Vanessa Durrieu

Alessandro Gandini

Mohamed Naceur Belgacem

Anne Blayo

Gilles Eiselé

Jean - Luc

Gilles Eisele

Jean-Luc Putaux

Preparation of Aqueous Anionic Poly-(urethane-urea) Dispersions: Influence of the Nature and Proportion of the Urethane Groups on the Dispersion and Polymer Properties

Keywords: poly(urethane-ureas), aqueous dispersions, oligoester and oligoether diols, aromatic and aliphatic diisocyanates, particle size, stability

Preparation of aqueous anionic poly-(urethane-urea) dispersions: Influence of the nature and proportion of the urethane groups on the dispersion and polymer properties

INTRODUCTION

The continuous need for reductions in both production costs and volatile organic compound emissions has speared an intense activity of research on the development of water-based polymer systems, particularly for coating and adhesive formulations. Aqueous polyurethane dispersions (PUD) constitute one of the major families of such materials [1][2][3][4][5] because of their remarkable properties, which have led to successful industrial applications. Most of the investigations related to PUDs were confined to a confidential industrial context, but recent scientific publications have provided interesting information on various aspects of both the synthetic stages and the properties of the ensuing dispersions. 4 The aim of the present study is similar to that of the work carried out by Price and co-workers 4 (and was indeed conducted in parallel) in terms of both synthetic approach (two-stage prepolymer mixing process) 6 and the search for relationships between prep-aration parameters and properties of the ensuing dispersions and solid polymers. However, on one hand, no organic solvent was used at any stage in our procedures and, on the other hand, the emphasis of our study was placed on the role of moieties surrounding the urethane groups and on their frequency along the polymer chain, with respect to the urea functions. The structure of the diisocyanate used plays an important role in the properties of the ensuing polymer. Thus, a symmetric molecular structure will favor: [START_REF] Wirpza | Polyurethanes Chemistry, Technology and Applications[END_REF] (i) the polymer capacity to crystallize; (ii) the separation of the microphases; and (iii) the increase of the mechanical properties, including the elastic modulus, the tensile strength, and the resistance to abrasion. Additionally, the presence of aromatic moieties will result in a higher rigidity of the final polymer. Finally, given the substantial difference in reactivity between aromatic and aliphatic isocyanates, their competitive condensation with water versus amines in the second stage of the preparation can influence the course of this chain extension.

The NCO/OH molar ratio determines the molecular weight of the prepolymer in the first stage of the synthesis and consequently the relative proportion of the urethane and urea groups in the final polymer chain. As an ultimate result, this ratio will therefore affect both the properties of the dispersions, such as particle size, and the dry polymer thermal and mechanical properties.

The present article deals with numerous PUD formulations, used with variable NCO/OH ratios and involving three aliphatic and an aromatic diisocyanates, two diols, a single diamine, and the same anionic emulsifying agent. The characterization of both the aqueous dispersions and the dry polymers enabled the establishment of criteria related to the various compositions used to prepare them.

EXPERIMENTAL Materials

The following commercial reagents were used without further purification: 1,4,1Ј,4Ј-diphenylmethane diisocyanate (MDI, Bayer, containing a small amount of the 1,3-derivative), ␣,␣, ␣', ␣'-tetramethyl-1,3-xylylene diisocyanate (TMXDI, Cytec), isophorone diisocyanate (IPDI, Bayer), 1,4,1Ј,4Ј-dicyclohexylmethane diisocya-nate (H 12 MDI, Bayer, mixture of boat/chair stereoisomers), ␣,-poly(propylene glycol) (PPG, Mn ϭ 1000, Shell), ␣,-poly(caprolactone) (PCL, Mn ϭ 530, Daicel), containing two oxyethylene central groups, dimethylol propionic acid (DMPA, Perstorp), dibutyltin dilaurate (DBTL, Aldrich), triethyl amine (TEA, Acros), and 1,2-ethylene diamine (EDA, Acros). The high purity, functionality, and molecular weight of all these products were verified by spectroscopic techniques and vapor pressure osmometry.

Synthesis

Prepolymer formation

The synthesis of the polyurethane anionomers was carried out in bulk, without any intervention of an organic solvent using the "prepolymer mixing pro-

Dispersion in water and chain extension

Once the prepolymer had reached the final expected NCO content, it was slowly poured into an aqueous TEA solution kept at 25-30°C and stirred at 280 rpm. The amount of TEA was calculated so as to ensure the complete neutralization of the carboxylic group of DMPA, more specifically in a 20% molar excess. Immediately after the completion of this addition, the chain extender (EDA) was introduced slowly (to avoid a sudden increase in viscosity, which would interfere with the formation of the good dispersion) and thereafter stirring was increased to 500 rpm and the medium was left to react (chain extension) for 90 min.

The quantity of EDA added was calculated to obtain a 1:1 NCO:NH 2 stoichiometry. The ensuing aqueous polyurethane-urea dispersions were characterized by a solid content of about 30% w/w. Scheme 1 summarizes the different mechanistic steps involved in the overall preparation of these dispersions.

Characterization

The aqueous dispersions were spread on a Teflon mold and dried to a constant weight in an oven at 60°C. FTIR and 1 H-NMR spectra were taken with a Perkin-Elmer Paragon 1000 and a Bruker 300 instrument, respectively. The thermal analyses (DSC and TGA) were carried out using a Setaram DSC-TGA 92 calorimeter with a heating rate of 10°C/min. The values given as degradation temperature in the text correspond to the temperature at which the sample lost 10% of its initial weight. GPC tracings were obtained with a Spectra Physics chromatograph equipped with two calibrated columns. Polymers were dissolved in a DMF-(0.1 M)NaNO 3 solution at a concentration of 1 g/L.

Static and dynamic contact angles on the polymer films were measured with a homemade goniometer equipped with a CDD camera working at up to 200 images/s. [START_REF] Aurenty | [END_REF] The results were collected in a video card and treated with image analysis software. Among the different approaches enabling the determination of the different contributions to the surface energy of the solid based on contact angle measurements, 9 we chose to work with the two most common ones, i.e., those proposed by Owens and Wendt and van Oss. The Owens-Wendt treatment is a refinement of Fowkes' approach to the polar and dispersive contribution to surface tension. The resulting equation is

W SL ϭ ␥ L (1 ϩ cos) ϭ 2 ͱ␥ L d ␥ S d ϩ 2 ͱ␥ L P ␥ S P ,
where W SL is the energy of adhesion, ␥ L is the liquid surface tension, is the contact angle, ␥ L l , ␥ L p are the dispersive and polar contributions to the liquid surface tension and ␥ S l , ␥ S p are the dispersive and polar contributions to the solid surface energy.

