Dr Dmitriy Dovzhenko
email: dovzhenkods@gmail.com

Maksim Lednev

Dr Konstantin Mochalov

Ivan Vaskan

Yury Rakovich

Igor Nabiev
email: igor.nabiev@univ-reims.fr

Maksim Lednev

Prof Mr Maksim Igor Lednev

Prof Igor Nabiev

Mr Ivan Vaskan

Prof Rakovich Yury

Polariton

Keywords: polaritons, tuneable microcavity, hybridization, fluorescent molecules, relaxation pathways, FRET

come

Resonant interaction between excitonic transitions of molecules and localized electromagnetic field allows the formation of hybrid light-matter polaritonic states. This hybridization of the light and the matter states has been shown to be able to significantly alter the intrinsic properties of molecular ensembles placed inside the optical cavity. Here, we have achieved strong coupling between the excitonic transition in typical oligonucleotide-based molecular beacons labelled with a pair of organic dye molecules, demonstrating an efficient donor-toacceptor resonance energy transfer, and the tuneable open-access cavity mode. The photoluminescence of this hybrid system under non-resonant laser excitation and the dependence of the relative population of light-matter hybrid states on cavity detuning have been characterized. Furthermore, by analysing the dependence of the relaxation pathways between energy states in this system, we have demonstrated that predominant strong coupling of the cavity photon to the exciton transition in the donor dye molecule can lead to such a large an energy shift that the energy transfer from the acceptor exciton reservoir to the mainly donor lower polaritonic state can be achieved, thus yielding the chromophores' donor-

Introduction

Strong light-matter coupling is a quantum electrodynamics phenomenon that takes place when the rate of resonant energy exchange (i.e., coupling strength) between the exciton transition in matter and the resonant localized electromagnetic field is higher than the competing decay and decoherence processes. Light-matter coupling then leads to the formation of two new "hybrid" light-matter (polaritonic) states with different energies, instead of the two original molecular and electromagnetic field energy states. Once the strong coupling regime is reached, the coupled system exhibits new properties possessed by neither the molecules nor the cavity [1]. Hence, by controlling the coupling strength, it is possible to modulate (or even control) various properties of the system, including the eigenenergy, excited state lifetime, efficiency and efficient distances of energy transfer, conductivity, etc. [2]. This paves the way to a wide variety of breakthrough practical applications, such as modification of chemical reactivity [3], enhanced conductivity [4], development of lowthreshold sources of coherent emission [5], and even polariton simulators and logic [6,7].

Recently it has been demonstrated that strong coupling can modulate both distance and efficiency of Förster resonance energy transfer (FRET) [8,9]. FRET is a process of nonradiative energy transfer from one fluorophore (donor) to another one (acceptor). The FRET effect only occurs when several conditions are satisfied: (i) the donor emission spectrum should overlap with the acceptor absorption spectrum; (ii) the donor and acceptor fluorophores should be in a favourable mutual orientation, and, (iii) since the FRET efficiency is inversely proportional to the sixth power of the distance between the fluorophores, the distance between the donor and the acceptor should not exceed the Förster limit (10 nm).

When these conditions are satisfied, the FRET effect results in a decreased donor fluorescence emission accompanied by a simultaneously increased acceptor fluorescence emission.

Under the strong coupling regime, both donor and acceptor excitonic states could be coupled to the same microcavity optical mode, which may act as a mediator. This mediation can make it possible not only to increase the efficient distances of the energy transfer to values ten times larger than the Förster limit (to more than 100 nm) [9], but also to ensure an up to sevenfold increase in the rate of energy transfer [8]. This increase in the energy transfer rate leads to a significant decrease in donor fluorescence in the presence of the acceptor. As a result, the energy transfer efficiency, which is characterized by the ratio between the intensities of the donor fluorescence in the presence and absence of the acceptor, will be also significantly increased. It has been reported recently that under the strong coupling regime the energy transfer efficiency may be increased from 0.55 to 0.90 [8].

The possibility of increasing and controlling the FRET efficiency is promising for the development of many photonic applications, specifically for biomedical research. In this regard, one of the most powerful photonic nanotools are oligonucleotide-based molecular beacons, which are used in biosensing and specific gene identification [10][11][12][13][14][15], RNA imaging [14,[16][17][18], revealing nucleic acid mutations [19], monitoring gene expression [20] and protein-protein interactions [21], nanomedicine [14,22], cell-surface glycosylation imaging [23], etc. All these applications are based on the same principle: a specifically designed molecular beacon is a circled oligonucleotide, with the donor and acceptor dye molecules conjugated to its ends and located in close vicinity to each other; hence, a strong donor-to-acceptor FRET occurs (Figure S1 in the SI). Due to this strong FRET effect, the donor fluorescence is completely quenched and, in this respect, the beacon is invisible. When the beacon oligonucleotide binds to its complementary oligonucleotide target, its circled structure is opening, the distance between the donor and acceptor increases, and this binding event is reported by the appearance of the originally quenched donor fluorescence signal. The possibility of controlling the FRET efficiency within the molecular beacon by making the originally invisible molecular beacons visible "on demand" may extend their applications.

