Phosphate solubilization by Penicillium spp. isolated from soil samples of Indian Himalayan region

A total of 246 fungal isolates representing 36 genera and 72 species were isolated from the soil samples collected from Indian Himalayan region. Twenty-one species belonged to the genus Penicillium alone. All the Penicillium species were screened for phosphate solubilizing activity on Pikovskaya agar at 21 °C. Eight species of Penicillium, exhibiting formation of halos (zone of solubilization) around the fungal colonies in qualitative plate assays, were selected for quantitative estimations. In quantitative estimations that were conducted upto day 30 (at 3 days interval), seven species of Penicillium brought maximum solubilization after day 15, while P. oxalicum showed maximum solubilization after day 21 of incubation. The increase in solubilization coincided with decrease in pH of the broth. Acid phosphatase activity was 1.5–2.0 times higher in comparison to alkaline phosphatase. Many of these species showed wide range of tolerance for temperature, pH and salt concentration.     

Piper regnellii extract biopolymer‐based microparticles: production,characterization and antifungal activity

Aims

This study aims to improve characteristics of Piper regnellii extract to make it applicable in formulations to treat dermatophytosis, also known as ringworm.

Methods and Results

Microparticles (MPs) were produced by spray drying with gelatin, alginate and chitosan as encapsulating agents; characterized by scanning electron microscopy, encapsulation efficiency, thermal analyses and X‐ray diffraction; and tested against Trichophyton rubrum by broth microdilution. Produced MPs had a mean diameter less than 2 μm, an increase in stability and release of the extract and good results for encapsulation efficiency, being 85·6% to gelatin MP, 71·3% to chitosan MP and 60·6% to alginate. MPs preserved the antifungal activity of P. regnellii extract T. rubrum.

Conclusion

Microencapsulation provided a significant improvement in the stability of the P. regnellii extract and better solubilization of chemical compounds, maintaining the antifungal effect against T. rubrum.

Significance and Impact of the Study

These results are useful for developing a formulation to treat fungal infections caused by dermatophyte species.     

A new method for estimating gross phosphorus mineralization and immobilization rates in soils

Phosphorus availability in soils is controlled by both the sizes of P pools and the transformation rates among these pools. Rates of gross P mineralization and immobilization are poorly known due to the limitations of available analytical techniques. We developed a new method to estimate P transformation rates in three forest soils and one grassland soil representing an Alfisol, an Ultisol, and Andisol, and a Mollisol. Three treatments were applied to each soil in order to separate the processes of mineral P solubilization, organic P mineralization, and solution P immobilization. One set of soils was retained as control, a second set was irradiated with -rays to stop microbial immobilization, and a third was irradiated and then autoclaved, also stop phosphatase activity. All three sets of samples were then incubated with anion exchange resin bags under aerobic conditions. Differences in resin P among the three treatments were used to estimate gross P mineralization and immobilization rates. Autoclaving did not affect resin-extractable P in any of the soils. Radiation did not alter resin-extractable P in the forest soils but increased resin-extractable P in the grassland soil. This increase was corrected in the calculation of potential P transformation rates. Effects of radiation on phosphatase activity varied with soils but was within 30% of the original values. Rates of P gross mineralization and immobilization ranged from 0.6–3.8 and 0–4.3 mg kg-soil^-1 d^-1, respectively, for the four soils. The net rates of solubilization of mineral P in the grassland soil were 7–10 times higher than the rates in forest soils. Mineralization of organic P contributed from 20–60% of total available P in the acid forest soils compared with 6% in the grassland soil, suggesting that the P mineralization processes are more important in controlling P availability in these forest ecosystems. This new method does not require an assumption of equilibrium among P pools, and is safer and simpler in operation than isotopic techniques.     

The activities of soil and root acid phosphatase in the nine tropical rain forests that differ in phosphorus availability on Mount Kinabalu, Borneo

Background and aims

Tropical rain forests on deeply weathered soils are increasingly thought to be limited by phosphorus (P), where plants and associated organisms would demonstrate adaptations to efficiently recycle P using acid phosphatase from organic matter. The activities of soil and root acid phosphatase were investigated in nine tropical rain forests that demonstrated a 20-fold difference in the soil organic P pool on Mt. Kinabalu, Borneo.

Methods

Acid phosphatase activity was measured at pH6.0 using p-nitrophenyl phosphate as substrate.