Thus, plotting the term

␥ L (1ϩcos)/ ͌ ␥ L d as a func- tion of ͌ ␥ L p / ͌ ␥ L d
, it is possible to determine ␥ S l from the intercept and ␥ S p from the slope. Table I gives the surface tension of the liquids we used in terms of their polar and dispersive contribution.

Van Oss' approach takes into account the acid-base interactions between the solid surface and the liquid drop, through the equation

W SL ϭ ␥ L (1 ϩ cos) ϭ ␥ LW ϩ ␥ AB ϭ 2 ͱ␥ S LW ␥ L LW ϩ 2 ͱ␥ L ϩ ␥ S Ϫ ϩ 2 ͱ␥ L Ϫ ␥ S ϩ , here:
␥ LW is the Lifshitz-Van der Waals contribution, ␥ AB is the acid-base contribution, ␥ ϩ is the acid contribution, and ␥ -is the basic contribution to the surface energy. Table I gives the corresponding contribution related to the five liquids we used in this context.

The observation of the suspended polymer particles in the poly(urethane-urea) dispersions was conducted using transmission electron cryomicroscopy. Thin vitrified films were prepared with a Leica EM CPC fastfreezing apparatus. A drop of highly diluted suspension was deposited on a commercial "lacey" carbon film (PELCO). The excess liquid was blotted out with a filter paper. Before evaporating the remaining fluid, the grids were quenched-frozen by liquid ethane cooled with liquid nitrogen at -171°C (Fig. 1). 10 The sample was then transferred into a Gatan 626 cryoholder kept at -180°C with liquid nitrogen and observed at a magnification of 11,500ϫ, using a Philipps CM200 cryomicroscope operating at a voltage of 80 kV. Pictures were recorded with an underfocus of about 1 m, using a Philipps Low Dose system, which allows the imaging conditions to be set up near the area of interest, to protect it from any irradiation prior to photographic exposure. [START_REF] Harris | Negative Staining Cryoelectron Microscopy: The Thin Films Techniques[END_REF][START_REF] Putaux | [END_REF] Negatives of the micrographs were digitized using a Kodac Megaplus CDD camera. Contrast enhancement and particle measurements were performed by NH-Image and optimas software.

The average particle size measurements of the aqueous dispersions were performed by light scattering with a Malvern Autosizer 2c.

The potential of these particles was measured by their electrophoretic mobility with a Malvern Zetasizer 2000.

The surface tension of the dispersions was determined using a digital tensiometer K10ST (Kru ¨ss) working with a Du Nouy platinum ring, whereas their viscosity was measured with a Carri-Med CSL 2 500 cone-plate rheometer using a fixed stress and working in a flow mode at a constant temperature (Ϯ0.1°C). For the sake of comparison, all viscosities given below were taken at a shear rate equal to 500 s Ϫ1 .

RESULTS AND DISCUSSION

Although they are more expensive and require a catalyst, aliphatic diisocyanates are generally preferred to their aromatic homologues for the preparation of polyurethane dispersions because: (i) they have low reactivity, particularly with respect to water; (ii) they enable the preparation of prepolymers at higher temperatures (100 -120°C against 60 -80°C for aromatic counterparts); (iii) they yield prepolymers with lower viscosity, which are therefore easier to disperse in water; and (iv) the NCO groups are not directly conjugated to a double bond, which confers a better light and aging resistance to the corresponding poly(urethane-ureas). However, in the present study, it was decided to compare their performances with that of MDI, viz. the aromatic diisocyanate most commonly used in solvent-based polyurethanes, but also in some aqueous polyurethane dispersions.

Effect of the diisocyanate structure

MDI

The reaction of MDI ϩ PPG ϩ DMPA gave a highly viscous product and was marred by the fact that DMPA was not totally solubilized. During the dispersion step, a gel formed, certainly because of the excessively high reactivity of the aromatic NCO groups, which promoted their side reaction with already formed urethane moieties, to give allophonate branching and eventually the formation of a polymer network. Moreover, during the dispersion step, some of the NCO groups would have reacted with water, thus increasing the number of urea linkages to the detriment of chain extension. These results confirmed the unsuitability of aromatic isocyanates for the preparation of aqueous PUD dispersions, at least with the present procedure, which did not call upon the use of organic solvents. Indeed, the use of MDI has been reported for aqueous dispersions, but only in the "so-called" acetone process, 13 in which both the prepolymer synthesis and the chain extension take place in organic solvent (so that the NCO functions can not react with water), before dispersion of the ensuing polymer solution in water.

HMDI

The prepolymer synthesis and the chain extension were carried out without any difficulty, but the final dispersion was not homogeneous and was unstable with time since a phase separation appeared with storage time. The characterization of these dispersions by cryomicroscopy gave evidence of particle encapsulation, as shown in Figure 2. This phenomenon was attributed to both the diisocyanate symmetry and the diol structural regularity, which would favor the formation of crystalline microphases in the prepolymer, enabling further reaction during the chain extension. Thus, if only the amorphous portions of the prepolymer could react with a chain extender, the resulting macromolecules would surround the inactive crystalline portions and generate the microcapsules. This morphology probably prevented an adequate distribution of the ionic moieties, which would explain the instability of the dispersion with time.

IPDI and TMXDI

To assess the relative reactivity toward OH functions of the NCO groups of these two diisocyanates, we studied the kinetics of their condensation with ethanol in dry acetone at room temperature. The initial concentration of both functions was 4 10 Ϫ2 M and the reactions were followed by the decrease of the infrared NCO peak at 2250 cm Ϫ1 .

With TMXDI, a good second-order behavior was obtained as shown in Figure 3, indicating that the two NCO functions displayed equal reactivity, as indeed expected because of the symmetry of the molecule bearing them. With IPDI, the asymmetric emplacement of the two NCO groups gave rise to two secondorder regimes as shown in Figure 4. The faster initial reaction dominated up to 40% conversion and was followed by a transition zone, leading to a slower second-order behavior starting at about 50% conversion. Table II gives the second-order rate constants corresponding to these kinetic studies. The reactivity of the TMXDI NCO groups was found to be intermediate between those of the two different counterparts attached to IPDI. We attribute the highest reactivity to the IPDI NCO attached to the methylene groups because of its lower steric screening and the higher reactivity of TMXDI NCO, compared with the second isocyanate group of IPDI, to its long-range activation by the aromatic ring. These results suggest that in the context of the preparation of PUD, IPDI was globally more reactive than TMXDI (particularly when using NCO/OH close to 2) during the prepolymer formation, whereas the NCO reactivity was reversed during the chain extension step.

Comparison between IPDI and TMXDI structures

The structures of the final polymers obtained from these two diisocyanates and the two macrodiols used in this study were examined by FTIR and 1 H-NMR spectroscopy, which displayed all the features related to regular linear poly(urethane-ureas). Figure 5 shows an example of two FTIR spectra of polymers prepared with the same macrodiol (PCL, Mn ϭ 530) and the two aliphatic diisocyanates, the NCO/OH ratio was kept constant (r ϭ 1.8). The common peaks were associated with relevant urethane, urea, and polycaprolactone moieties and differences were assigned essentially to the presence of the aromatic ring of TMXDI (bands at 3053 and 1085 and between 700 and 800 cm Ϫ1 ).

