Kevin M Smith

Rinaldo Poli

Jeremy N Harvey

Poli Harvey Smith

Ligand Dissociation Accelerated by Spin State Change

published or not. The documents may come

Ligand dissociation accelerated by spin state change: locating the minimum energy crossing point for phosphine exchange in CpMoCl2(PR3)2 complexes

The reactivity consequences of spin state changes in solution phase organometallic chemistry

are not yet fully understood. 1 Although it has been proposed that having to hop from the potential energy surface (PES) of one spin state to another might slow reactions down, several kinetic studies of ligand addition processes have found no such "spin flip barrier". [2][3][4] The influence of spin state changes on the reactivity of transition metal ions in the gas phase, however, is fairly well established, 5- 7 and these reactions provide useful models for condensed-phase processes. For example, studies of the reactions of gas phase metal oxide ions, such as that between FeO + and CH4, have recently led to new insights into the chemistry of cytochrome P-450 and of other metal-oxo species relevant to biochemistry and organic synthesis. [8][9][10][11][12][13][14] Several years ago, we reported that dissociative phosphine exchange reactions in spin doublet CpMoCl2(PR3)2 complexes were unexpectedly fast (equation 1), and proposed that this acceleration was due to a facile spin-forbidden dissociation to a quartet CpMoCl2(PR3) intermediate, occurring with a barrier lower than that for the pathway involving spin-allowed dissociation to the corresponding doublet. 15 Recent advances in computational techniques, particularly density functional theory (DFT), 16 have made it possible to assess the relative energies of unsaturated organometallic species in different spin states. Calculations on CpMoCl2(PH3)n (n = 1, 2) models confirmed that the proposed pathway would be thermodynamically favorable, with the CpMoCl2(PH3) intermediate predicted to have a quartet ground state. 17 Computational support for the all-important kinetic feasibility of the proposed mechanism was less forthcoming. Evaluation of rates of non-adiabatic reactions [START_REF] Yarkony | For an overview of non-adiabatic chemistry[END_REF] with spin state changes requires (a) an unambiguous determination of the of the strength of the spin-orbit-induced coupling between the two PESs at that point. In this Letter, we have calculated both points (a) and (b) for the CpMoCl2(PH3)2 system. The MECP is found to lie considerably lower in energy than the spin doublet dissociative intermediate, and there is substantial spin-orbit coupling at that point, in full support of the mechanistic hypothesis. For point (a), we have compared two approaches for locating the MECP: the partial optimization method, which has been frequently used in studies of transition metal chemistry; and the explicit full-dimensional optimization of the MECP, which had not until now been performed for such large, realistic polyatomic models such as that studied here. We believe that this latter method will prove to be more broadly useful for the theoretical investigation of spin state effects in transition metal chemistry in solution. 8,9,[19][20][START_REF] Harvey | Computational Organometallic Chemistry[END_REF] In the first place, we have studied the crossing between the doublet and quartet PESs of the CpMoCl2(PH3)2 system using the partial optimization method. This approach has already been frequently used to estimate the position of the MECP in various complex models of solution phase organometallic reactions. [START_REF] Keogh | [END_REF][23][24][25][26] It involves multiple geometry optimizations on both PESs whilst holding constant one carefully selected internal geometry parameter, until a value for the parameter is obtained at which both spin states have the same energy. The parameter chosen is usually a bond length, or an angle between two bonds, which has markedly different values at the minima corresponding to the two spin states. Because of the multiple partial geometry optimizations required, the method can be rather computationally expensive, but of more concern is the fact that it only provides an estimate of the lower energy limit to the MECP because all the other degrees of freedom are optimized separately on the two surfaces. Therefore, the "Crossing Point" located using this technique is in fact two different points, which, depending on which unique parameter is chosen, and on the features of the two PESs, may or may not be very similar to each other and to the real MECP.

A measure of how different the two points are can be obtained by computing single point energies on each PES at the partially optimized geometry of the other one. These energies can also be used as very rough upper energy limits for the MECP. The results of our DFT computations on the dissociation of PH3 from CpMoCl2(PH3)2 using this procedure are illustrated in Figure 1 and Table 1. As noted previously, 17 the dissociated 15electron CpMoCl2(PH3) species has a quartet ground state, calculated here to lie 8.4 kcal mol 1 below the doublet. The coordinate we have chosen to vary is the Mo-P distance, along which the interaction of doublet CpMoCl2(PH3) with the incoming phosphine ligand is purely attractive, whereas the corresponding quartet PES is purely repulsive. The two curves cross, near the point with a fixed MoP distance of 3.43 Å. At that distance, their relative energies (defined with respect to the CpMoCl2(PH3)2 doublet minimum) are both within 0.1 kcal mol 1 of 11.9 kcal mol 1 . Single point computations for each spin state at the partially optimized geometry of the other spin state give higher energies, of 18.9 and 20.2 kcal mol 1 , respectively, for the doublet and the quartet. This reflects the differences between the partially optimized geometries on the two PESs (Figure 1), which are significant if not enormous. [START_REF] Kubácek | As previously noted, quartet CpMoCl2(PH3) posseses a normal three-legged piano stool structure while doublet CpMoCl2(PH3) displays significant deformations. Erreur ! Signet non défini[END_REF] The estimated lower and upper limits to the MECP's energy obtained in this way can be seen (Fig. 1) to lie, respectively, well below and just above the doublet dissociation asymptote of 16.9 kcal mol 1 . Although the lower limit is the more reliable of the two, there does remain a certain ambiguity as to the position of the MECP, and therefore, as to whether or not the pathway for spin-forbidden dissociation to the quartet will be kinetically favored over the spinallowed doublet route. The importance of locating MECPs explicitly when trying to understand spin-forbidden reaction mechanisms is well recognized, and several groups have done so in the study of mediumsized organic and main-group inorganic systems. Recent examples include the interconversion of singlet and triplet phenyl cation, 28,29 the unimolecular dissociations of triplet methoxy cation 30 and of ethylenedione, 31 and the two combustion reactions, HC + N2 32 and O + C2H2. 33 Some applications to small transition-metal containing systems have also been reported, such as the gas-phase dehydrogenation of methane by the Scandium cation. 34 However, many of the methods available for finding MECPs 35,36 are based on the use of complete active space self-consistent field (CASSCF) computations, whose expense generally scales very badly with system size. Although many useful results can be obtained with carefully executed CASSCF computations on transition metal systems, 37 this expense factor does in part explain why no MECP for a reaction of a large organometallic compound has yet been optimized. However, except when the wavefunction of one or both of the spin-states is intrinsically multi-referential in nature, which is not the case here, there is no fundamental reason why CASSCF-based computations are needed to locate MECPs. Instead, any ab initio method, provided it describes both states in a sufficiently accurate way, can be used for this purpose. 26,38 Our approach, 26 which is a simple modification of two previously described ones, 36,38 relies on combining the separately computed energies and gradients on the two PESs so as to produce an effective gradient which is then followed downhill towards the MECP. Its compatibility with DFT computations, the method of choice at the present time for computations on large transition metal systems, 16 makes it ideal for the present work.