Results

The specific phosphatase activity of tree roots on a soil-surface-area basis was significantly positively related with P-use efficiency of above-ground productivity, suggesting a physiological linkage between above and below-ground systems in the adaptation to P deficiency. The phosphatase activities of soils and roots were significantly negatively correlated with the pool size of soil organic P fractions, suggesting that demand for P determines phosphatase activities.

Conclusions

It is suggested that tree roots and soil microbes develop more active phosphatases in response to the chronic shortage of soil P, which forms the basis for an important functional role for the efficient acquisition of P from soil organic matter.     

Phosphatase activity of extra-radical arbuscular mycorrhizal hyphae: A review

Phosphatase activity of arbuscular mycorrhizal (AM) fungi has attracted attention in three fairly distinct domains: intracellular enzymes with defined metabolic functions that have been studied in intraradical hyphae, histochemical staining of alkaline phosphatase as an indicator of fungal activity measured both intra- and extraradically, and extracellular activity related to mineralization of organic P (P_o) compounds that may enhance mycorrhizal utilization of an important nutrient pool in soil. This review focuses on the latter subjects with emphasis on extraradical mycelium (ERM), while it draws on selected data from the vast material available concerning phosphatases of other organisms. We conclude that histochemical staining of alkaline phosphatase is a sensitive and suitable method for monitoring the effect of adverse conditions encountered by ERM both as a symbiotically functional entity in soil, and in vitro without modifying interference of soil or other solid substrates. Furthermore, the quantitative importance of extracellular enzymes for P nutrition of AM plants is estimated to be insignificant. This is concluded from the low quantitative contribution extracellular hyphae of AM fungi give to the total phosphatase activity in soil, and from estimations of which processes that may be rate limiting in organic P mineralization. Maximum values for the former is in the order of a few percent. As for the latter, solubilization of P_o seems to be far more important than P_o hydrolysis for utilization of P_o by AM fungi and plants, as both endogenous soil phosphatase activity and phosphatases of other soil organisms are ubiquitous and abundant. Our discussion of mycorrhizal phosphatases supports the view that extracellular phosphatases of roots and micro-organisms are to a large extent released incidentally into soil, and that the source has limited benefit from its activity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phosphatase activity in the heterotrophic dinoflagellate Pfiesteria shumwayae

The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function.     

Moderate salinity improves the availability of soil P by regulating P-cycling microbial communities in coastal wetlands

Accelerated sea-level rise is expected to cause the salinization of freshwater wetlands, but the responses to salinity of the availability of soil phosphorus (P) and of microbial genes involved in the cycling of P remain unexplored. We conducted a field experiment to investigate the effects of salinity on P cycling by soil microbial communities and their regulatory roles on P availability in coastal freshwater and brackish wetlands. Salinity was positively correlated with P availability, with higher concentrations of labile P but lower concentrations of moderately labile P in the brackish wetland. The diversity and richness of microbial communities involved in P cycling were higher in the brackish wetland than the freshwater wetland. Salinity substantially altered the composition of the P-cycling microbial community, in which those of the brackish wetland were separated from those of the freshwater wetland. Metagenomic sequence analysis indicated that functional genes involved in the solubilization of inorganic P and the subsequent transport and regulation of P were more abundant in coastal soils. The relative abundances of most of the target genes differed between the wetlands, with higher abundances of P-solubilization (gcd and ppa) and -mineralization (phoD, phy, and ugpQ) genes and lower abundances of P-transport genes (pstB, ugpA, ugpB, ugpE, and pit) in the brackish wetland. A significant positive correlation between the concentration of labile P and the abundances of the target genes suggested that salinity may, at least in part, improve P availability by regulating the P-cycling microbial community. Our results suggest that the P-cycling microbial community abundance and P availability respond positively to moderate increases in salinity by promoting the microbial solubilization and mineralization of soil P. Changes in microbial communities and microbially mediated P cycling may represent microbial strategies to adapt to moderate salinity levels, which in turn control soil function and nutrient balance.     

PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation

Autophagosome formation is promoted by the PI3 kinase complex and negatively regulated by myotubularin phosphatases, indicating that regulation of local phosphatidylinositol 3‐phosphate (PtdIns3P) levels is important for this early phase of autophagy. Here, we show that the Caenorhabditis elegans myotubularin phosphatase MTM‐3 catalyzes PtdIns3P turnover late in autophagy. MTM‐3 acts downstream of the ATG‐2/EPG‐6 complex and upstream of EPG‐5 to promote autophagosome maturation into autolysosomes. MTM‐3 is recruited to autophagosomes by PtdIns3P, and loss of MTM‐3 causes increased autophagic association of ATG‐18 in a PtdIns3P‐dependent manner. Our data reveal critical roles of PtdIns3P turnover in autophagosome maturation and/or autolysosome formation.     

Carry-over effects of soil inoculation on plant growth and health under sequential exposure to soil-borne diseases

Aim

To investigate the effects of biochar on biological and chemical phosphorus (P) processes and identify potential interactive effects between P fertilizer and biochar on P bioavailability in the rhizosphere of maize.

Methods

We conducted a pot-experiment with maize in a sandy loam soil with two fertilizer levels (0 and 100 mg P kg ^?1) and three biochars produced from soft wood (SW), rice husk (RH) and oil seed rape (OSR). Sequential P fractionation was performed on biochar, bulk soil, and rhizosphere soil samples. Acid and alkaline phosphatase activity and root exudates of citrate, glucose, fructose, and sucrose in the rhizosphere were determined.

Results

RH and OSR increased readily available soil P, whereas SW had no effect. However, over time available P from the biochars moved to less available P pools (Al-P and Fe-P). There were no interactive effects between P fertilizer and biochar on P bioavailability. Exudates of glucose and fructose were strongly affected by especially RH, whereas sucrose was mostly affected by P fertilizer. Alkaline phosphatase activity was positively correlated with pH, and citrate was positively correlated with readily available P.

Conclusion

Biochar effects on biological and chemical P processes in the rhizosphere are driven by biochar properties.

     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

Total and active microbial communities and <Emphasis Type="Italic">phoD</Emphasis> as affected by phosphate depletion and pH in soil

Background and Aims

Soil microbial communities contribute to organic phosphorus cycling in a variety of ways, including secretion of the PhoD alkaline phosphatase. We sampled a long-term grassland fertilization trial in Switzerland characterized by a natural pH gradient. We examined the effects of phosphate depletion and pH on total and active microbial community structures and on the structure and composition of the total and active phoD-harboring community.

Methods

Archaeal, bacterial and fungal communities were investigated using T-RFLP and phoD-harboring members of these communities were identified by 454-sequencing.

Results

Phosphate depletion decreased total, resin-extractable and organic phosphorus and changed the structure of all active microbial communities, and of the total archaeal and phoD-harboring communities. Organic carbon, nitrogen and phosphorus increased with pH, and the structures of all total and active microbial communities except the total fungal community differed between the two pH levels. phoD-harboring members were affiliated to Actinomycetales, Bacilliales, Gloeobacterales, Planctomycetales and Rhizobiales.

Conclusions

Our results suggest that pH and associated soil factors are important determinants of microbial and phoD-harboring community structures. These associated factors include organic carbon and total nitrogen, and to a lesser degree phosphorus status, and active communities are more responsive than total communities. Key players in organic P mineralization are affiliated to phyla that are known to be important in organic matter decomposition.

     

Microbial interactions associated with secondary cucumber fermentation

Aims

To evaluate the interaction between selected yeasts and bacteria and associate their metabolic activity with secondary cucumber fermentation.

Methods and Results

Selected yeast and bacteria, isolated from cucumber secondary fermentations, were inoculated as single and mixed cultures in a cucumber juice model system. Our results confirmed that during storage of fermented cucumbers and in the presence of oxygen, spoilage yeasts are able to grow and utilize the lactic and acetic acids present in the medium, which results in increased brine pH and the chemical reduction in the environment. These conditions favour opportunistic bacteria that continue the degradation of lactic acid. Lactobacillus buchneri, Clostridium bifermentans and Enterobacter cloacae were able to produce acetic, butyric and propionic acids, respectively, when inoculated in the experimental medium at pH 4·6. Yeast and bacteria interactions favoured the survival of Cl. bifermentans and E. cloacae at the acidic pH typical of fermented cucumbers (3·2), but only E. cloacae was able to produce a secondary product.

