The application of safe for humans and the environment Polyversum antifungal agent containing living cells of Pythium oligandrum for biotransformation of prochiral ketones.

This report presents the whole-cell biotransformation of benzofuranyl-methyl ketone derivatives with the application of Polyversum antifungal agent containing Pythium oligandrum microorganism. Stereochemistry of the reduction of prochiral substrates was modified by the bioconversion conditions (concentration of reagents, a source of the carbon atom, biotransformation medium). In optimized conditions enantioselective process was noted. Secondary alcohols with excellent enantiomeric purity and high yields were obtained. The enantiomeric excess and conversion degree of 1-(benzofuran-2-yl)ethanol, 1-(7-ethylbenzofuran-2-yl)ethanol and 1-(3,7-dimethylbenzofuran-2-yl)ethanol were 99%/98.1%, 94%/94.4% and 99%/72.6%, respectively. In the presence of P. oligandrum, one of the enantiotopic hydrides of the dihydropyridine ring coenzyme is selectively transferred to a re side of the prochiral carbonyl group to give products with S configuration. This study demonstrates an inexpensive, eco-friendly approach in synthesis of optically pure benzofuran derivatives and can be an interesting alternative to organocatalysis. Furthermore, this method can be used in biotechnology processes due to its good chemical performance and a high degree of product isolation.

Influence of temperature on in vitro zoosporogenesis of Pythium insidiosum.

This study verified the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. P. insidiosum isolates (n = 26) were submitted to zoosporogenesis and incubated at 5°C, 15°C, 20°C and 37°C (1st stage). Grass fragments were evaluated under optical microscopy at 4, 8, and 24 hours of incubation. Afterward, all isolates were incubated at 37°C and assessed at the same periods of time (2nd stage). The development of hyphae, presence of vesicles, zoosporangia and zoospores were checked. Only the presence of short hyphae was observed at 5°C. At 15°C, the hyphae were either under development or elongated and two isolates produced zoospores. When the isolates were submitted to 20°C for 4 hours, the presence of long and mycelial hyphae, vesicles, zoosporangia and zoospores was observed, which also happened at the other periods evaluated. In the second stage, the isolates which were initially at 5°C and 15°C evidenced long developing hyphae with the presence of vesicles, zoosporangia, and zoospores within 4 hours of incubation, and these characteristics were kept at the other evaluated periods. The isolates kept at 37°C showed evident zoosporogenesis in the first 4 hours of evaluation. It was concluded that temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. On the other hand, 5°C and 15°C temperatures do not kill the microorganism.

Synthesis and antifungal activity of 2-allylphenol derivatives against fungal plant pathogens.

2-Allylphenol (2-AP) is an effective fungicide against a number of plant pathogens, which can be metabolized and bio-transformed to four chemical compounds by Rhizoctonia cerealis. To determine if its degradation affects antifungal activity, two major metabolites derived from 2-AP including 2-(2-hydroxypropyl) phenol and 2-(3-hydroxypropyl) phenol were synthesized. Inhibition of mycelial growth of several plant pathogens by the metabolites was evaluated, and structures of two metabolites were determined by hydrogen nuclear magnetic resonance (1H NMR). Among these metabolites, only 2-(2-hydroxypropyl) phenol inhibited test pathogens effectively. EC50 values of 2-(2-hydroxypropyl) phenol for inhibition of mycelial growth of R. cerealis, Pythium aphanidermatum, Valsa mali and Botrytis cinerea ranged from 1.0 to 23.5µg/ml, which were lower than the parental fungicide 2-AP that ranged from 8.2 to 48.8µg/ml. Hyphae of R. cerealis and P. aphanidermatum treated with 2-(2-hydroxypropyl) phenol were twisted. Newly developed hyphae were slender, twisted and swollen on the tip, while old hyphae were hollow and ruptured. This is the first report indicating the formation of 2-(2-hydroxypropyl) phenol may have contributed to toxicity of 2-allylphenol in control of plant pathogens.