The characterization of the four polymers arising from these combinations, using NCO/OH ratios (r close to 2) is given in Table III. With both diols the glass transition temperature was higher when TMXDI was used, given the enhanced polymer stiffness associated with its aromatic ring. Conversely, the thermal stability was higher with IPDI-based polymers, probably because of its totally aliphatic structure. [START_REF] Wirpza | Polyurethanes Chemistry, Technology and Applications[END_REF] The values of the total surface energy were very similar among the four polymers and moreover the two different determination techniques gave concordant results. A modest difference was found in the values of the polar contribution in favor of the TMXDI-based polymer, which was attributed to the aromatic ring -bonds.

The properties of the corresponding four aqueous dispersions are given in Table IV. As expected, no significant variations were encountered in terms of pH (essentially determined by the excess TEA added in the neutralization step), surface tension (independent of the polymer structure), and potential (which is directly related to the negative charge density on the polymer chains, which was kept constant through the constant amount of DMPA). The only parameter that was sensitive to the specific changes in the conditions applied to these four combinations was the average particle size. The major differences occurred when the macrodiol was changed, since the use of PCL induced a substantial increase in particle size. We interpreted this trend on the basis of the higher hydrophobic character of this structure with respect to that associated with propylene oxide units, which would enhance water repellence at the dispersion stage and thus limit the extent of polymer fragmentation. The nature of the diisocyanate did not play a detectable role on particle size when PPG was used, whereas with PCL, TMXDI produced a moderate increase with respect to IPDI. It seems that with the higher hydrophobic systems based on PCL, the structure of the diisocyanate can also intervene. Here, the increase in particle size associated with the use of TMXDI was attributed to its higher molecular stiffness compared with that of IPDI.

It can be concluded that the structure of the diisocyanate can affect significantly the course of the synthesis, the stability of the ensuing dispersions, and the properties of the final polymers, without intervening, in a major way, with modification on the other properties of the aqueous dispersions.

The cryomicroscopy images revealed that the actual shape of the particles depended on the structure of the diisocyanate used, as shown in Figures 6 and7. In fact, whereas the particles obtained from dispersions based on TMXDI were on average very nearly spherical, the shape of the counterparts prepared with IPDI was more irregular, most probably because of the softer character of the PU containing the latter, i.e., the more flexible molecular structures, which facilitated particle deformation under shear at the dispersion stage.

Effect of the [NCO]/[OH] molar ratio

This parameter is mostly responsible for the variations in the prepolymer molecular weight and its relative content of urethane and urea groups. To assess its role on the properties of both aqueous emulsions and final polymers, we studied three systems based on TMXDI and PPG in which its value varied between 1.2 and 2.0, respectively, (corresponding to average DPn of 11 to 3). In a first approach, we carried out only the synthesis of three prepolymers, with r ϭ 1.2, 1.6, and 2, in the presence of DMPA, as indicated under Experimental, and neutralized the terminal NCO groups with an excess of t-butylamine, to simulate the chemistry of the chain extension process without actually increasing the molecular DP. The ensuing products were isolated and characterized. Figure 8 shows the FTIR spectra of these three materials, which give the expected identical qualitative features related to the moieties arising from each monomer and those associated with the formation of urethane and terminal urea groups. The relevant quantitative differences among these spectra arose from the increasing proportion of terminal urea groups, associated with the corresponding decrease on the DP, which are clearly visible in the peaks around 3340 (N-H), 1650 (C ϭ O), and 1530 cm Ϫ1 (C-N). Table V gives the glass transition temperatures and the viscosity of these prepolymers. The changes in T g were modest, but in the right direction, viz. an increase with increasing DP. The presence of two opposing structural trends explained the modest increase, namely a decrease in interchain hydrogen bond (stiffening effect) as a function of increasing DP, counterbalanced by the classical increase in T g associated with oligomer chain length. In the case of the viscosity values (which were obtained at 80°C because of the high consistency of these prepolymers), the intermolecular cohesive forces generated by the hydrogen bonding among urea terminal groups (which were the larger the lower the DP) dominated largely over the opposite effect associated with a viscosity increase with increasing chain length. This difference in behavior is understandable, because the flow properties of oligomers are primarily determined by the extent of intermolecular interactions, whereas T g is less sensitive to these interactions and more affected by chain length. A crystalline product insoluble in the bulk of the viscous oligomers was produced in the experiments carried out with r ϭ 2. It was characterized by FTIR and NMR spectroscopy and shown unambiguously (Table VI) to be the t-butylamine diurea of TMXDI.

We then proceeded to prepare several dispersions using DMPA at a fixed content and TMXDI or IPDI in variable proportions. Thus, instead of blocking the NCO end groups of the prepolymers of a monoamine, we carried out the chain extension with EDA, as already described.

Figure 9 compares the FTIR spectra of three of the isolated polymers (prepared with PPG (Mn ϭ 1000) and TMXDI) as a function of r. As in the case of the urea terminated prepolymer, the urea N-H, CϭO, and C-N peaks increased markedly with increasing excess of NCO, to the detriment of the corresponding bands pertaining to the urethane moieties.

As expected, the molecular weight of these polymers increased with decreasing value of r and their distribution widened. Figure 10 shows a comparison of GPC tracings of two of these products, which illustrate the above trend. The narrow distribution associated with r ϭ 2 is particularly relevant since the chain extension was accomplished in this case starting with a prepolymer of an average DP of 3 and therefore a very sharp size distribution.

The other relevant properties of these polymers are given in Table VII. With both isocyanates, the glass transition temperature increased with increasing r values (Fig. 11), indicating that now the determining parameter was the proportion of strong H-bonding arising from the corresponding increasing proportion of urea moieties. In other words, interchain cohesive energy factors played a more important role than molecular weight.

The thermal stability of these polymers showed a slight improvement with increasing urea moiety content, confirming previous results, which showed systematically that these functions resist better than urethane ones against thermal treatment. [START_REF] Wirpza | Polyurethanes Chemistry, Technology and Applications[END_REF] Finally, the surface energy of these polymer films gave the same values, as measured by the two methods. Whereas the dispersive contribution remained essentially constant in terms of both the diisocyanate used and the value of r, the polar contribution increased regularly as r increased, indicating that the urea functions displayed a higher polarity than their urethane counterparts. All these features are clearly displayed in Figure 12.

Table VIII gives the characterization of the corresponding dispersions. Once again their pH, surface tension, and potential were not significantly affected by the change in diisocyanate and the variation in the [NCO]/[OH] ratio. These observations are in tune with the fact that as long as the nature, the quantity, and the total neutralization of the emulsifying agent are unchanged, there is no reason justifying any variation in those properties.