Conclusions

The methodology used in this study confirmed that a complex microbiota is responsible for the changes observed during fermented cucumber secondary fermentation and that certain microbial interactions may be essential for the production of propionic and butyric acids.

Significance and Impact of the Study

Understanding the dynamics of the development of secondary cucumber fermentation aids in the identification of strategies to prevent its occurrence and economic losses for the pickling industry.     

Soil Phosphorus Availability and Transformation Rates in Relictic Pinsapo Fir Forests from Southern Spain

Abies pinsapo fir forests are remnant of temperate-like coniferous forests currently subjected to the typical seasonal constraints of Mediterranean-type climates. We have examined for the first time, P availability and cycling in A. pinsapo forests from southern Spain by using both, measurements of concentrations of soil P fractions (resin extractable P, bicarbonate extractable P and NaOH extractable P) and rates of P supply to (P solubilization, P mineralization and phosphomonoesterases (PMEase activity) and P immobilization from the soil solution. Soils from A. pinsapo stands differing in lithology (serpentinitic and calcareous substrates) and successional status (from young-agradative to old-growth stages) were chosen for this study. Labile organic P fractions, PMEase activity and rates of P gross mineralization and immobilization were significantly higher in agradative stands on serpentines than in successionally comparable calcareous stands. This suggests an important role of the organic P subcycle in the A. pinsapo stand on serpentine. On calcareous lithology, PMEase activity and all soil P transformation rates significantly increased throughout the successional series. Similarly, all organic and inorganic P fractions measured in old-growth forests showed the maximum values of the series. These trends fit the predictions of standard patterns of P cycling changes along with succession, in which P supply to plants greatly depends on solubilization from mineral forms at early-to-mid successional stages, whereas the importance of processes related to the organic P subcycle increases as succession progresses.     

DIFFERENTIAL ACTIVITIES OF ACID PHOSPHATASE FROM ADAXIAL AND ABAXIAL REGIONS OF LUPINUS LUTEUS (FABACEAE) COTYLEDONS

Acid phosphatases of abaxial and adaxial regions in the cotyledons of the Lupinus luteus which possess structurally distinct protein bodies were examined. Acid phosphatase activity was investigated by enzyme assays and by gel electrophoresis and was localized by cytochemical methods in the cotyledons of Lupinus luteus L. during germination and seedling development. Acid phosphatase activity was significantly higher in the adaxial (heterogeneous protein body) region as compared to the abaxial (homogeneous protein body) region of the cotyledon. The pH optimum of acid phosphatase from the abaxial region and from the adaxial region was 4.5 and 5.0, respectively. There were significant differences in substrate specificity and isoenzymic composition of the enzyme between the two regions. Isoenzymic composition changed during the course of germination and seedling development. Acid phosphatase was localized in the matrix of the homogeneous protein bodies and in the globoids of the heterogeneous protein bodies at imbibition. After germination (d 3, d 4, d 7) acid phosphatase was localized primarily in the inner cell walls and intercellular spaces of both regions. These results show that different isoenzymes of acid phosphatase show differential localization and the rate of acid phosphatase activation or synthesis differs in cells from the two regions of the cotyledon.     

Isolation of Phosphate Solubilizing Bacteria from Maize Rhizosphere and Their Potential for Rock Phosphate Solubilization–Mineralization and Plant Growth Promotion