We have accordingly optimized the geometry (Figure 2) of the MECP between the doublet and quartet PESs of CpMoCl2(PH3)2. The structure obtained is similar to those derived using the partial optimization method. Interestingly, the main difference is in the "special" Mo-P distance, which is over 0.2 Å shorter in the fully optimized structure. The relative energy at the MECP is of 12.1 kcal mol 1 , i.e. 4.8 kcal mol 1 lower than the doublet dissociation asymptote. We have also used computations to estimate the root-mean square (rms) doublet-quartet spin-orbit coupling at the MECP, and find it to be on the order of 250 cm 1 . This value is large enough to ensure facile surfacehopping at the MECP. 39 Combining these results on the energy of the MECP and the spin-orbit coupling provides full support for the mechanistic hypothesis, whereby dissociation of doublet Selected bond lengths and angles for partially optimized doublet (plain text), partially optimized quartet (italics), and MECP (bold). To compare the two methods for characterizing MECPs, one can see that the energy of the fully optimized structure is rather close to that of the crossing point of the partial optimization curves. Also, the geometries are reasonably close, with the exception of the Mo-P distance, where there is a quite significant difference. The overall good agreement obtained is probably due to the nature of the reaction: for the phosphine exchange process, the choice of the Mo-P bond as a "special" coordinate for the partial optimization process is relatively straightforward, and the differences in the optimized values of the other parameters on the two surfaces, whilst significant, are not major. The fact that the explicit MECP localization does not require a "special" coordinate, and can be started from an arbitrarily chosen geometry, 28,36,38 is potentially valuable for cases where identifying such a parameter is less obvious. Before explicitly optimizing the MECP, it was not immediately apparent that the differences between the optimized values for the other degrees of freedom on the two PESs would not exert a more severe influence. While the only criterion available for judging the quality of the partial optimization result, the single-points upper limit calculations, provide little help, the explicit MECP localization is unambiguous. It is also a matter of interest to note that fully optimizing the MECP is the less computationally demanding of the two approaches. Overall, therefore, apart from availability issues, there appears to be no reason to prefer partial optimization in studies of crossings of PESs of different spin.

In contrast to gas phase studies of transition metal ions, much work remains to be done to investigate the effect of spin state changes on reactivity in solution phase organometallic chemistry.

Recently, spin state effects in solution have been addressed in the reactions of metal oxo compounds, and in CH and SiH activation by unsaturated CpM(CO)n species. In these cases, different spin states exhibit distinct reactivity patterns, and the factors which influence the spin crossover process play a dramatic role in dictating the observed reactivity. It is hoped that the computational determination of MECPs in transition metal systems, using the explicit technique shown to be possible and preferable in this Letter, will prove useful in the identification and examination of further examples of this phenomenon. More generally, we believe this will contribute to an enhanced awareness and understanding of the effects of spin state changes in organometallic reactions.

Computational Details

The ab initio computations descibed in this Letter were mostly performed with Gaussian 94, [START_REF] Frisch | Gaussian 94[END_REF] using the B3LYP hybrid density functional, together with the standard LanL2DZ basis sets on all atoms, and the associated effective core potentials on the Mo, P and Cl centers. Unrestricted "wavefunctions" were used throughout, with the expectation value of S 2 being very close to its ideal value in all cases.

CS symmetry was assumed for all structures. For the calculation of the spin-orbit coupling, the semiempirical effective nuclear charge-one electron operator approach 41 was applied, using the Gamess-USA program. [START_REF] Schmidt | [END_REF] Full CI wavefunctions in the space of the Mo 4d orbitals were used to describe the doublet and quartet. Due to computational restrictions, these were expanded using ROHF orbitals optimized for one of the two states, with a standard double-zeta all-electron basis set on all the lighter atoms, together with the standard SBK relativistic ECP and basis set on Mo. The effective charges used were those of ref. 41(a), except for Mo, where a value of 200 was optimized to fit the experimental splitting of Mo 3+ ( 4 F). The low quality of the wavefunctions used is shown by the different values obtained using the quartet (rms SOC 314 cm 1 ) and doublet (213 cm 1 ) orbitals in the calculations. Based on experience with other systems, 2631 however, we believe that as an estimate, our proposed value of 250 cm 1 should not be too far wrong.

Ligand

  Dissociation Accelerated by Spin State Change p. 3 / 11 energy at the minimum energy crossing point (MECP) between the relevant PESs, and (b) calculation

Smith, Poli, Harvey Ligand 11 Figure 1 .

 Harvey111 Figure 1. Energies of partially optimized doublet and quartet CpMoCl2(PH3)2 relative to the overall doublet minimum, at various fixed MoP distances, including dissociated CpMoCl2(PH3) (dashed lines) and estimated upper limit of crossing point energy (+).

CpMoCl2(PH3) 2

 2 proceeds through the quartet intermediate, CpMoCl2(PH3). Thus, far from being a hindrance, the spin state change actually facilitates ligand dissociation by lowering the activation enthalpy compared to an alternative reaction pathway that remains confined to the doublet PES.15

Figure 2 .

 2 Figure 2. Optimized geometry of MECP between doublet and quartet CpMoCl2(PH3)2.

Table 1 .