One of promising ways to control the FRET efficiency is to use light-matter interaction in various regimes [8,24].

Polariton-assisted energy transfer between spatially separated molecules has been extensively studied in various configurations [8,9,25]. In Ref. [25], hybrid polaritonic states have been demonstrated to be an efficient energy transfer pathway between two spatially separated J-aggregates with an initially negligible direct energy transfer via dipole-dipole coupling. However, for many practical applications that we have mentioned above, it is even more intriguing to have a way to alternate the energy relaxation in a mixed media where the donor and acceptor molecules are located in close vicinity to each other and the FRET effect is mediated by direct dipole-dipole coupling between them. In Ref. [26], hybridization of the light and the matter states in a microcavity filled with a blend of two BODIPY fluorescent dyes with similar properties has been investigated. Here, the electromagnetic microcavity modes of light have been found to be coupled, at the same extent, with both donor and acceptor exciton transitions. It has been shown that, in this system, direct dipole-dipole coupling is more efficient than the energy transfer via strong coupling. Interestingly, the strong coupling to only one of the two excitonic states of the system has also been shown to be promising if the control over relaxation pathways in FRET systems has to be obtained. In Ref. [27], the authors have theoretically demonstrated that, whereas exclusive strong coupling of the cavity photon to the donor states can enhance the energy transfer to the acceptors, the reverse is not true. On the other hand, it has been shown that sufficiently strong coupling exclusively to the acceptor can modify the energy levels of the system in such a way that the transfer from the acceptor to the donor states mediated by polariton states can occur, leading to the chromophore role reversal or "carnival effect" [27]. It is interesting to investigate the possibility of combining these effects by means of strong coupling of exclusively the donor state to the cavity photon with a large coupling strength. This should lead to the formation of a polariton state with a relatively large fraction of the donor exciton and a small fraction of the acceptor exciton, with the energy lower than that of the original acceptor state. However, such a modification will require very strong coupling to ensure significant alteration of energy levels.

To date, most FRET studies using the strong coupling regime have employed only simple Fabry-Perot microcavities, which have relatively large mode volumes and, hence, rather moderate light-matter coupling strengths, which have significantly limited the experimental observations of the theoretically described effects [27]. Recently, we have engineered a tuneable microcavity with a lateral mode localization characterized by a drastically decreased mode volume, which allows obtaining a considerably larger coupling strength [28], and have employed this microcavity in the present study. In order to ensure a sufficiently strong coupling of the microcavity photon modes exclusively to the molecular beacon's donor state, we used the 6-carboxyfluorescein (FAM) dye, which has a large dipole moment and a high quantum yield as a donor, and the rhodamine derivative carboxytetramethylrhodamine (TAMRA) with much lower dipole moment and quantum yield as a molecular beacon's acceptor (see the Sample preparation section and the Supporting Information (SI) for details). The distance between these donor and acceptor dye molecules conjugated with the opposite termini of the molecular beacon was determined by the diameter of the DNA double helix (about 2 nm), which was short enough to ensure efficient direct dipole-dipole coupling between them (see Figure S1 in the SI). This system made it possible to investigate the polariton-assisted energy transfer in a tuneable open-access microcavity containing oligonucleotide-based molecular beacons with a donor-acceptor pair of closely located FAM and TAMRA organic dyes exhibiting an efficient FRET outside the microcavity.

In this study, we have measured the dependence of the photoluminescence (PL) properties of the molecular beacon solution in a microcavity and have analysed the dependence of the polaritonic state population on the detuning of the optical microcavity mode. We have used the Jaynes-Cummings model to calculate the eigenfunctions of the strongly coupled three-component system and have estimated the mixing of excitons and photon fractions in hybrid states. We have also estimated the possibility of changing the relaxation pathways by varying the degree of the exciton-photon mixing in polaritonic states. Furthermore, we have explored the particular situation when the donor is much stronger coupled with the optical mode than the acceptor. For the best of our knowledge, we are the first to experimentally demonstrate the possibility to reverse the donor and acceptor roles ("carnival effect") within a donor-acceptor pair of organic dyes, which has been theoretically investigated by the group of Joel Yuen-Zhou [29].

Results and Discussion

In order to investigate the feasibility of controlling the resonant energy transfer in a donor-acceptor pair of closely located organic dyes, we have employed a tuneable microcavity with a relatively small mode volume previously developed in our group [30,31].