Molecular and morphological characterization of three new species of Pythium from Iran: P. ershadii, P. pyrioosporum, and P. urmianum.

Three new species of Pythium: P. ershadii, P. pyrioosporum, and P. urmianum from soils of various regions in Iran are described and illustrated. These species are morphologically distinct from all other known species. Pythium ershadii is morphologically characterized by pyriform ornamented oogonia and rarely production of pyriform oospores. Pythium pyrioosporum differs from other species of the genus by the production of pyriform oospores and smooth walled oogonia, oospores with a tapering elongation toward a hypogynous antheridium and intercalary hypogynous antheridia. Pythium urmianum is distinguished by the presence of intercalary hypogynous antheridia, smooth walled oogonia formed laterally on hyphae or on short side branches and peanut-shaped oospores. Phylogenetic relationships of these new taxa with other Pythium species were investigated using internal transcribed spacers rDNA and partial coxI sequence data. The three species reside in clade E1 and are separated from closely related species.

Chlorophyll fluorescence imaging as a tool to monitor the progress of a root pathogen in a perennial plant.

MAIN CONCLUSION: The chlorophyll fluorescence parameter ΦNO is an excellent metric for the non-destructive monitoring of disease progression, measured over a broad range of light intensities. The suitability of the slow induction chlorophyll fluorescence parameters ΦPSII, ΦNPQ, and ΦNO to monitor in vivo disease progression in a host-root pathogen pathosystem was evaluated and compared to the established method of monitoring disease by measuring Fv/Fm. Using the infection of ginseng plants (Panax quinquefolius L.) with Pythium irregulare Buisman as a model, light response curves were used to establish the optimal irradiance for the resolution of differences between fluorescence parameters ΦPSII, ΦNPQ and ΦNO. As infection progressed only changes in ΦNO remained consistent with increased irradiance, and increased as infection progressed. Furthermore, ΦNO showed a high sensitivity for distinguishing increased disease load. In contrast, the magnitude in change of ΦPSII and ΦNPQ were sensitive to irradiance levels. The magnitude of increase in ΦNO per unit disease score was equivalent to the corresponding decline in Fv/Fm values. Thus ΦNO is as sensitive as Fv/Fm in monitoring biotic stress. The ability to measure ΦNO under a wide range of light intensities, including natural light, potentially without the need for dark adaptation, means that it can be used in the development of a general protocol for non-invasive, in vivo monitoring of plant health, from the laboratory to the field scale.

Pythium cederbergense sp. nov. and related taxa from Pythium clade G associated with the South African indigenous plant Aspalathus linearis (rooibos).

The genus Pythium consists of more than 120 species and is subdivided into 11 phylogenetic clades (A-K) based on internal transcribed spacer (ITS) region sequence data. Pythium clade G contains only seven known species, with most not being well described. Our study characterized 12 Pythium isolates from Aspalathus linearis (rooibos) that fit into clade G. Phylogenetic analyses of the ITS region and a combined phylogeny of four gene regions (ITS, ß-tubulin, COX1 and COX2 [cytochrome c oxidase subunits I, II]) identified five clade G subclades. The rooibos isolates formed two groups, Pythium Rooibos I (RB I) and II (RB II), that clustered into two separate clades within subclade 1. The nine Pythium RB I isolates formed a distinct clade from P. iwayamai and is described here as a new species, Pythium cederbergense sp. nov. The three Pythium RB II isolates had P. canariense and P. violae as their closest relatives and were genetically diverse, suggesting the presence of several new species or a species complex that cannot be resolved with the current data, thus precluding a species description of this group. Morphological analyses showed that P. cederbergense and Pythium RB II were indistinguishable from each other but distinct from known clade G species. Clade G studies are being hampered by imprecise morphological descriptions of P. violae, P. canariense and P. iwayamai and each species being represented by only one isolate. The P. cederbergense and Pythium RB II isolates all were nonpathogenic toward rooibos, lupin and oats seedlings. One oligonucleotide was developed for each of P. cederbergense and Pythium RB II, which was able to differentiate the isolates with DNA macro-array analyses.