 1 MoP bond lengths (Å) and relative energies (in kcal mol 1 , relative to optimized doublet

	CpMoCl2(PH3)2) of spin doublet and quartet CpMoCl2(PH3)2 complexes.
	Complex	MoP	Doublet	Quartet
	CpMoCl2(PH3)2	2.580	0.0	
	CpMoCl2(PH3) + PH3		+16.9	+8.5
	partial optimization	3.430	+11.9	+11.8
	estimated upper limits	3.430	+18.9	+20.2
	MECP	3.201	+12.1	+12.1

Acknowledgement. R. P. thanks the Région Bourgogne for supporting this research, J. N. H. thanks Prof. G. G. Balint-Kurti for computational resources, and Mike Schmidt for providing the Gamess-USA program, and K. M. S. thanks the European Commission for a TMR Marie Curie


Jacques Andrieu
email: andrieu@u-bourgogne.fr

Clara Baldoli

Stefano Maiorana

Rinaldo Poli
email: poli@u-bourgogne.fr

Philippe Richard

Chiral α-P,N Ligands From a Diastereoselective Ph2PH Addition to (η6-Benzaldimine)tricarbonylchromium Complexes

HAL is

Introduction

Since the discovery that bifunctional P,N ligands increase considerably the activity and/or the selectivity of palladium, ruthenium or rhodium catalysts, [1][2][3][4] the preparation of this type of ligand has been the subject of extensive investigations. As an example, pyridyl diphenylphosphine (2-PyPPh2) crucially controls the activity and selectivity of alkyne methoxycarbonylation catalysts. [1] Other -aminophosphine ligands are rare: compound Ph2PCH2N(CH3)2, having a tertiary amine function, is stable in the solid state and in solution, [5] while Et2PCH2NH t Bu and Ph2PCH(Ph)NHPh have only been described in the solid state. [5,6] Recent studies by some of us have demonstrated in solution a reversible P-C bond cleavage of -aminophosphine ligands with a secondary amine function (Scheme 1). [7] It was found, for instance, a K = 50 for R 1 = R 2 = Ph and that electronwithdrawing substituents R 1 and R 2 or phosphorus coordination to Cu(I) favor the formation of the P-C bond. This behavior rationalises previously reported unexpected P-C bond cleavage reactions. [6,8,9] We now report a stereoselective extension of this synthetic method leading to the preparation of chiral -P,N ligands.

Scheme 1

Results and Discussion

The synthetic strategy consists in using chiral ortho-substituted benzaldimine Cr(CO)3 complexes (Scheme 2). As it is well known, in these complexes the coordination of the aromatic ring to the bulky Cr(CO)3 fragment makes one imine diastereoface inaccessible to the phosphine attack. In addition the presence of the ortho-substituent favours the conformation in which the ortho-group is anti with respect to the imino moiety. These stereochemical features of planarchiral chromium tricarbonyl complexes [START_REF] Solladie´-Cavallo | Advances in Metal-Organic Chemistry[END_REF][START_REF] Davies | Comprehensive Organometallic Chemistry II[END_REF] and of Cr(CO)3 complexed imines [START_REF] Maiorana | [END_REF][13][14][15] in particular, have been previously exploited in stereoselective synthesis. to the formation of equilibrium quantities of the desired aminophosphine ligand (2-6) as a single diastereoisomer, within the detection limits of 31 P NMR spectroscopy (Table 1). For the sake of semplicity, Scheme 2 shows this transformation for only one of the two enantiomers. The relative concentrations at equilibrium are determined by integration of the 1 H NMR resonances of the imine and product methyne protons and/or the Ph 2 PH and product 31 P NMR resonances. With the exception of complex 6, these constants are smaller relative to that previously reported for the formation of 1 (R 1 = R 2 = Ph, Scheme 1). [7] Given the previously established electronic control on the equilibrium of Scheme 1, the presence of the Cr(CO) 3 moiety would be expected to enhance the formation of the P-C bond, while the presence of the Y substituent would provide an effect which depends on the relative  and π donor/acceptor properties. It can therefore be concluded that, when R = Ph, the combination of the arene-coordinated Cr(CO) 3 group and the Cl substituent is sufficient for a quantitative P-C bond formation. For compound 5, the electron withdrawing effect of the Cr(CO) 3 moiety is not sufficient to compensate the overall donor properties of the two methoxy substituents. The individual effect of the Cr(CO) 3 moiety has been evaluated by comparing the formation of 6 with corresponding reaction of the Cr(CO) 3 -free imine, forming product 7. It can be observed that the constant K increases by at least a factor of 3 upon Cr(CO) 3 coordination. The comparison between compounds 3 and 5 on one side and compounds 2 and 4 on the other side shows that K increases by changing R from CH 3 to p-CH 3 OC 6 H 4 and CH 2 COOCH 3 , respectively. These variations are consistent with the previously established electronic control on the stability of the P-C bond. [7] The comparison between compounds 2 and 3, on the other hand, shows that K increases by changing Y from CH 3 to CH 3 O, indicating that the methyl group is an even stronger donor than the methoxy group. While the nature of R and Y has an important effect on the equilibrium, the rate at which the equilibrium is achieved is not significantly changed (for instance, t1/2 = 12 min for the formation of 6, close to that observed for the formation of 1). [7] It is interesting to remark that the 31 P NMR chemical shifts of the aminophosphine products 2-6 qualitatively correlate with their stability. The equilibrium constant increase as the 31 P NMR resonance of the product shifts downfield. Both changes are related to the electronic properties of the substituents R and Y, while steric differences may be considered negligible. The strong variation of 31 P NMR chemical shift between compounds 6 and 7, on the other hand, may be attributed to the combination of a weak electronic effect (K changes only by a factor of ca. 3) and a larger steric effect.

The aminophosphine 6 was isolated by cristallization from CHCl 3 in 70% yield as yellow crystals and its absolute configuration has been confirmed by X-ray crystallography. The asymmetric unit contains both enantiomers and one CHCl 3 solvent molecule. It is interesting to note a dimeric structure (see Figure 1), the two monomer units being held together by two rare intermolecular P ... H-N bridges. The P ... N* and P* ... N separations are 3.638 and 3.702 Å, respectively, which are similar to those found for compound Ph 2 P-C(S)-NHMe (3.607 Å). [16] It is to be noted that an R configuration for the (arene)tricarbonylchromium(0) unit induces an R configuration for the new stereocenter in the product (and vice-versa), as predicted on the basis of the stereochemical considerations presented above. In conclusion, we have described the use of an arene-coordinated tricarbonylchromium template for the diastereoselective addition of Ph2PH to an imine, leading to chiral -P-C-N ligands. Further information on the electronic control of the reversible P-C bond formation havs been obtained by the use of various Cr(CO) 3 -complexes aromatic imine substrates. In addition to promoting the asymmetric induction, the Cr(CO) 3 moiety exerts an electronic contribution favouring the formation of the P-C bond. The coordination chemistry of the -aminophosphine ligands obtained is currently under investigation in order to evaluate their potential in catalytic asymmetric reactions.