On the other hand, the average particle size was significantly affected by the changes in the values of r in that an increase of this ratio produced a corresponding decrease in particle size with both diisocyanates (Fig. 13). This trend was related to the higher hydrophilic properties of the urea moieties compared with those of their urethane counterpart. It can be concluded that the introduction of a higher proportion of urea groups by increasing the [NCO]/[OH] ratio can play a similar role as that of the emulsifying agent in favoring a decrease in the average particle size.

In general, a decrease in the average particle size produces a corresponding increase in the viscosity of

Effect of [NCO]/[OH] ratio on the average particle size of dispersions (see Table VIII). their aqueous dispersions, because the liquid-solid interactions are enhanced by the increase in the specific area of the particles. The present results confirmed this trend, but only up to a certain [NCO]/ [OH] ratio, after which a decrease in viscosity was observed (Fig. 14). This decrease was less pronounced than the initial increase and was attributed to possible particle aggregation (not seen in the average size measurements because of the dilution required to carry them out) at the lower range of their size.

CONCLUSION

This investigation provided some useful indications on how to optimize the properties of polyurethaneurea dispersions. In particular, it showed that aliphatic diisocyanates like TMXDI and IPDI are much more appropriate than aromatic structures or symmetric aliphatic ones and that the [NCO]/[OH] ratio plays a fundamental role and gives the most promising products in terms of small particle size in the dispersions and physical properties of the final polymer films, when it approaches a value of 2. Work is in progress to assess the role of several macrodiols and the emulsifying agent.

Figure 1

 1 Figure1Principle of the fast-freezing protocol to prepare thin vitrified specimens for cryo-TEM.

  Scheme 1

Figure 2

 2 Figure 2 Cryo-TEM image of nanoparticles prepared from H 12 MDI and PPG 1000 (scale bar: 100 nm).

Figure 3

 3 Figure3Second-order plot for the reaction of ethanol and TMXDI.

Figure 4

 4 Figure 4 Second-order plot for the reaction of ethanol and IPDI.

Figure 5

 5 Figure 5 FTIR spectra of PUDs prepared with PCL and the indicated diisocyanate.

Figure 6

 6 Figure 6 Cryo-TEM image of nanoparticles prepared from PPG and TMXDI (scale bar: 100 nm).

Figure 7

 7 Figure 7 Cryo-TEM image of nanoparticles prepared from PPG and IPDI (scale bar: 100 nm).

Figure 8

 8 Figure 8 FTIR spectra of prepolymers prepared with PPG and TMXDI, at different [NCO]/[OH] ratios, and neutralized with t-butylamine.

Figure 9

 9 Figure 9 FTIR spectra of polymers based on PPG and TMXDI at different [NCO]/[OH] ratios and chain extended with EDA.

Figure 10

 10 Figure 10 GPC tracings of two polymers based on PPG and TMXDI (r ϭ 1.6 or 2) and chain extended with EDA.

Figure 11

 11 Figure 11 Effect of r on the glass transition temperatures of different isolated polymers (see TableVII).

Figure 12

 12 Figure 12 Effect of the [NCO]/[OH] ratio on the surface energy of polymer films (see TableVII).

  Figure 12 Effect of the [NCO]/[OH] ratio on the surface energy of polymer films (see TableVII).

TABLE I Surface Tension, Polar and Dispersive Contributions, and Acid-Base Characteristics (mJ/m 2 ) of the Liquid Probes Used in This Study

 I

		␥ T	␥ D	␥ P	␥ LW	␥ ϩ	␥ Ϫ
	Water	72.8	21.8	51.0	21.8 25.5	25.5
	Glycerol	63.4	37.0	26.4	-	-	-
	Formamide	58.2	39.5	18.7	39.2	2.3	39.6
	Diodomethane	50.8	48.5	3.3	-	-	-
	Hexadecane	27.5	27.5	0.0	27.5	0.0	0.0

TABLE II Second-Order Rate Constants and Domains of Validity Diisocyanate

k (kg/mol.s) Domain of validity (%) k 1 k 2 TMXDI 0.095 85 IPDI 0.16 0.038

TABLE III Effect of the Diisocyanate Structure on the Properties of the Final Polymers

 III

					Diol
				PPG (r ϭ 2)	PCL (r ϭ 1.8)
			Diisocyanate	TMXDI	IPDI	TMXDI	IPDI
	Glass transition temperature	°C		Ϫ3	Ϫ38	68	36
	Degradation temperature	°C		305	310	310	325
		mJ/m 2	␥ D	21.7	17.6	19.2	17.7
	Surface energy (Owens-Wendt method)	mJ/m 2	␥ P	17.6	18.8	20.5	20.5
		mJ/m 2	␥ T	39.3	36.4	39.7	38.2
	Surface energy (Van Oss method)	mJ/m 2	␥ T	38.5	37.0	39.0	38.2

TABLE IV Effect of the Diisocyanate Structure on the Properties of Aqueous Dispersions

 IV

				Diol
			PPG (r ϭ 2)	PCL (r ϭ 1.8)
		Diisocyanate	TMXDI	IPDI	TMXDI	IPDI
	pH		7.7	8.2	8.0	9.1
	Surface tension	mJ/m 2	38.8	39.2	47.8	42.9
	Average particle size	Nm	35.6	34.7	101.1	72.1
	potential	MV	Ϫ40.2	Ϫ40.7	Ϫ36.7	Ϫ39.0

TABLE V Effect of the [NCO]/[OH] Ratio on the Prepolymer Properties

 V

	Diisocyanate		TMXDI
	[NCO]/[OH] ratio	2	1.6	1.2
	Glass transition temperature (°C)	Ϫ30	Ϫ27	Ϫ24
	Viscosity at 80°C (mPa.s)	577	231

TABLE VI Spectral Characterizations of the Diurea Crystals

 VI

	FTIR between NaCl faces in cm Ϫ1
	3362	N-H
	3012	Aromatic ring (TMXDI)
	2972-2929	CH3
	1640	C¢O of the urea function
	1559	C-N of the urea function
	782	Aromatic ring (TMXDI)
	1 H-NMR in pyridine-d 5 (ppm)
	5.79	H