Pythium polare, a new heterothallic oomycete causing brown discolouration of Sanionia uncinata in the Arctic and Antarctic.

Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis.

Microbial-induced carbon competition in the spermosphere leads to pathogen and disease suppression in a municipal biosolids compost.

The aim of this study was to understand whether competition for fatty acids in plant seed exudates by compost-derived seed-colonizing microbial communities could explain the suppression of plant infections initiated by sporangia of Pythium ultimum. The germination behavior of P. ultimum sporangia in response to cucumber seeds was measured to determine the impact of seed-colonizing microbes on pathogen suppression. Seed-colonizing microbial communities from municipal biosolids compost utilized cucumber seed exudates and linoleic acid in vitro, reducing the respective stimulatory activity of these elicitors to P. ultimum sporangial germination. However, when sporangia were observed directly in the spermosphere of seeds sown in the compost medium, levels of germination and sporangial emptying did not differ from the responses in sand. The percentage of aborted germ tubes was greater after incubating sporangia in compost medium for 12-h than the level of germ tube abortion when sporangia were incubated in sand. Abortion did not occur if previously germinated sporangia were supplemented with cucumber seed exudate. Furthermore, removal of cucumber seed exudate after various stages of germ tube emergence resulted in an increase in aborted germ tubes over time. Adding increasing levels of glucose directly to the compost medium alleviated germ tube abortion in the spermosphere and also eliminated disease suppression. These data fail to support a role for linoleic acid competition in Pythium seedling disease suppression but provide evidence for general carbon competition mediated by seed-colonizing microbial communities as a mechanism for the suppression of Pythium seed infections in municipal biosolids compost.

Larval Bradysia impatiens (Diptera: Sciaridae) potential for vectoring Pythium root rot pathogens.

A series of laboratory experiments were conducted to investigate the capacity of Bradysia impatiens (Johannsen) larvae to ingest propagules from two strains each of Pythium aphanidermatum (Edson) Fitzp. and P. ultimum Trow and transmit the pathogens to healthy geranium seedlings on a filter-paper substrate in petri dishes. The capacity of fungus gnat larvae to transmit P. aphanidermatum to seedlings rooted in a commercial peat-based potting mix and germination of Pythium oospores and hyphal swellings before and after passage through the guts of larval fungus gnats were also examined. Assays revealed that Pythium spp. transmission by larval fungus gnats varied greatly with the assay substrate and also with the number and nature of ingested propagules. Transmission was highest (65%) in the petri dish assays testing larvae fed P. aphanidermatum K-13, a strain that produced abundant oospores. Transmission of strain K-13 was much lower (<6%) in plug cells with potting mix. Larvae were less efficient at vectoring P. ultimum strain PSN-1, which produced few oospores, and no transmission was observed with two non-oospore-producing strains: P. aphanidermatum Pa58 and P. ultimum P4. Passage of P. aphanidermatum K-13 through larval guts significantly increased oospore germination. However, decreased germination of hyphal swellings was observed following larval gut passage for strains of P. ultimum. These results expand previous studies suggesting that larval fungus gnats may vector Pythium spp.

Two new species of Pythium, P. senticosum and P. takayamanum,isolated from cool-temperate forest soil in Japan.

Pythium senticosum and P. takayamanum spp. nov. were isolated from cool-temperate forest soil in Japan. P. senticosum can grow at 5 C and is fast growing at 25 C with a radial growth of 22.2 mm 24 h(-1). The species is morphologically characterized by ovoid to ellipsoid sporangia with apical papilla, ornamented oogonia with acute conical spines, and antheridia with broad attachment to oogonia. P. takayamanum is very different and can grow at 35 C. This species is morphologically characterized by its wavy antheridial stalks and ellipsoidal oogonia with constricted areas. Phylogenetic analyses of the ITS rDNA region and the partial COX2 gene showed that the two species are genetically distinct from each other and from their closest relatives. P. senticosum is closely related to P. dimorphum and P. undulatum whereas P. takayamanum is closely related to P. rhizosaccharum and P. parvum.