Experimental Section

General Remarks: All manipulations were carried out under an atmosphere of purified argon and in the dark using standard Schlenk techniques. All solvents were dried and deoxygenated prior to use. 1 H and 31 P{ 1 H}-NMR measurements were carried out on a Bruker AC200 spectrometer. The peak positions are reported with positive shifts in ppm downfield of TMS as calculated from the residual solvent peaks ( 1 H) or downfield of external 85% H3PO4 ( 31 P). The IR spectra were reconderd on a Bruker IFS 66V spectrophotometer with KBr optics. The elemental analysis was carried out by the analytical service of the Laboratoire de Synthèse et d'Electrosynthèse Organométalliques with a Fisons Instruments EA1108 analyzer. The starting complexes (CO)3Cr[ 6 -o-C6H4(Y)(CH=NR)] used in this study were obtained as previously described in the literature. [START_REF] Maiorana | [END_REF][13][14]17] Aminophosphines 2-5: All experiments were conducted with identical operating procedures and concentrations. The equilibrium formation of product 5 is described in detail. Ph2PH (50 l, 0.287 mmol) was added to a solution of the benzaldimine chromium tricarbonyl complex (CO)3Cr[ 6 -o-C6H4(OCH 3 )(CH=N-p-C 6 H 4 OCH 3 )] (0.108 g, 0.287 mmol) in CDCl3 (0.5 ml). The resulting solution was stirred at room temperature for 3 hrs. 31 The equilibrium constant was determined by integration of the H 1 NMR signals, whose intensity did not change over the subsequent 48 hours.

Aminophosphine 6: Ph2PH (50 l, 0.287 mmol) was added to a solution of the complex (CO)3Cr[ 6 -o-C6H4(Cl)(CH=NPh)] (0.181 g, 0.474 mmol) in CHCl3 (10 ml). The resulting solution was stirred at room temperature for 3 hrs, filtered and concentrated to half volume. Addition of pentane afforded the complex 6 as yellow crystals (0,188 g, 70%). g cm -3 ,  = 0.744 mm -1 . The structure was solved by Patterson methods and subsequent difference Fourier analysis. [18] Due to the weak diffracting power of the crystal, the refinement was performed on the 2338 observed reflections (I > 2(I)) among the 5365 unique reflections collected. The model was refined isotropically except for the Cr, Cl and P atoms. Except one hydrogen atom bonded to a nitrogen atom located in the final Fourier difference map, hydrogen atoms were included in their calculated positions and refined with a riding model. A final refinement on F 2 with 2338 intensities > 2(I) and 292 parameters converged at wR(F 2 ) = 0.208 and R(F) = 0.081. Crystallographic data (excluding structure factors) for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-127927. Copies of the data can be obtained free of charge on application to CCDC. 12 Union Road, Cambridge CB2 1EZ, UK [Fax : int. code + 44(1223)336-033 ; E-mail : deposit@ccdc.cam.at.uk].

  Scheme 2

Figure 1 .

 1 Figure 1. ORTEP view of compound 6 with ellipsoids shown at the 30% probability level. All hydrogen atoms are omitted for clarity, except those on the N, N*, C(7) and C(7)*, highlighting the hydrogen-bonding interactions and the absolute configuration at C(7) and C(7)*.

  P NMR (81.03 MHz, CDCl3): = 12.5 (s) and -36.6 (s) ppm respectively for 5 and the free Ph2PH. IR (CO, CHCl3): 1970 (b), 1904 (vb) cm -1 .

CHCl 3 ,

 3 Mw = 1195.12. Enraf-Nonius CADA diffractometer, Mo-K radiation ( = 0.71073 Å), T = 293 K ; monoclinic, P21/c, a = 11.267(2), b = 28.202(4), c = 18.014 (3) Å,  = 104.808(14)°, V = 5533.9(16) Å 3 , Z = 4, D x = 1.434

Table 1 .

 1 Results of the reaction between (CO)3Cr[ 6 -o-C6H4(Y)(CH=NR)] and Ph 2 PH. a

		Y	R	d.e. (%)	% P-C b	K	 P (ppm)
	2	CH 3	CH 3	> 98	44	3.1	10.4
	3	CH 3 O	CH 3	> 98	54	5.8	10.9
	4	CH 3	CH 2 COOCH 3	> 98	62	10.7	11.5
	5	CH 3 O p-CH 3 OC 6 H 4	> 98	73	17	12.4
	6	Cl	C 6 H 5	> 98	>99	>1000	13.7
	7	Cl	C 6 H 5 c	-	92	330	4.7
		a Solvent = CDCl 3 ; [imine] 0 = [Ph 2 PH] 0 = 0.57 M. b Calculated by integration of the 1 H and
	31 P resonances. c Without the Cr(CO) 3 fragment.

Acknowledgments

We acknowledge the Conseil Régional de Bourgogne, the MENRT, the CNRS and CNR for their financial support to this work.


Dr Jacques Andrieu

Jochen Dietz

Rinaldo Poli
email: rinaldo.poli@u-bourgogne.fr

Philippe Richard

Prof Rinaldo Poli

Reversible P-C bond formation

come

Summary. The saturated -aminophosphine ligands containing a secondary amine function Ph 2 PCH(R')NHR" establish a solution equilibrium with Ph 2 PH and R'CH=NR" and are stabilized by electron-withdrawing substituents R' and R" and by coordination of the phosphorus donor to Cu(I).

Since the discovery that bifunctional P,N ligands increase considerably the activity and/or the selectivity of palladium, ruthenium or rhodium catalysts, [1][2][3][4] the preparation of this type of ligands has been the subject of extensive investigations. Amongst these ligands, -aminophosphines crucially control the activity and selectivity of alkyne methoxycarbonylation catalysts. 1 Unsaturated ligands of this type (having an sp 2 -hybridized N donor) are exemplified by 2-PyPPh 2 (Py = C 5 H 4 N). Saturated versions of this class, i.e. Ph 2 PCH(R)NR'R" have received little attention. While the Mannich reaction between Et 2 PH, CH 2 O and the Me 2 NH compounds leads to the stable (both in the solid state and in solution) Ph 2 PCH 2 N(CH 3 ) 2 ligand having a tertiary amine function, 5 aminophosphines prepared by addition of Et 2 PH to CH 2 =N t Bu 5 or Ph 2 PH to PhCH=NPh 6 without solvent have only been described in the solid state. The subsequent reaction with iodomethane leads to an unexpected P-C bond cleavage. 6 Anionic -P,N ligands prepared by nucleophilic addition of Ph 2 P -Li + to PhCH=NPh or RLi to 2-PyPPh 2 have also showed instability in solution. 7,8 We now wish to present NMR and synthetic studies that enable us to rationalise the solution instability of -P,N ligands with secondary amine functions and to understand the electronic factors that favor their stabilisation. ligand and of its related anionic form in solution is due to a reversible P-C bond formation, with proton migration for the neutral species.