Phosphate solubilizing bacteria (PSB) play a significant role in plant P nutrition by their effect on soil P dynamics and their subsequent ability to make P available to plants via solubilization and mineralization processes. This study aimed to evaluate the effect of separate and combined use of indigenous PSB, poultry manure (PM) and compost on solubilization and mineralization of rock phosphate (RP) and their subsequent effect on growth and P accumulation of maize (Zea mays L.). A group of fifty seven bacteria were isolated from the rhizosphere/rhizoplane of maize that had been grown in soils collected from varying altitudes (655–2,576 m) of the mountain region of Rawalakot, Azad Jammu and Kashmir, Pakistan. After screening, the capacity of eleven isolates to solubilize mineral phosphate was quantitatively evaluated using insoluble Ca₃(PO₄)₂ in culture medium as a time course study through spectrometer. The growth hormone producing (IAA) capacity of the isolates was also determined. Furthermore, five potential isolates were tested for their ability to increase P release capacity (mineralization) of insoluble RP in an incubation study. The effect of PSB inoculation on maize was determined in a completely randomized greenhouse experiment where root and shoot biomass and P accumulation in plants were assessed. The P solubilization index of selected isolates varied from 1.94 to 3.69, while the P solubilization efficiency ranged between 94.1% and 269.0%. The isolates MRS18 and MRS27 displayed the highest values. The P solubilization in the liquid medium was maximum at 6 and 9 days of incubation ranging between 9.91 and 44.04 µgmL^?1 and the isolates MRS27 and MRS34 exhibited the highest solubilization. Six isolates showed additional capability of producing IAA ranging between 2.66 and 28.41 µgmL^?1. Results of the incubation study indicated that P release capacity (P mineralization) of RP-amended soil varied between 6.0 and 11.8 µgPg^?1 that had been significantly increased to 30.6–36.3 µgPg^?1 (maximum value) when PSB were combined with RP. The combined application of PSB and organic amendments (PM, compost) with RP further increased P mineralization by releasing a maximum of 37.7 µgPg^?1 compared with separate application of RP (11.8 µgPg^?1) and organic amendments (21.5 and 16.5 µgPg^?1). The overall effect of PSB (as a group) with RP over RP alone on maize growth showing a relative increase in shoot length 21%, shoot fresh weight 42%, shoot dry weight 24%, root length 11%, root fresh weight 59%, root dry weight 35% and chlorophyll content 32%. This study clearly indicates that use of PSB, and organic amendments with insoluble RP could be a promising management strategy to enhance P availability in soil pool and improve plant growth in intensive cropping systems.     

The effects of warming and nitrogen addition on soil nitrogen cycling in a temperate grassland, northeastern China

Background

Both climate warming and atmospheric nitrogen (N) deposition are predicted to affect soil N cycling in terrestrial biomes over the next century. However, the interactive effects of warming and N deposition on soil N mineralization in temperate grasslands are poorly understood.

Methodology/Principal Findings

A field manipulation experiment was conducted to examine the effects of warming and N addition on soil N cycling in a temperate grassland of northeastern China from 2007 to 2009. Soil samples were incubated at a constant temperature and moisture, from samples collected in the field. The results showed that both warming and N addition significantly stimulated soil net N mineralization rate and net nitrification rate. Combined warming and N addition caused an interactive effect on N mineralization, which could be explained by the relative shift of soil microbial community structure because of fungal biomass increase and strong plant uptake of added N due to warming. Irrespective of strong intra- and inter-annual variations in soil N mineralization, the responses of N mineralization to warming and N addition did not change during the three growing seasons, suggesting independence of warming and N responses of N mineralization from precipitation variations in the temperate grassland.

Conclusions/Significance

Interactions between climate warming and N deposition on soil N cycling were significant. These findings will improve our understanding on the response of soil N cycling to the simultaneous climate change drivers in temperate grassland ecosystem.     

Characterization of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5

Background  

Protein Ser/Thr phosphatase 5 (PP5) and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p) each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level.     

Contrasting nitrogen and phosphorus dynamics across an elevational gradient for subarctic tundra heath and meadow vegetation

Aims

This study explores soil nutrient cycling processes and microbial properties for two contrasting vegetation types along an elevational gradient in subarctic tundra to improve our understanding of how temperature influences nutrient availability in an ecosystem predicted to be sensitive to global warming.

Methods

We measured total amino acid (Amino-N), mineral nitrogen (N) and phosphorus (P) concentrations, in situ net N and P mineralization, net Amino-N consumption, and microbial biomass C, N and P in both heath and meadow soils across an elevational gradient near Abisko, Sweden.

Results

For the meadow, NH₄ ⁺ concentrations and net N mineralization were highest at high elevations and microbial properties showed variable responses; these variables were largely unresponsive to elevation for the heath. Amino-N concentrations sometimes showed a tendency to increase with elevation and net Amino-N consumption was often unresponsive to elevation. Overall, PO₄-P concentrations decreased with elevation and net P immobilization mostly occurred at lower elevations; these effects were strongest for the heath.

Conclusions

Our results reveal that elevation-associated changes in temperature can have contrasting effects on the cycling of N and P in subarctic soils, and that the strength and direction of these effects depend strongly on dominant vegetation type.     

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum,Syngens 1–5, P. jenningsi,P. caudatum,and the P. aurelia Complex1

ABSTRACT. Forty-eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.