Morphological and phylogenetic analyses of Pythium species in South Africa.

The genus Pythium is important in agriculture, since it contains many plant pathogenic species, as well as species that can promote plant growth and some that have biocontrol potential. In South Africa, very little is known about the diversity of Pythium species within agricultural soil, irrigation and hydroponic systems. Therefore, the aim of the study was to characterise a selection of 85 Pythium isolates collected in South Africa from 1991 through to 2007. The isolates were characterised morphologically as well as through sequence and phylogenetic analyses of the internal transcribed spacer regions (ITS) and the 5.8S gene of the nuclear ribosomal DNA. Phylogenetic analyses showed that the isolates represented ten of the 11 published Pythium clades [Lévesque & De Cock, 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycological Research 108: 1363-1383]. Characterisation of isolates in clade D and J suggested that the phylogenetic concept of Pythium acanthicum and Pythium perplexum respectively, needs further investigation in order to enable reliable species identification within these clades. Our phylogenetic analyses of Pythium species in clade B also showed that species with globose sporangia group basal within this clade, and are not dispersed within the clade as previously reported. The 85 South African isolates represented 34 known species, of which 20 species have not been reported previously in South Africa. Additionally, three isolates (PPRI 8428, 8300 and 8418) were identified that may each represent putative new species, Pythium sp. WJB-1 to WJB-3.

Pythium delawarii--a new species isolated from soybean in Ohio.

A new species of Pythium isolated from soybean in Ohio is described. Pythium delawarii sp. nov. is characterized by globose internally proliferating sporangia, aplerotic oospores and diclinous antheridia that make broad lengthwise contact. Sporangia produce conspicuous papilla and germinate indirectly by producing zoospores via a vesicle and proliferate internally or the sporangia germinate directly with either one or more germ tubes. Pythium delawarii is pathogenic on soybean causing damping-off of seedlings. This oomycete can grow at 10-34 C with an optimum of 28 C. The sequence of the ITS1, 5.8S and ITS2 region of the rDNA did not match the sequence of any known Pythium species but was similar to P. citrinum, P. litorale and P. sterilum. P. delawarii can be distinguished from these three species based on the presence of aplerotic oospores and diclinous antheridia and the absence of hypogynous antheridia. Therefore biological, morphological and molecular data support the recognition of a new species.

Pythium recalcitrans sp. nov. revealed by multigene phylogenetic analysis.

A new species of Pythium collected from grapevine roots (Vitis vinifera) in South Africa and roots of common beet (Beta vulgaris) in Majorca, Spain, is described. The phylogenetic position of the new species was investigated by multigene sequence analyses of the internal transcribed spacers (ITS1 and ITS2) of the rDNA region, as well as three other nuclear and three mitochondrial coding genes. Maximum likelihood phylogenetic analyses based on ITS rDNA and concatenated beta-tubulin and cytrochrome c oxidase II alignment place Pythium recalcitrans together with P. sylvaticum and P. intermedium. Pythium recalcitrans sp. nov. is morphologically almost indistinguishable from other Pythium species that only form hyphal swellings in culture. However its species status is justified by the distinctiveness of the DNA sequences in all the genes examined. In culture P. recalcitrans exhibits fast radial growth, abundant spherical to subglobose hyphal swellings but produces no zoosporangia. Sexual structures are not seen in agar media but form in autoclaved grass blades floated on water. Multiple antheridia (1-7) are encountered with most of them diclinous and crook-necked. Oospores are thin-walled and either aplerotic or plerotic. P. recalcitrans was pathogenic to seedlings of Beta vulgaris and Solanum lycopersicum.