The generalisation to a wider group of imines establishes the substituent effect on the equilibrium position (see Scheme II). In particular, the presence of an electron-withdrawing group has a beneficial effect on the ligand formation when this is located either on the nitrogen atom (d>a>b>c) or on the carbon atom (e>d). This study shows that the careful choice of substituents may allow the synthesis of stable (in solution) -P,N saturated ligands.

Scheme 2

As we are interested in the exploration of the coordinating properties of these P,N ligands, we wished to examine whether coordination to a suitable metal could also affect the stability of the P-C bond. The above mentioned 2-PyPPh 2 ligand is known to act as an assembling ligand (adopting a - 2 coordination mode) in dinuclear copper(I) and in numerous other polynuclear complexes. 4,9 Addition of one equivalent of [Cu(NCMe)4](BF4) 10 Compound 3 is highly unstable, even in neat MeCN, leading to the slow precipitation of metallic copper. Addition of Me 2 NCH 2 CH 2 NMe 2 (tmeda), on the other hand, yields stable [Cu(tmeda)(Ph2PCHPhNHPh)](BF4) 4, with NMR properties quite similar to those of 3. White single crystals of 4 were obtained from CHCl3. The geometry of the cation, shown in Figure 1, reveals a rare example of three-coordination for a Cu(I) complex. 11,12 The -P,N ligand is only P-coordinated and the amino function remains dangling, whereas the analogous 2-PyPPh 2 ligand always adopts a -P,N coordination mode in dinuclear copper complexes. 9,11 Note that a fluxional behaviour interconverting P-and N-coordinated ligands in solution is inconsistent with the observed variation of the 31 P chemical shift from the free to the coordinated ligand, which is similar to that observed for the 2-PyPPh 2 system. In conclusion, we have shown that the instability of -P,N ligands with a secondary amine function and their corresponding anions in solution is due to reversible P-C bond formation, which can be reduced or suppressed by a decrease of electron density. This can be accomplished by either electron withdrawing substituents on the nitrogen or carbon atoms, or by coordination of the phosphorus donor. These results allow an easy and rapid access to new complexes with saturated aminophosphine ligands. Further studies are in progress in order to control the chirality of the central carbon atom (P-C*-N) and to explore the coordination properties of -P,N ligands toward early and late transition metals.

We acknowledge the Conseil Régional de Bourgogne and the CNRS for support of this work and the European Commission for an Eramus exchange undergraduate student program concerning Jochen Dietz from the University of Kaiserslautern, Germany.

Experimental

All manipulations were carried out under an atmosphere of purified nitrogen using standard Schlenk techniques. All solvents were dried and deoxygenated prior to use. All chemical shifts are given in ppm.

Reversible formation of compounds 2a-e. To a solution of PhCH=NPh (0.312 g, 1.72 mmol) in CDCl3 (10 ml) was added Ph2PH (0.300 ml, 1.72 mmol). The mixture was stirred for 2 hrs, yielding an equilibrium of 2a with the starting materials. 1 H NMR (CDCl 3 ):  = 7.97-6.68 (m, 20 H aromatics), 5.16 (dd, 1H, PCH, 2 J(P,H) = 3.2 Hz, 3 J(H,H) = 6.5 Hz), 4.41 (dd, 1H, NCH, 3 J(P,H) = 3.8 Hz, 3 J(H,H) = 6.5 Hz, exchange readily with D2O). 31 P NMR (CDCl 3 ): = 3.70 ppm (s). [START_REF] Sheldrick | SHELXS and SHELXL97[END_REF] C NMR (CDCl 3 ):  = 140.00-113.72 (m, 24C aromatics), 56.92 (d, 1C, PCH, 1 J(PC) = 14.0 Hz). By analogous reactions, equilibrium mixtures of compounds 2b-e with the corresponding starting materials were obtained (equilibrium constants as indicated in Table II).

to the equilibrium mixture containing 2 ,

 2 Ph2PH and PhCH=NPh in CDCl3 produces a solution with a major 31 P NMR resonance at  = -3.90 and a minor one at  = -35.2 corresponding to the copper complexes [Cu(NCMe)x(Ph2PCHPhNHPh)](BF4) 3, and [Cu(NCMe)x(PPh2H)](BF4), respectively (Scheme I). Only traces of coordinated Ph 2 PH and free PhCH=NPh have been detected in the 1 H NMR spectrum (K 3 = 800 M -1 ), indicating an almost quantitative shift of the equilibrium. This result shows that removal of electron density from the phosphine end of the molecule also stabilizes the P-C bond.

Fig. 1 .

 1 Fig. 1. An ORTEP view of the cation of compound 4. Selected bond distances (Å) and angles

  1 H NMR (CDCl 3 ). PCHN proton: 4.12 and 4.10 (2b, d, 2 JPH = 9.8 Hz, and 2 JPH = 5.6 Hz respectively ), 4.22 (2c, d, 2 JPH = 9 Hz), 5.20 (2d, dd, 2 JPH = 7.0 Hz, 3 JHH = 2.9 Hz), 4.63 and 4.27 (2e, d, 2 JPH = 9.8 Hz and 2 JPH = 5.6 Hz respectively); NH proton: 2.35 (2b, s, br), 4.28 (2c, d, 3 JPH = 5.8 Hz), 5.05 (2d, dd, 3 JPH = 6.7 Hz, 3 JHH = 2.9 Hz), 2.03 (2e, s, br). 31 P NMR (CDCl 3 ): 1.49 and 0.89 (2b), 1.20 (2c), 5.78 (2d), 4.85 and 4.61 (2e). Compounds 2b and 2e are present as both possible diastereoisomers.

Acknowledgments

The reaction of Ph 2 P -Li + with one equivalent of N-benzylideneaniline, followed by quenching with water and extraction in toluene affords the desired product 2a, see scheme I.

Scheme 1

The 31 P and 1 H NMR spectroscopic properties confirm the nature of the product. However, the NMR spectra of the redissolved crystallised product show the presence of the diphenylphosphine and imine starting materials in amounts that increase with time, indicating a reversible P-C bond cleavage. NMR monitoring of this process in CDCl 3 (integration of the 1 H NMR imine CH=N and product P-CH-N signals) reveals the establishment of a stable equilibrium position (t1/2 = 10 min; K 2 = 50 M -1 after > 2 days), which was independently confirmed by NMR monitoring of the reaction between Ph 2 PH and PhCH=NPh. A closer examination shows that an analogous equilibrium is also established for the anionic species, although this is shifted to a weaker extent toward the PCN product 1 relative to the neutral system (K 1 = 10 M -1 by 31 P NMR integration in THF/C 6 D 6 ). Thus, our NMR experiments show that the instability of the -P,N Preparation of compound 4. The equilibrium solution of ligand 2a obtained as described above was transferred to a Schlenk tube containing [Cu(NCMe)4](BF4) (0.540 g, 1.72 mmol) and the mixture was stirred for 1 hr. 1 H NMR (CDCl 3 ):  = 7.46-6.46 (m, 20 H aromatics), 5.23 (s, b, 1H, PCH), 4.61 (s, b, 1H, NCH), 2.00 (s, b, 12H, CH3CN, free and coordinated were not separated). 31 P NMR (CDCl 3 ): = -3.90 (s). [START_REF] Sheldrick | SHELXS and SHELXL97[END_REF] X-ray structure analysis of compound 4. Crystal dimensions: 0.3x0.3x0.2 (mounted in capillary).

