of these epigenetic marks, thus contributing to the knowledge of composition and structure of the genome/epigenome of Brachiaria species and hybrids. These data can be useful for speciation and genome evolution studies in genus Brachiaria, and represent important markers to explore relationships between genomes.

     

Autecology of a new species of carnivorous marsupial,the endangered black-tailed dusky antechinus (<Emphasis Type="Italic">Antechinus arktos</Emphasis>), compared to a sympatric congener,the brown antechinus (<Emphasis Type="Italic">Antechinus stuartii</Emphasis>)

The carnivorous marsupial Antechinus are one of the few mammal genera known to exhibit the phenomenon of semelparous reproduction, where all males die at the end of a frenetic annual mating period. The genus Antechinus has recently been revised, seeing four new species named and one existing subspecies raised to species status. Here, we present the first ecological assessment of one new species, the endangered black-tailed dusky antechinus (Antechinus arktos), based on a 2-year mark-recapture study of two proximate sites within the cloud forest of Springbrook National Park, south-east Queensland, Australia. We also present comparative ecological data from a sympatric congener, Antechinus stuartii. In total, 103 A. arktos (49 male; 54 female) and 2125 A. stuartii (1229 male; 896 female) captures were made over 16,630 trap nights at the two sites. The occurrence and synchrony of reproductive events observed in A. arktos closely parallel the semelparous reproductive strategy exhibited by all congeners studied to date. The A. arktos populations mate during mid-September, with birth of up to six young occurring during mid-October. The low abundance and fecundity, together with a limited and apparently retracting distribution, suggest A. arktos is under threat of climate-induced extinction in the coming decades.     

Hope springs eternal in the starfish gonad: preserved potential for sexual reproduction in a single-clone population of a fissiparous starfish

Among echinoderms, asexual reproduction by fission occurs in few species. This strategy is considered a temporary response to stressful conditions and usually alternates with sexual reproduction events; thus, monoclonal populations are extremely rare. The occurrence of a single-clone population of the starfish Coscinasterias tenuispina at Llançà (NW Mediterranean) allowed us to study intra-clonal variation of the reproductive cycle during a two-year study. The few developed gonads (all male) were found in winter months, coinciding with the minimum photoperiod (ρ = ?0.82; P < 0.001) and lowest temperatures (ρ = ?0.75; P < 0.001), only in best-fed individuals, indicating that food availability influences individual ability for gonad development. Fissiparity happened throughout all the sampled period, but its rate increased with warm temperatures (ρ = 0.68; P < 0.0001). In contrast to what has been reported in other species, no correlation between fission rates and population density was found. The population was maintained over time by asexual reproduction and remained monoclonal. Although sexual reproduction has probably not occurred in this all-male population for a long time, the ability to yearly produce mature gonads is retained by some individuals, indicating that potential to reproduce sexually may be preserved, even in the case of strictly asexual populations.     

Genetic structure of diploid-polyploid complex of spined loach genus <Emphasis Type="Italic">Cobitis</Emphasis> (<Emphasis Type="Italic">Cypriniformes,Cobitidae</Emphasis>) in the Lower Danube

The extremely high diversity of spined loach biotypes in the Lower Danube has been detected by biochemical genetic investigation and cytometric analysis of 358 specimens collected in the riverbed and shallow channels. Along with two diploid species (C. elongatoides and C. “tanaitica”), six hybrid forms were revealed, namely, diploid C. elongatoides-“tanaitica”; triploid C. 2 elongatoides-“tanaitica,” C. elongatoides-2 “tanaitica,” and C. 2 elongatoides-species-1; and tetraploid C. 3 elongatoides-“tanaitica” and C. elongatoides-species-2-2 “tanaitica.” In addition, specimens with recombinant genotypes were also found. In spite of the apomictic mode of reproduction, the polyploids did not possess clonal structure, but according to the level of polymorphism and the genotype distribution, they were isomorphous to parental diploid species. Thus, in contrast to the polyploidy in Cobitids of the Dnieper, which have appeared in the basin due to the expansion, the polyploids of the Lower Danube are autochthonous and were derived by crossing with local diploid species. The process is apparently proceeds without any limitations.     