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae).

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.

Morphological and molecular characterization of an equine isolate of Pythium insidiosum and comparison with the first human isolate from the same geographic region.

Pythium insidiosum causes pythiosis, a life-threatening disease that occurs in tropical regions and affects man and animals. Although pythiosis in Brazil had been described in various animal species, the first human case was only recently reported. The present study aimed to characterize the morphologic and molecular characteristics of a new equine isolate of P. insidiosum and compare them with those of the first Brazilian human isolate. Both isolates were recovered from the same region of the country. Macroscopic and microscopic features were evaluated in two culture media. Sporangia formation and zoospore release were obtained after culturing the isolates with fragments of grasses and crops in an appropriate liquid induction medium. The molecular analysis of the isolates consisted of the complete sequencing of the ITS-5.8S rDNA region and sequences of both showed identical composition of 836 bp and 99% similarity with the isolates M16, 65, M12, 339 and 394 deposited at GenBank. Simple mycological procedures such as the production of sporangia and zoospores may distinguish P. insidiosum from zygomycetes. The rDNA sequencing indicates that, in Brazil, both humans and animals might be infected by a common genotype of the pathogen.

Pythium cryptoirregulare, a new species within the P. irregulare complex.

Pythium identification is based on several characteristics with considerable variation, particularly in Pythium irregulare Buis. as currently recognized. Thirty-one isolates of Pythium irregulare Buis. from various hosts and geographic regions were compared by genetic analysis of multiloci DNA fingerprints, sequence analysis of nuclear and mitochondrial genes and morphological and growth rate studies. Previous research indicated two distinct groupings within the species, P. irregulare sensu stricto and a clade referred to here as Pythium sp. Parsimony analyses of 338 AFLP markers divided P. irregulare s.l. into two clades. Comparison of the allele frequencies of 236 polymorphic AFLP loci revealed significant differences between them. The two clades differed in the frequencies of 182 (77%) alleles. P. irregulare s.s. had 122 (52%) polymorphic loci while Pythium sp. had 205 (87%). Pythium sp. had one fixed allele and 79 polymorphic loci absent in P. irregulare s.s. P. irregulare s.s. displayed 16 polymorphic loci absent in Pythium sp. Parsimony and distance analyses of the ribosomal intergenic transcribed spacers (ITS) and the cox II gene sequences support the separation of P. irregulare s.s. and Pythium sp. Amplicon length in P. irregulare s.s. ITS sequences were 936-938 bp and 936-949 bp in Pythium sp. The two clades were separated by two fixed insertion/deletion mutations, nine fixed nucleotide substitutions in the ITS region and three fixed single nucleotide substitutions in the cox II sequences. Average growth rates of the groups differed at 10, 30 and 36 C but not at 15, 21 or 25 C. Statistically significant differences were found in oogonium, oospore and ooplast diameters, antheridial cell length and in ooplast index. We propose that a new species, Pythium cryptoirregulare, be delineated from Pythium irregulare sensu stricto.

Effect of fungal hyphae on the access of bacteria to phenanthrene in soil.

The effect of fungal hyphae on the mobilization of soil-dwelling bacteria and their access to hydrophobic phenanthrene in soil was tested in columns containing air-filled agricultural soil. The experimental design included a spatial separation between zones of bacterial inoculation and contamination. Motile Pseudomonas putida PpG7 (NAH7) and fast-growing, hydrophilic Pythium ultimum were used as the model phenanthrene-degrading and vector organisms, respectively. Efficient translocation of strain PpG7 in the range of centimetres in presence of P. ultimum indicated that the fungal mycelia bridged air-filled pores and thereby provided a continuous network of water-paths that enabled bacteria to spread in the soil. Biodegradation of the soil-associated phenanthrene was found only in the presence of the fungal mycelia, hence proving that the fungal network facilitated the access of the bacteria to the contaminant. Our data suggest that the specific stimulation of indigenous fungi is a promising method to mobilize pollutant degrading bacteria and thereby improve soil bioremediation in-situ.