The data collection was carried out on a CAD4 Enraf-Nonius goniometer. The structure was solved by interpretation of the Patterson map and subsequent difference Fourier techniques. All nonhydrogen atoms were refined anisotropically. [START_REF] Sheldrick | SHELXS and SHELXL97[END_REF] Crystal and refinement data: C32H38N3PF4Cu, Mr


J P I L G R I M

S S I O Z I O S

M B Ay L I S

G V E N T E R

C G A R R

Identifying potential candidate Culicoides spp. for the study of interactions with Candidatus Cardinium hertigii

Keywords: Cardinium, Culicoides, Rickettsia, symbiosis

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors responsible for the transmission of several viruses of veterinary importance. Previous screens of Culicoides have described the presence of the endosymbiont Candidatus Cardinium hertigii (Bacteroidetes). However, any impacts of this microbe on vectorial capacity, akin to those conferred by Wolbachia in mosquitoes, are yet to be uncovered and await a suitable system to study Cardinium-midge interactions. To identify potential candidate species to investigate these interactions, accurate knowledge of the distribution of the endosymbiont within Culicoides populations is needed. We used conventional and nested PCR assays to screen Cardinium infection in 337 individuals of 25 Culicoides species from both Palearctic and Afrotropical regions. Infections were observed in several vector species including C. imicola and the Pulicaris complex (C. pulicaris, C. bysta, C. newsteadi and C. punctatus) with varying prevalence. Phylogenetic analysis based on the Gyrase B gene grouped all new isolates within 'group C' of the genus, a clade that has to date been exclusively described in Culicoides. Through a comparison of our results with previous screens, we suggest C. imicola and C. sonorensis represent good candidates for onward study of Cardinium-midge interactions.

Worldwide, biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are known to transmit more than 50 arboviruses [START_REF] Mellor | Culicoides biting midges: their role as arbovirus vectors[END_REF]. Notably, bluetongue virus (BTV) and Schmallenberg virus (SBV) pose a great threat to livestock welfare and have caused serious economic damage to the European livestock industry. African horse sickness virus (AHSV) causes a highly lethal disease of equids, with past epizootic outbreaks in Africa, the Middle East and southern Europe. Vaccines have been very effective in halting the transmission of Culicoides-borne viruses but take significant time and resources to get into at-risk populations. This creates a need to develop alternative mitigation methods and tools to counter a continued threat of emergence of BTV and other Culicoides-borne pathogens.

Correspondence: Gregory D. D. Hurst, Institute of Infection, Veterinary and Ecological Sciences, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, U.K. Tel.: +44 151 7954520. E-mail: g.hurst@liverpool.ac.uk

The microbiome of arthropods is known to modify host biology in a number of ways, ranging from nutritional provisioning to parasite protection. These effects are particularly pronounced in the case of endosymbionts [START_REF] Su | Symbiont-mediated functions in insect hosts[END_REF]. For example, the endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) leads to an arbovirus blocking effect in Aedes aegypti mosquitoes, which has led to their successful deployment as a dengue fever control strategy in field trials [START_REF] Nazni | Establishment of Wolbachia strain wAlbB in Malaysian populations of Aedes aegypti for dengue control[END_REF]. The potential to harness viral blocking effects in wild populations is enabled by Wolbachia-induced cytoplasmic incompatibility (CI)-embryo death in mating between infected males and uninfected females. CI is exploited as a mechanism to drive the Wolbachia into a population to distribute this virus blocking phenotype into a naïve population; as Wolbachia pres-ence in a population reduces the fitness of uninfected females, the bacterium is able to reach high frequencies of infection. Importantly, this protective phenotype appears to be effective against a broad range of RNA viruses, supporting the potential use of endosymbiont-based biocontrol in major midge-borne pathogens such as BTV, SBV and AHSV.

The endosymbiont Candidatus Cardinium hertigii (Bacteroidetes) has been found to be widely associated with Culicoides [START_REF] Morag | Prevalence of the symbiont Cardinium in Culicoides (Diptera: Ceratopogonidae) vector species is associated with land surface temperature[END_REF][START_REF] Mee | Detection of low-level Cardinium and Wolbachia infections in Culicoides[END_REF][START_REF] Pagès | First detection of Wolbachia-infected Culicoides (Diptera: Ceratopogonidae) in Europe: Wolbachia and Cardinium infection across Culicoides communities revealed in Spain[END_REF]. Although the biological role of Cardinium in biting midges is undetermined, data from different host species demonstrate various Cardinium-dependent reproductive alterations including parthenogenesis-induction and CI [START_REF] Zchori-Fein | A newly discovered bacterium associated with parthenogenesis and a change in host selection behavior in parasitoid wasps[END_REF][START_REF] Hunter | A bacterial symbiont in the Bacteroidetes induces cytoplasmic incompatibility in the parasitoid wasp Encarsia pergandiella[END_REF], with the latter providing a potential drive mechanism for future introductions into Culicoides populations. Importantly, potential effects relating to pathogen-blocking and fitness (e.g., fecundity) remain underexplored.

A key requirement for studying Cardinium impacts in midges stems from the need for a model system. The main barrier to such a model is the difficult laboratory mass-rearing of midges, with only C. nubeculosus and C. sonorensis colonies currently in existence. Despite this, vector competence studies of field-caught Culicoides are still possible, such that identifying populations containing both Cardinium infected and uninfected individuals (for negative controls) is also of interest. In light of this, we investigated the distribution and prevalence of Cardinium infections in Culicoides populations from Palearctic and Afrotropical regions, using a conventional and nested PCR screen approach. These data are then added and compared to previous screen data to assess what we know about the distribution of Cardinium in field and laboratory populations and the suitability of specific midge species for further investigation pertaining to the endosymbiont.