1 and H 2 , m, i ϭ 4 1.44 H 3 and H 4 , m, i ϭ 30 1.25 H 5 , m, i ϭ 3.6

TABLE VII Effect of the [NCO]/[OH] Molar Ratio on the Polymer Properties

 VII

								Diisocyanate
						TMXDI					IPDI
			r	2	1.8	1.6	1.4	1.2	2	1.8	1.6	1.4
	Glass transition temperature	°C		Ϫ3	Ϫ26	Ϫ28	Ϫ31	Ϫ34	Ϫ38	Ϫ43	Ϫ44	Ϫ48
	Degradation temperature	°C		305	305	295	270	-	310	315	-	295
	Surface energy
	(Owens-Wendt method)	mJ/m 2	␥ D	21.7	19.7	17.4	15.6	14.3	17.6	16.3	14.2	13.0
		mJ/m 2	␥ P	17.6	17.7	19.2	19.1	18.2	18.8	17.8	19.3	19.3
		mJ/m 2	␥ T	39.3	37.4	36.5	34.7	32.6	36.4	34.1	33.5	32.3
	Surface energy
	(Van Oss method)	mJ/m 2	␥ T	38.5	37.8	36.7	34.8	33.0	37	35.7	34.3	31.7

TABLE VIII Effect of the [NCO]/[OH] Ratio on the Properties of Different Dispersions

 VIII

						Diisocyanate
					TMXDI				IPDI
		r	2	1.8	1.6	1.4	1.2	2	1.8	1.6	1.4
	pH		7.7	9.0	8.8	7.5	8.1	8.2	9.3	8.3	7.5
	Surface tension	mJ/m 2	38.8	38.6	38.2	37.4	38.7	39.2	37.8	37.3	38.5
	Average particle size	nm	35.6	52.2	75.4	79.3	83.7	34.7	46.2	57.3	71.1
	potential	mV	Ϫ40.2	Ϫ39.5	Ϫ37.4	Ϫ36.1	Ϫ38.7	Ϫ40.7	Ϫ39.1	Ϫ45.3	Ϫ41.7
	Viscosity	MPa.s	47.5	56.1	78.2	33.7	14.4	91.9	96.3	52.5

The authors thank SICPA Co. for the financial and technological support generously attributed to this project.


Apiochytrium Granulosporum

Nov Gen Et Sp

Sergey A Karpov

David Moreira

Maria A Mamkaeva

Olga V Popova

Vladimir V Aleoshine

Purificación López-García

New member of Gromochytriales (Chytridiomycetes)

Keywords: chytrids, molecular phylogeny, ultrastructure

Molecular phylogenetic analysis of 18S rRNA gene sequences of nearly any species of Chytridiomycota has typically challenged traditional classification and triggered taxonomic revision. This has often led to the establishment of new taxa which, normally, appear well supported by zoospore ultrastructure as diagnostic character. To construct a meaningful and comprehensive classification of Chytridiomycota, the combination of molecular phylogeny and morphological studies of traditionally defined chytrid species is needed. In this work, we have studied morphological and ultrastructural features based on light and transmission electron microscopy as well as molecular phylogenetic analysis of a parasite of the yellow-green alga Tribonema gayanum (strain X-124 CCPP ZIN RAS) morphologically similar to Rhizophydium granulosporum. Phylogenetic analysis of the 18S rRNA gene sequence of this strain supports that it represents a new genus and species affiliated to the recently established order Gromochytriales. The ultrastructure of X-124 confirms its phylogenetic position sister to Gromochytrium and serves as the basis for the description of the new genus and species Apiochytrium granulosporum.

INTRODUCTION

The systematics of Chytridiomycota has been under continuous revision after a global molecular phylogenetic tree of Fungi based on six gene markers was published some years ago (James et al. 2006a). From that time, the phylogenetic investigation of 18S rRNA gene sequences of nearly any species or strain has triggered the revision of neighboring taxa, often leading to the establishment of new higher rank taxa, e.g. family, order and class, divisions normally supported by zoospore ultrastructure. In the past few years, chytrid taxonomy has experienced considerable changes. For instance, James et al. (2006b) described the new phylum Blastocladiomycota; [START_REF] Letcher | Ultrastructural and molecular phylogenetic delineation of a new order, the Rhizophydiales[END_REF] redescribed the Rhizophydium clade (James et al. 2000(James et al. , 2006a) ) as the order Rhizophydiales; [START_REF] Mozley-Standridge | Cladochytriales -a new order in Chytridiomycota[END_REF] established the order Cladochytriales for the Cladochytrium clade (James et al. 2006a) and [START_REF] Simmons | Lobulomycetales, a new order in the Chytridiomycota[END_REF] redescribed the clade formerly represented in phylogenetic trees by Chytriomyces angularis (James et al. 2006a) as the order Lobulomycetales. New orders have also been proposed: Polychytriales [START_REF] Longcore | The Polychytriales ord. nov. contains chitinophilic members of the rhizophlyctoid alliance[END_REF], Gromochytriales and Mesochytriales (Karpov et al. 2014) and Synchytriales [START_REF] Longcore | Synchytrium microbalum sp. nov. is a saprobic species in a lineage of parasites[END_REF]. Moreover, some genera with typical chytrid morphology and hence thought to branch early in the fungal tree, such as Olpidium [START_REF] Sekimoto | A multigene phylogeny of Olpidium and its implications for early fungal evolution[END_REF], Amoeboradix and Sanchytrium (Karpov et al. 2017b), appeared to branch among more apical fungal lineages, out from the Chytridiomycota clade. It is clear that we need molecular data for each traditionally described species of chytrids to construct a meaningful and comprehensive classification of Chytridiomycota (see also [START_REF] Powell | Chytridiomycota[END_REF]. Transmission electron microscopy (TEM) sometimes reveals peculiarities that can be used as new taxonomic characters or, on the contrary, may show the unimportance of some commonly accepted ultrastructural features. However, zoospore ultrastructure has so far provided useful discriminatory characters for taxonomy in agreement with the molecular phylogeny of fungi. Some chytrids have been found to contain insertions in ribosomal DNA, which may or may not be present in close relatives, but that are rather common in Dikarya and green algae (Karpov et al. 2017b). Here we present light microscopy observations, ultrastructure and molecular phylogenetic analysis of strain X-124 (CCPP ZIN RAS), a parasite of algae isolated from freshwater samples using Tribonema gayanum as a host. Phylogenetic analysis of 18S rRNA gene sequences of this strain supports that it represents a new genus and species affiliated to the recently established order Gromochytriales. The ultrastructure of X-124 confirms its phylogenetic position sister to Gromochytrium and serves as the basis for the description of this new genus and species.

MATERIALS AND METHODS

Collection and isolation

Strain X-124 was isolated by M.A. Mamkaeva in 2012 from the freshwater sample x-22 collected in a small pond in Kuty village, Kingisepp District, Leningrad Province, Russia. A strain was maintained in culture on Tribonema gayanum (strain 20 CALU) as a host as described inKarpov et al. (2016).

Light and transmission electron microscopy

Light and DIC microscopy observations of living cultures were carried out on a Zeiss Axioplan microscope (Leica Microsystems, St. Petersburg, Russia) equipped with black and white MRm Axiocam (Leica Microsystems, St. Petersburg, Russia).