<Emphasis Type="Italic">Angiostoma namekuji</Emphasis> n. sp. (Nematoda: Angiostomatidae) from terrestrial slugs on Oshiba Island in the Seto Inland Sea,Japan

A new species of nematode, Angiostoma namekuji n. sp. (Angiostomatidae: Rhabditida), is described from the intestinal lumen of the terrestrial slug Philomycidae gen. sp. collected from Oshiba Island in the Seto Inland Sea, Hiroshima Prefecture, Japan. The new species is recognized by the following characteristics: body length 2,782–3,599 (mean 3,240) μm (male); 4,666–5,532 (5,030) μm (female); lateral field present; pharyngeal corpus with valves in the bulb; male with short tail, c = 35–57 (48), with one denticle; and seven pairs of genital papillae arranged as 1+2/3+1; female with tail having small denticles on distal tip; uterus c.50% of the body size; each ovary long, starting near vulva, not coiled, reflexed and reaching uterus; ovaries not crossing each other. Our phylogenetic tree based on sequences of the nuclear 28S ribosomal RNA gene supported the generic allocation of the new species in Angiostoma Dujardin, 1845.     

Population demographics and trade-offs to reproduction of an invasive and noninvasive species of <Emphasis Type="Italic">Rubus</Emphasis>

Do trade-offs between growth and reproduction differ between an invasive and noninvasive plant species and how do such trade-offs relate to population demographics? To help address these questions, we compared demographics for an invasive plant species, Rubus discolor, with a noninvasive congener, R. ursinus, in several populations of varying density. Removal of floral buds from reproductive canes increased the size of juvenile canes that arose from clonal sprouting in R. ursinus, suggesting a trade-off between current reproduction and growth. Removal of floral buds had no effect on growth of R. discolor. R. ursinus displayed trade-offs between reproduction (sexual and vegetative) and future growth based on negative correlations between leaf area production and both clonal sprouting and seedling production during the previous year. R. discolor did not exhibit these trade-offs. Both species had high population growth rates in low-density populations, but exhibited little or no growth in high-density populations. A life table response experiment was used to determine the underlying cause for the effect of density on population growth. For R. ursinus, lack of population growth in high-density populations was due primarily to increased mortality of clonally sprouting canes, while for R. discolor, it was due to decreased clonal cane production. Elasticity analysis revealed that clonal growth was more important than sexual reproduction for population growth of both species. However, elasticity values for sexual reproduction in R. discolor were greater in high- than low-density populations. This suggests an increased reliance on sexual reproduction in populations that had reached stable sizes, which could increase the capacity of R. discolor to disperse to new sites. Elasticity analyses were also used to simulate the efficacy of various control strategies for R. discolor. Control of this species could be attained by reducing clonal production within existing populations while reducing seed production to limit establishment of new populations.     

Seasonal variations in the fucoidan content of brown algae from Peter the Great Bay,Sea of Japan

The seasonal dynamics in the level of fucose-containing sulfated polysaccharides (fucoidans) have been studied in three brown alga species from Peter the Great Bay, Sea of Japan: Saccharina japonica, Sargassum pallidum, and Stephanocystis crassipes. In the latter two species, seasonal variations in the fucoidan content have been analyzed for the first time. During the year, the level of fucoidan in S. japonica varied from 0.98 to 4.19% dry weight; in S. crassipes, from 2.50 to 5.23%; in S. pallidum, from 1.86 to 6.31%. The maximum amount of this polysaccharide is accumulated in algae during reproduction: in August–October for S. japonica; in June and July for S. crassipes; and in July for S. pallidum. No significant correlation has been found between the level of fucoidan in algal tissues and the annual dynamics in sea water temperature, salinity, and concentration of biogenic elements. The obtained data can be helpful in determining the time to harvest algae for fucoidan extraction.