Culicoides specimens were collected between 2007 and 2016 from sites spanning France, South Africa and the United Kingdom (Table 1). Details of Culicoides collection methods, storage and species identification can be found in [START_REF] Pilgrim | Torix group Rickettsia are widespread in Culicoides biting midges (Diptera: Ceratopogonidae), reach high frequency and carry unique genomic features[END_REF]. DNA extracts from a previous study by [START_REF] Lewis | First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus, important Palaearctic vectors of bluetongue virus[END_REF], describing positive Cardinium infection in C. pulicaris and C. punctatus from the United Kingdom, were used to validate screening assays.

Amplification of the host COI gene was initially assessed as a means of quality control by conventional PCR assay. COI amplicons from the Pulicaris complex were Sanger sequenced to determine cryptic species, which are difficult to distinguish morphologically. To this end, individuals were designated as belonging to a particular species if their COI haplotype had >99% sequence identity to previously designated DNA barcodes. DNA extracts, which passed quality control, were then screened for Cardinium using conventional and nested primers amplifying partial sequences of the Gyrase B (GyrB) gene (Primer sequences and PCR cycling conditions in File S1). Prevalence estimates were defined by the nested PCR data and categorised into no infection (0%), polymorphic infection (>0.1% and < 100%) and fixation (100%). Collection sites of Culicoides populations were then plotted against Cardinium infection prevalence based on the nested PCR assay and mapped geographically. For populations containing both males and females, Fisher's exact test (two-tailed) was used to investigate possible associations between endosymbiont and sex with a significance cut-off of P < 0.01.

The relatedness of Cardinium strains from different host species was analysed using nucleotide sequences of amplicons derived from conventional PCR. The Gyrase B gene (GyrB) was chosen for phylogenetic analysis because it has a higher level of divergence when compared to the conserved 16S rRNA gene, another gene used in Cardinium phylogeny reconstruction. Amplicons were purified enzymatically (ExoSAP) before being sequenced using a BigDye ® Terminator v3.1 kit (Thermo Scientific, Waltham, MA, U.S.A.), and capillary sequenced through both strands on a 3500 xL Genetic Analyser (Applied Biosystems, Austin, TX, U.S.A.). Sequences were aligned using the LINSI algorithm in MAFFT v7 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF]. A maximum likelihood (ML) phylogeny was then inferred with RAxML v8 [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] using 1000 rapid bootstrap replicates and using the GTR + I + G model, which was selected with jModelTest 2 [START_REF] Darriba | jModelTest 2: more models, new heuristics and parallel computing[END_REF] using the Akaike information criterion, with the topology search taking the best of Subtree Pruning and Nearest Neighbour Interchange rearranging.

Collection and screening of 337 specimens of 35 midge populations consisting of 25 species from both Palearctic and Afrotropical regions indicated varying prevalence of Cardinium between species (Table 1). All specimens were female apart from some males collected for C. imicola, C. punctatus, C. impunctatus and C. bolitinos (File S1). Most populations (23/35) did not show any signs of Cardinium infection, although low collection size in some cases tempers against a hard conclusion that infection is absent from the population. Of the remaining 12 populations, seven were at fixation for Cardinium (although a few of these had low sample sizes) with the other five showing signs of polymorphism, with a mix of infected and uninfected individuals. This prevalence range is similar to the study of Australian Culicoides by [START_REF] Mee | Detection of low-level Cardinium and Wolbachia infections in Culicoides[END_REF], in which 3/26 positive species had a fixed infection, with the remainder being of intermediate prevalence. Notably, Mee's study [START_REF] Mee | Detection of low-level Cardinium and Wolbachia infections in Culicoides[END_REF] detected at least one Cardinium positive individual in each population screened, which is at odds to our findings and another study by [START_REF] Pagès | First detection of Wolbachia-infected Culicoides (Diptera: Ceratopogonidae) in Europe: Wolbachia and Cardinium infection across Culicoides communities revealed in Spain[END_REF], investigating Cardinium distribution in Culicoides from Spain.

It is possible that the apparent 'hotspot' in Australia is as a result of a more sensitive assay, as the qPCR method used by [START_REF] Mee | Detection of low-level Cardinium and Wolbachia infections in Culicoides[END_REF] is sometimes preferred to nested PCR in screening. Alternatively, variation in thermal environment could explain the prevalence discrepancies observed between these bioclimatic zones. [START_REF] Morag | Prevalence of the symbiont Cardinium in Culicoides (Diptera: Ceratopogonidae) vector species is associated with land surface temperature[END_REF] found a positive correlation between land surface temperature (LST) and Cardinium prevalence in Culicoides imicola in Israel, with higher prevalence at higher mean LST. Furthermore, there is a known relationship between Cardinium presence and latitude more globally, with Cardinium infection being more common for hosts near the equator [START_REF] Charlesworth | Wolbachia, Cardinium and climate: an analysis of global data[END_REF].

In order to investigate Cardinium-Culicoides interactions, candidate model midge species must be identified, which combine the ability to vector etiological agents with either lab culturability or a natural polymorphism in endosymbiont presence that permits comparison between infected and uninfected Phylogenetic analysis based on a 1300 bp region of the GyrB gene confirmed a monophyletic clade of Cardinium, grouping all Culicoides Cardinium isolates from this study in group C of the genus [START_REF] Nakamura | Prevalence of Cardinium bacteria in planthoppers and spider mites and taxonomic revision of "Candidatus Cardinium hertigii" based on detection of a new Cardinium group from biting midges[END_REF], in which all previously described sequences of Culicoides clustered (Fig. 1) (Genbank accession numbers [https://www.ncbi.nlm.nih.gov/genbank/]: LR877462-LR877465). The C. imicola GyrB sequence obtained from South Africa in this study (LR877465) was identical to both of those obtained from Kenya (KR026927) and Israel (JN166963), indicating a global infection. Likewise, the Cardinium sequences obtained from C. punctatus from Wolverhampton, U.K. (LR877464) had 100% identity to the sequence reported in the same species by [START_REF] Lewis | First detection of endosymbiotic bacteria in biting midges Culicoides pulicaris and Culicoides punctatus, important Palaearctic vectors of bluetongue virus[END_REF]HG380244). [START_REF] Mee | Detection of low-level Cardinium and Wolbachia infections in Culicoides[END_REF] have previously suggested that Cardinium strains group by geography; by contrast, we find a sporadic distribution of strains with respect to location. For example, the C. bysta and C. newsteadi strains (LR877462 and LR877463) identified from Corsica, group with strains from Israel and Japan, respectively (JN166964 and AB506792). Furthermore, no clear pattern of Cardinium distribution was observed between the three geographical regions of this study, though the small sample size for some species forbids any formal test of the presence or absence of heterogeneity (File S2).