Electron microscopy

Infected algal filaments were fixed with 0.3% OsO4 in 0.1M cacodylate buffer during two minutes, then, glutaraldehyde was added to 2% final concentration for 30 min in the dark. Then the fixative was removed and 2% glutaraldehyde in 0.1M cacodylate buffer was added for 1 hour, rinsed in 0.1M cacodylate buffer and postfixed in 1% OsO4 in the same buffer for one hour. All fixation stages were carried out on ice. After rinsing in 0.1M cacodylate buffer for 5 min, the filaments were dehydrated in alcohol series and in propylen oxide, and finally embedded in Spurr resin (Sigma Aldrich, St Louis, USA). Ultrathin sections were prepared using a Leica Ultracut ultratome with a diamond knife. After double staining, the sections were observed using a JEM 1400 microscope (Jeol, Japan) equipped with the digital camera Olympus Veleta (Japan).

18S rRNA gene sequencing and molecular phylogeny

Cells from infected cultures were pelleted by centrifugation and their DNA was purified using the PowerSoil DNA purification kit from MoBio (Carlsbad, USA). 18S rRNA gene fragments were then amplified by PCR using the fungal specific primers Fun_UF1 (5'-CGAATCGCATGGCCTTG) [START_REF] Kappe | Universal fungus-specific primer systems and group-specific hybridization oligonucleotides for 18S rDNA[END_REF]) and Fun_AU4

(5'-RTCTCACTAAGCCATTC) [START_REF] Vandenkoornhuyse | Arbuscular mycorrhizal community composition associated with two plant species in a grassland ecosystem[END_REF]. PCR reactions were carried out in a final volume of 25 μl. The reaction mix contained 1 μl of the eluted DNA, 1.5 mM MgCl2, dNTPs (10 nmol each), 20 pmol of each primer, and 0.2 U TaqPlatinum DNA polymerase (Invitrogen). After a first denaturation step at 94 °C for 2 min, the PCR reaction consisted of 35 cycles of the following iterative cycle: denaturation at 94 °C for 15s, annealing at 50°C for 30 sec, and polymerization at 72 °C for 2 min. A final elongation at 72 °C for 7 min was carried out. We cloned the resulting 18S rRNA amplicons to identify individual fungal sequences using the Topo TA Cloning System (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Clone inserts were then amplified by PCR using flanking vector primers and the inserts of expected sizes were sequenced bidirectionally with vector primers (Beckman Coulter Genomics, Takeley, UK). Two closely related chytrid sequences were identified and compared with sequences in the GenBank nr database using BLAST [START_REF] Altschul | Basic local alignmentsearch tool[END_REF]). The new sequences and those retrieved by BLAST were aligned using MAFFT with the accuratelinsi option [START_REF] Katoh | MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform[END_REF]. The multiple alignment was trimmed with TRIMAL [START_REF] Capella-Gutierrez | trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[END_REF]) and a Bayesian phylogenetic tree was reconstructed using MrBayes [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF] applying the GTR + G + I model with four chains and 10,000,000 generations. After checking for convergence, the first 15,000 trees were eliminated as burn-in, and a consensus tree was constructed by sampling every 100 trees. The new Apiochytrium granulosporum strain X-124 18S rRNA sequences have been deposited in GenBank under accession numbers XXXXX.

Intron S943 phylogeny

Intron localization was defined by comparing assembled contig with other species from NCBI nr database using BLAST. Insertion positions were identified using the 16S gene reference of Escherichia coli [START_REF] Cannone | The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs[END_REF]; ttp://www.rna.icmb.utexas.edu/SAE/2C/rRNA_Introns). Additional SSU sequences containing intron S943 were downloaded from GenBank. Sequences were aligned with MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF]) and manually adjusted in BioEdit [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. Bayesian inference was calculated with MrBayes applying the GTR + G + I model with four chains and 10,000,000 generations. After checking for convergence, the first 50% trees were eliminated as burn-in, and a consensus tree was constructed. The resulting Bayesian tree was visualized using the iTOL web tool [START_REF] Letunic | Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees[END_REF].

RESULTS

Morphology Zoospores

This fungus produces slightly elongated or spherical zoospores 2.3-2.7 μm in diameter with a posterior single lipid globule and a comparatively short flagellum about 8-9 μm in length having a short acroneme of approximately 1 μm (Fig. 1A,B). In released zoospores, the single prominent lipid globule locates at a posterior position, near the point of flagellum emergence (Fig. 2A-D). The lipid globule is associated with a flat microbody and covered with fenestrated cisternae facing the plasma membrane (Fig. 2D). The elongated nucleus is slightly curved and oriented in posterior-anterior direction. Its posterior end is located near the microbody-lipid complex (MLC) (Fig. 2D), the lateral cavity of the nucleus is filled with small vesicles and rare cisternae of the Golgi apparatus (not shown). Mitochondrial profiles with flat cristae are scattered in the cytoplasm, and the ribosomes do not aggregate (Fig. 2A,D). Mature intrasporangial zoospores are predominantly of amoeboid appearance, and in general exhibit the same location of MLC, nuclear cavity, Golgi apparatus, dispersed ribosomes and mitochondria (Fig. 4B-D). A non-functional kinetosome (centriole) is located at sharp angle (ca. 60°) with the flagellar kinetosome and connected to it with a broad fibrillar bridge (Fig. 4C,D). The flagellar transition zone contains a spiral fiber, or cylinder (Fig. 2B,C). A short posterior root of two microtubules passes, between kinetosome and centriole, to the plasma membrane (Fig. 4D). Traces of one more root (probably anterior) of 2-3 microtubules can be found between the plasma membrane and the fenestrated cisternae (Fig. 2A), but its origin could not be fully tracked.

Cyst

Zoospores encyst on the surface of the host alga and produce a rhizoid penetrating the host cell wall (Figs 1A; 2E, F). In addition to the chitin wall, the cyst is covered with a rather thick layer of electron dense amorphous material (Fig. 2E). Each cyst contains a nucleus with a large central nucleolus and a lipid globule that can be visible in the thin sections (Fig. 2E). The rhizoids were not visible on the living material, but the ultrastructural observation revealed a very thin rhizoidal stem penetrating the algal cell wall and its thickened part just under the wall of the host (Fig. 2E,F). Such a narrow hole (less than 200 nm as measured in serial sections) in the algal cell wall for thin rhizoids has been also found in the mature sporangium (not shown). The rhizoid distal part branches inside the cell, sometimes enveloping the host nucleus, which looks deformed (Fig. 3B).