Orbital pythiosis: a non-fungal disease mimicking orbital mycotic infections, with a retrospective review of the literature.

In the past decade there have been four well-documented cases of orbital pythiosis caused by Pythium insidiosum. All were recorded in apparently healthy children. Although pythiosis seems to be a rare infection in humans, we recently conducted a review of the medical literature to investigate misdiagnosed cases of orbital pythiosis in the past 100 years in children. To track putative cases of orbital pythiosis, we first identified orbital cases initially diagnosed as fungal infections. We were particularly interested in cases (a) involving apparently young healthy hosts, (b) the presence of hyaline, aseptate hyphal elements in the infected tissues, (c) the morphological features of the hyphal elements, (d) the presence of an eosinophilic granulomatous reaction with the Splendore-Hoeppli phenomenon around the mycelial elements, (e) resistance to antifungal therapy, (f) outcome after therapy, if any, and (g) cultural strategies. This study showed that indeed, there had been five other recorded cases of orbital infections, all in young children in the USA, with characteristics consistent with infections caused by P. insidiosum. The reports had described those cases of orbital-cranial-arterial diseases as patients with aspergillosis (one case), penicilliosis infection (one case), and zygomycosis (three cases). We reviewed those anomalous cases and discuss details about their clinical, pathologic, therapeutic, and etiologic evidence used to reclassify them as putative cases of orbital pythiosis.

Molecular phylogeny and taxonomy of the genus Pythium.

The phylogeny of 116 species and varieties of Pythium was studied using parsimony and phenetic analysis of the ITS region of the nuclear ribosomal DNA. The D1, D2 and D3 regions of the adjacent large subunit nuclear ribosomal DNA of half the Pythium strains were also sequenced and gave a phylogeny congruent with the ITS data. All the 40 presently available ex-type strains were included in this study, as well as 20 sequences of recently described species from GenBank. Species for which no ex-type strains were available were represented by either authentic strains (6), strains used in the 1981 monograph of the genus by van der Plaats-Niterink (33), or strains selected on morphological criteria (17). Parsimony analysis generated two major clades representing the Pythium species with filamentous or globose sporangia. A small clade of species with contiguous sporangia was found in between the two main clades. A total number of 11 smaller clades was recognized, which often correlated with host-type or substrate and in several cases with a subset of morphological characters. Many characters used in species descriptions, such as antheridium position, did not correlate with phylogeny. A comparison of the ex-type and representative strains with all ITS sequences of Pythium in GenBank revealed limited infraspecific variation with the exception of P. rostratum, P. irregulare, P. heterothallicum, and P. ultimum. The total number of species examined was 116 (including 60 ex-type strains). Twenty-six species had ITS sequences identical or nearly identical to formerly described species, suggesting possible conspecificity. The importance of comparing ITS sequences of putative new species to the now available ITS database in order to avoid unwarranted new species names being introduced.

Pythium regulare sp. nov., isolated from the Canary Islands, its taxonomy, its region of rDNA, and comparison with related species.

Pythium regulare (CI-34) was isolated from some soil samples taken in the Canary Islands (Spain). This new species is very closely related to P. irregulare isolated from pea roots in The Netherlands by Buisman in 1927. The species of Pythium are members of the kingdom Chromista. Pythium regulare is characterized by its ornamented oogonia bearing blunt or digitate spines, and its non-sporulating type of sporangia or hyphal bodies, its aplerotic oospores, its monoclinous and diclinous antheridia that at times crowd around the oogonia. The taxonomic description of this oomycete, the PCR of the internal transcribed region (spacers ITS1, ITS2, and the gene 5.8 S) of its ribosomal nuclear DNA as well as the nucleotide sequences, and its comparison with related species are given here.