By contrast with other endosymbiont-insect associations, the biological significance of Cardinium in Culicoides remains unknown and requires further research. Despite a likely vertical transmission route, our observed lack of sex-bias in infection observed in this study (C. punctatus; Fisher's exact, P = 0.31 and C. imicola; Fisher's exact, P = 0.68) corroborates previous studies [START_REF] Morag | Prevalence of the symbiont Cardinium in Culicoides (Diptera: Ceratopogonidae) vector species is associated with land surface temperature[END_REF][START_REF] Mee | Detection of low-level Cardinium and Wolbachia infections in Culicoides[END_REF][START_REF] Pagès | First detection of Wolbachia-infected Culicoides (Diptera: Ceratopogonidae) in Europe: Wolbachia and Cardinium infection across Culicoides communities revealed in Spain[END_REF] and suggests the induction of parthenogenesis, feminisation or male-killing is unlikely to be associated with Cardinium. Other Cardinium strains have been implicated in cytoplasmic incompatibility (CI), including in the wasp Encarsia pergandiella [START_REF] Hunter | A bacterial symbiont in the Bacteroidetes induces cytoplasmic incompatibility in the parasitoid wasp Encarsia pergandiella[END_REF]. A recent publication of the C. punctatus Cardinium genome by [START_REF] Siozios | The draft genome of strain cCpun from biting midges confirms insect Cardinium are not a monophyletic group and reveals a novel gene family expansion in a symbiont[END_REF] has suggested possible unique genes related to CI. Overall, these observations suggest potential Cardinium-induced CI should be a priority of investigation in the future due to its possible role in driving the endosymbiont (and any desired effects) into midge populations.

The major Palearctic vector of BTV, C. obsoletus, has previously been shown to contain Cardinium at 0.4% prevalence [START_REF] Pagès | First detection of Wolbachia-infected Culicoides (Diptera: Ceratopogonidae) in Europe: Wolbachia and Cardinium infection across Culicoides communities revealed in Spain[END_REF]. We did not observe the endosymbiont in the one C. obsoletus population screened in this study, although the sample size was too small to conclude the absence of infection, and lack of DNA barcodes in this case prevents matched comparison. Nevertheless, a low prevalence indicates Cardinium is likely not relevant to vector biology at a population level and thus this species complex does not appear to be a suitable candidate for investigation.

By contrast, two other important vector species, C. imicola and C. sonorensis, have been reported to carry Cardinium infection [START_REF] Morag | Prevalence of the symbiont Cardinium in Culicoides (Diptera: Ceratopogonidae) vector species is associated with land surface temperature[END_REF][START_REF] Möhlmann | Mosquitoes, midges and microbiota: European vector diversity and the spread of pathogens[END_REF]. Our results demonstrated the presence of Cardinium in one population of C. imicola from South Africa (Onderstepoort), whereas specimens of two populations from Corsica were negative for the presence of Cardinium, a pattern likely associated with properties of founder individuals [START_REF] Pilgrim | Cardinium symbiosis as a potential confounder of mtDNA based phylogeographic inference in Culicoides imicola (Diptera: Ceratopogonidae), a vector of veterinary viruses[END_REF]. Thus, these species appear to be the most promising candidate species for investigating Cardinium effects on vectorial capacity. As certain C. imicola and C. sonorensis populations are polymorphically infected [START_REF] Morag | Prevalence of the symbiont Cardinium in Culicoides (Diptera: Ceratopogonidae) vector species is associated with land surface temperature[END_REF][START_REF] Möhlmann | Mosquitoes, midges and microbiota: European vector diversity and the spread of pathogens[END_REF], the two species both present naturally occurring negative controls without the need for the confounding effects of curing through antimicrobials. In favour of C. imicola as a model is the ease of obtaining large field catches, but barriers in laboratory cultivation are still to be overcome. However, C. sonorensis laboratory colonies already exist, suggesting this species is the most promising candidate for investigating symbiont-virus interactions and symbiont-mediated reproductive effects. It will be worthwhile, additionally, to screen other Culicoides species, which may become colonised in the near future, such as C. stellifer [START_REF] Erram | Laboratory rearing of Culicoides stellifer (Diptera: Ceratopogonidae), a suspected vector of orbiviruses in the United States[END_REF].

Fig. 1 .

 1 Fig. 1. Maximum likelihood phylogeny of Cardinium strains. Phylogeny is based on a 1300 bp region of the GyrB gene generated in RAxML using 1000 rapid bootstrap replicates and using the GTR + I + G substitution model. Groups A (blue) B (red) and C (yellow) are designated based on Cardinium taxonomic convention. Stars represent sequences generated in this study. Brackets indicate subgenus and country of origin. [Colour figure can be viewed at wileyonlinelibrary.com].

Table 1 .

 1 GyrB conventional and nested PCR results of Cardinium identification in Culicoides spp. collected in different Palearctic and Afrotropical regions.

					Proportion of	Proportion of
					Cardinium positive	Cardinium
					conventional	positive nested
					PCR (n)	PCR (n)
					[95% confidence	[95% confidence
	Subgenus	Culicoides species	Locality (site name)	Year of collection	interval]	interval]

pulicaris, compared to Lewis et al. (2014) (the higher frequency, 10/29, in Lewis et al. represents a laboratory artefact; see doi: 10.6084/m9.figshare.13187069).

© 2021 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society, Medical and Veterinary Entomology, doi: 10.1111/mve.12517

Acknowledgements

We thank Kenneth Sherlock, Georgette Kluiters, Steve Price, Lukasz Lukomski, the Direction générale de l'alimentation (DGAL) and the national veterinary services (France) for their support with the collection of midge samples. We are grateful to Jan Chirico and Mats Ander for the identification of Culicoides from Sweden, Ignace Rakotoarivony (CIRAD) for the identification of Culicoides from France and Karien Labuschagne for the identification of Culicoides from South Africa. This work was supported by a BBSRC Doctoral Training Partnership studentship (BB/M011186/1) awarded to JP. The authors declare no conflict of interest.

Data availability statement

The data that support the findings of this study are openly available in figshare at: http://doi.org/10.6084/m9.figshare. 13187069, http://doi.org/10.6084/m9.figshare.13142684, and http://doi.org/10.6084/m9.figshare.13130306. Accession numbers generated in this study: LR877462-LR877465.

Supporting Information

Additional supporting information may be found online in the Supporting Information section at the end of the article.

Author contributions

JP, GDDH, SS and MB assisted in the conception and design of the study. Field work was undertaken by JP, CG and GV. Lab work was undertaken by JP. Analyses and interpretation of the data were undertaken by JP, GDDH, MB, SS, CG and GV, as well as drafting of the manuscript.