Sporangium

Monocentric epibiotic sessile slightly broaden pear-shaped sporangia up to 10-12 μm in height with a broad apical papilla for zoospore releasing locate on the Tribonema filament (Fig. 1C-E). The immature sporangia have characteristic granulated contents (Fig. 1 E) with several nuclei and one big lipid globule (Fig. 3A). At a later stage of sporangium maturation (Fig. 4A), this globule disappears, likely as a consequence of its use as an energy source for zoospore formation. Interestingly, in spite of the fact that the plasmodium division just has started and the flagella are not yet formed, the papilla is already open (Fig. 4A). In the mature sporangium, each newly formed zoospore contains a single big globule immediately prior to zoospore release. Thus, each intrasporangial zoospore already contains a posterior lipid globule near the flagellar base (Fig. 4B-D).

18S rRNA phylogeny

The two 18S rRNA gene sequences of the strain X-124 were very similar (99,4% identical) and formed a clade sister to the cluster containing Gromochytrium mamkaevae and some environmental sequences with high support (Fig. 5). We ignore if the two X-124 sequences correspond to two closely related chytrid strains present in our culture or to two slightly divergent 18S rRNA genes present in a single species. According to our tree, this strain represents a new deep-branching lineage within the order Gromochytriales, clearly distinct from Gromochytrium. In addition, the strain X-124 sequences contains a large (1476 bp) insertion absent in the other Gromochytriales sequences. The presence of this large insertion may have impacted the whole 18S rRNA gene structure of X-124, which may explain the relatively long branch of this strain compared with the other Gromochytriales. Taking into account its phylogenetic distance, we consider that strain X-124 represents a new genus and species within the order Gromochytriales. Our tree also supports strongly the sister relationship between the Gromochytriales and the Mesochytriales, and the position of the recently described Polyphagales as sister of the Gromochytriales+Mesochytriales cluster (Fig. 5), in agreement with previous analyses (Karpov et al. 2016).

S943 intron phylogeny

The 18S rRNA gene of the strain X-124 contains a group I intron with an insertion position at S943 according to E. coli reference [START_REF] Cannone | The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs[END_REF]. The intron locates on the antisense strand of P8 and carries a homing endonuclease gene coding His-Cys box protein 244 amino acid residues long. Homing endonuclease genes are rare but widely distributed among S943 introns [START_REF] Haugen | The Evolution of Homing Endonuclease Genes and Group I Introns in Nuclear rDNA[END_REF]). The predicted homing endonuclease protein of the strain X-124 does not have at its C-terminus with the 2nd zinc-binding motif and dimerization tail which are common for His-Cys box family proteins [START_REF] Chevalier | Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility[END_REF]. However, zinc-binding conservative motif can be detected if multiple frame shifts are introduced into the nucleotide sequence. Previously, pseudogenes/remnants of homing endonuclease genes were found in S943 introns [START_REF] Haugen | The Evolution of Homing Endonuclease Genes and Group I Introns in Nuclear rDNA[END_REF]). Within chytridiomycetes homing endonuclease genes in 18S rRNA genes were also found in Gonapodya sp. (intron insertion position S943) and Phlyctochytrium planicorne (intron insertion position S508). According to tblastn search against nr/nt database of NCBI, the endonuclease from Gonapodya sp. is the most similar toward endonuclease from the strain X-124. S943 is one of the most common positions among all group I intron insertion sites in 18S rRNA genes [START_REF] Haugen | The Evolution of Homing Endonuclease Genes and Group I Introns in Nuclear rDNA[END_REF]Karpov et al. 2017b). S943 introns are widely distributed in different fungi, amoeba, algae, cercozoans and ciliates. Most fungi group together in the Bayesian phylogenetic tree of S943 introns, forming a monophyletic Dikarya group (Supplementary Figure S1). With a few exceptions, ascomycetes form a large cluster, which include one basidiomycete and one zygomycete species. Basidiomycetes form three paraphyletic groups and one group inside the ascomycetes cluster. Viridiplantae form a monophyletic group which include two amoeba species. Strain X-124 intron branches off from the tree right before the dikaryan radiation. There are other species branch around X-124 with unclear branching order -two uncultured chytrid clones, five monoblepharids, the aphelid Amoeboaphelidium occidentale, and some oomycetes and cercozoans.

DISCUSSION

Phylogeny and ultrastructure

The 18S rRNA gene sequences of the X-124 strain form a monophyletic cluster with some environmental sequences (Fig. 5) that corresponds to the recently described order Gromochytriales (Karpov et al. 2014). This order includes one cultured chytrid, Gromochytrium mamkaevae, having zoospores with posterior ribosome aggregates unbounded by endoplasmic reticulum and one MLC with anterior location with a rather thick microbody. By contrast, X-124 shows dispersed ribosomes and a posterior MLC with a thin microbody. Both species exhibit a single lipid globule with fenestrated cisternae facing towards the exterior, posterior and probably anterior microtubular roots, a cylinder or spiral filament in the flagellar transition zone and several dispersed mitochondria. G. mamkaevae has a centriole at an angle of approx. 30° with respect to the kinetosome vs. 60° in X-124. We did not observe a centriole in released zoospores; nonetheless, we can propose that the angle of centriole location in the X-124 intrasporangial zoospore may probably be changed during complete maturation in the released zoospore. Although a centriole-kinetosome bridge was certainly present, our material did not allow studying it in detail. Some zoospore characters of X-124, i.e. dispersed ribosomes and posterior MLC, are similar to those of Mesochytrium penetrans [START_REF] Karpov | Phylogenetic position of the genus Mesochytrium (Chytridiomycota) based on zoospore ultrastructure and 18S and 28S rRNA gene sequences[END_REF], which belongs to the closely related order Mesochytriales. However, other zoospore characters of M. penetrans essentially differ from those of X-124: the ER bounding MLC associated with a single big mitochondrion, fenestrated cisterna facing the flagellar base, and the presence of a vacuole and several fibrils associated with the centriole and kinetosome [START_REF] Karpov | Phylogenetic position of the genus Mesochytrium (Chytridiomycota) based on zoospore ultrastructure and 18S and 28S rRNA gene sequences[END_REF]. The flagellar apparatus of the M. penetrans zoospore contains a spiral fiber in the transition zone, but does not have any microtubular roots [START_REF] Karpov | Phylogenetic position of the genus Mesochytrium (Chytridiomycota) based on zoospore ultrastructure and 18S and 28S rRNA gene sequences[END_REF]. In summary, the zoospore morphology and, in particular, the flagellar apparatus of X-124 are more similar to those of G. mamkaevae than to those of M. penetrans, confirming the affiliation to the Gromochytriales. The partial zoospore similarity of X-124 to M. penetrans is in agreement with a somewhat intermediate phylogenetic position of X-124 between Gromochytrium and Mesochytrium. In fact, the X-124 18S rRNA sequences branch deep in the Gromochytriales (Fig. 5), which might explain that X-124 has retained some features of the last common ancestor of Gromochytriales and Mesochytriales, sharing characters with both orders. From a taxonomy perspective, our data provide additional material for revision the key diagnostic characters defining the order Gromochytriales. However, at this stage of investigation, such a revision is obviously immature. More molecular and ultrastructural data from other representatives of the order will be needed to attempt that task.

S943 intron phylogeny

In general, the results of the phylogenetic analysis of S943 intron sequences are similar to those of the 18S rDNA phylogeny of these groups (Supplementary Figure S1), as previously seen in [START_REF] Haugen | The Evolution of Homing Endonuclease Genes and Group I Introns in Nuclear rDNA[END_REF]. High level taxa such as Viridiplantae, Dikarya, Ascomycota and Basidiomycota appear monophyletic, which supportsthat vertical gene transfer is the main mechanism of S943 intron heredity among these taxa. However, the incongruent localization of a few branches might suggest some cases of horizontal transfer, as also seen in [START_REF] Haugen | The Evolution of Homing Endonuclease Genes and Group I Introns in Nuclear rDNA[END_REF]. For example, a transfer between ascomycetes and basidiomycetes might have happened in the case of Ballistosporomyces sasicola. The majority of taxa include nonintronized and intronized species, although the former situation predominates across the phylogenetic tree. The observed mosaic distribution of introns might argue against the hypothesis of their vertical heredity. Whether vertical inheritance followed by losses or horizontal gene transfer from preferred sources are the cause of this pattern will require further study.

Taxonomy

According to light microscopic observations, strain X-124 is similar to Rhizophydium granulosporum Scherff., a parasite of Tribonema with a broadly pyriform sporangium of close dimensions (7-14 μm), delicate rhizoids arising from a short main axis and ovoid zoospores of nearly the same dimensions (3 μm long and 2 μm diameter) with a large colorless globule [START_REF] Sparrow | Aquatic Phycomycetes[END_REF][START_REF] Letcher | A taxonomic summary and revision of Rhizophydium (Rhizophydiales, Chytridiomycota)[END_REF]. However, the globule in Rh. granulosporum zoospore is slightly eccentric, not posterior as in X-124, and the flagellum of Rh. granulosporum seems to be longer than in X-124, although the flagellar length was not shown in the diagnosis of Rh. granulosporum. The resting spore was not found in X-124. Thus, zoospores of X-124 strain differ essentially from those of Rh. granulosporum and belong to a new genus. Apiochytrium Karpov et Moreira gen. nov. MycoBank MB 827963 Zoospores contain posterior MLC with single lipid globule and flat microbody near the flagellar base, dispersed ribosomes and several scattered mitochondria. Kinetid structure as for the order. Etymology: Apio (Greek) -pear, referring to the pyriform sporangium. Type species A. granulosporum Karpov et Moreira sp. nov. Apiochytrium granulosporum Karpov et Moreira sp. nov (Fig. 1).

MycoBank MB 827964

Monocentric epibiotic sessile pyriform sporangia up to 12 μm in height and 9 μm broad with 1 broad apical papilla for zoospore releasing. Branching rhizoid is invisible on the living material. Elongated to spherical zoospores 2.3-2.7 μm in diameter or up to 3 μm in length with posterior single lipid globule and flagellum 8-9 μm in length. A non-functional kinetosome (centriole) is located at sharp angle to the flagellar kinetosome. Parasite of yellow-green alga Tribonema gayanum.

Etymology: after the name Rh. granulosporum -most similar to the strain X-124. GenBank Accession Number XXXXXXX. Type: Fig. 1. Karpov et al. this [START_REF] Malysheva | Culture collection of parasitic protists at the Zoological Institute RAS (CCPP ZIN RAS)[END_REF] under No: X-124.

ACKNOWLEDGMENTS

The isolation, cultivation, morphological study of strain X-124 and the manuscript writing have been supported by RSF grant no. 16-14-10302, and the molecular analysis by ERC Advanced Grant No.

Fig. 1 .

 1 Fig. 1. DIC images of living zoospores (A, B) and sporangia (C-H) of Apiochytrium granulosporum sp. nov. (X-124 CCPP ZIN RAS). A -released zoospores, cyst (cy) and empty sporangium (sp), B -zoospores at high magnification. Arrows on B show posterior lipid globule. C, D -young sporangia, E -premature sporangium. Scale bars: A -5 μm, B -3 μm, C-E -5 μm.

Fig. 2 .

 2 Fig. 2. Transmission electron micrographs of Apiochytrium granulosporum sp. nov. (X-124 CCPP ZIN RAS). A-D -sections of free-swimming zoospores, B, C -consecutive sections of flagellar transition zone with spiral filament (arrowheads), E -encysted zoospore penetrating the algal cell wall (aw) with a rhizoid (rh), F -rhizoid (rh) inside the alga. Abbreviations: aw-algal cell wall, chl-chloroplast, fc-fenestrated cisterna, flflagellum, hc-host cytoplasm, l-lipid globule, mmitochondrion, mi-microbody, n-nucleus. Arrowheads on A show microtubules of the anterior root. Scale bars: A -500 nm, B, C -200 nm, D -300 nm, E -800 nm, F -400 nm.

Fig. 3 .

 3 Fig. 3. Transmission electron micrographs of Apiochytrium granulosporum sp. nov. (X-124 CCPP ZIN RAS). A -young sporangium with several nuclei and a big lipid globule, B -host nucleus (hn) surrounded by rhizoids (rh). Abbreviations: aw-algal cell wall, hnhost nucleus, l-lipid globule, nnucleus, nu-nucleolus, sw-sporangial wall. Scale bars: A -2 μm, B -1 μm.

Fig. 4 .

 4 Fig. 4. Transmission electron micrographs of Apiochytrium granulosporum sp. nov. (X-124 CCPP ZIN RAS). A -immature sporangium with open papilla, several nuclei and without lipid globule. Arrowheads show developing cell borders, B -sporangium with mature and releasing zoospores, C -intrasporangial zoospore. Arrowhead shows a fibrillar bridge between centriole (c) and kinetosome (k). D -longitudinal section of centriole adjacent to kinetosome which is obliquely sectioned here. Arrowheads show a posterior root of two microtubules. Abbreviations: aw-algal cell wall, fl-flagellum, l-lipid globule, m-mitochondrion, n-nucleus. Scale bars: A -2.5 μm, B -2 μm, C -200 nm, D -200 nm.

Fig. 5 .

 5 Fig. 5. 18S rRNA gene Bayesian phylogenetic tree based on 1456 conserved sites. The tree is rooted using sequences of Lobulomycetales and Polychytriales. Numbers at branches are Bayesian posterior probabilities (only values higher than 0,7 are shown).

  Figure 1.

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Figure

  Figure 5.

  publication. RUSSIA, Leningrad Province, Kingisepp District, small pond in Kuty village (59°48'62'' N, 28°94'66'' E). Sample collected by Maria Mamkaeva in April 2012. Ex type culture deposited in Culture Collection of Parasitic Protists of Zoological Institute of Russian Academy of Science (CCPP ZIN RAS)