Briefly, the tuneable microcavity unit was composed of plane and convex mirrors that form an unstable λ/2 Fabry-Perot microcavity (Figure 1a, see the SI for details). The upper mirror was made convex in order to satisfy the plane-parallelism condition at one point, thus minimizing the mode volume. The plane bottom mirror was mounted on top of a Z-piezo positioner, which provided fine tuning of the microcavity length in the range of up to 10 µm with a nanometre precision. The plane-convex design of the tuneable microcavity is characterized by rather high quality factors (up to several hundred units), whereas the mode volumes can be as low as tens of thus combining the advantages of both optical and plasmonic cavities [31]. In this study, the quality factor of the microcavity mode was about 35

and the mode volume was about 15 ´ for all the detunings used. Previously, we have demonstrated the advantages of the developed tuneable microcavity, such as a controllable distance between the mirrors with a nanometre accuracy and a small mode volume, which result in a much higher Rabi splitting energies compared to the standard optical microcavities [28]. In particular, we have demonstrated a strong coupling of the ensemble of Rhodamine 6G molecules with a Rabi splitting as high as 225 meV at room temperature, which has been previously shown only for the case of surface plasmon-polaritons. A drawback of the tuneable setup developed is that the effect of strong coupling cannot be simply observed in the transmission spectra, because the concentration of the molecules inside the cavity is relatively low and the number of photons from the white LED used for measurements of the transmission considerably exceeds the number of generated excitons. However, the developed by us microcavity combining the advantages of optical microcavity (low energy dissipation) and plasmonic cavity (small mode volume) significantly increases the number of materials suitable for operation in a strong coupling regime, thus paving the way to plenty of new practical applications. In this study, we employed this set-up to achieve a high Rabi splitting energy of hybrid states formed by the exciton transitions in donor-acceptor pairs of organic dyes exhibiting the FRET effect and the localized resonant electromagnetic field. acceptor. The distance between the donor and acceptor dye molecules conjugated with the opposite termini of the oligonucleotide-based molecular beacon is determined by the diameter of the DNA double helix (about 2 nm), which is small enough to ensure efficient direct dipole-dipole coupling between them (Figure S1) [32]. The details on the chemical structures of the oligonucleotide-based molecular beacon and dye molecules, as well as on the optical properties of the dyes, are presented in the SI.

Figure 1b

shows the PL spectra of oligonucleotide-based molecular beacons labelled with FAM alone, TAMRA alone, or both FAM and TAMRA dyes placed onto the bottom mirror of the setup that were measured in the absence of the upper mirror; i.e., the beacons were located outside the microcavity.

FAM is a well-known organic dye with a quantum yield as high as 97% [33], with the main exciton transition characterized by a relatively large transition dipole moment ranging from 7 to 12 D [33] and the main emission peak maximum at about 2.36 eV. The TAMRA dye has a much lower quantum yield of about 22% [34], as well as lower values of the transition dipole moment [35]. The PL emission maximum of the TAMRA dye solution is about 2.13 eV. However, it is noteworthy that the transition dipole moments and PL quantum yields of the dyes may be changed upon their conjugation with the oligonucleotide due to the possible appearance of new intermolecular interactions in the conjugated samples. Figure 1b shows that the PL spectra of the FAM-TAMRA donor-acceptor pair excited at 450 nm could be obtained by linear superposition of the PL spectra of these dyes measured separately, which indicates the absence of direct ground-state interaction between these dye molecules in the molecular beacon. Despite the low quantum yield, the PL emission from TAMRA was stronger than that from FAM in the case of the donor-acceptor pair operating in the FRET regime. The efficiency of the resonance energy transfer from FAM to TAMRA molecules was estimated to be about 80% (see the SI for details).

We further analysed the PL spectra of the dyes conjugated with the oligonucleotide of the molecular beacon alone and in the form of a donor-acceptor pair operating in the FRET regime and placed into the tuneable microcavity at different cavity detunings (Figure 2). The cavity mode tuning has been performed by changing the distance between the microcavity mirrors from 735 to 945 nm at 15-nm steps. The corresponding PL emission spectra are shown in Figures 2a-2c. It is worth mentioning that the specific properties of our microcavity allowed us to detect the PL emission from molecules weakly coupled to the transverse modes of both the lowest and higher orders of the cavity (see the SI for details). This PL associated with the enhanced emission peak following the cavity mode, slightly blue-shifted relative to the cavity photon spectral position corresponding to the lowest-order transverse mode of the cavity.

Qualitatively, this blue shift can be understood considering the difference in signal collection efficiency between the transmission and PL measurements (see the SI for details). The resulting emission corresponding to this peak is satisfactorily fitted in the weak coupling approximation. Figure 2d presents the experimentally measured dependence of the spectral position of the emission maximum associated with the weakly coupled molecules on the cavity mode detuning extracted from Figure 2c and the corresponding model calculations. It can be seen that the calculated dependence is in good agreement with the experimental data, the difference not exceeding the full-width-at-half-maximum (FWHM) of the cavity mode. It is also important to note that the large shift of the emission peak from the cavity mode in the region of low mode energies was due to the poor spectral overlap of the dye PL spectra with the lowest-order transverse cavity mode. Similar PL emissions from weakly coupled molecules were observed for the FAM and TAMRA dyes separately (Figures 2a,2b). It is noteworthy that, due to the non-resonant excitation through the lower mirror of the microcavity, the power of excitation inside the cavity depended on the cavity detuning.

Although the intensities of the measured PL spectra presented in Figure 2 are not calibrated against the excitation power, the necessary corrections have been made in the analysis presented below.

The molecular beacons labelled with the TAMRA dye alone and placed into the microcavity exhibited only the emission from weakly coupled molecules (Figure 2a), which can be explained by higher losses, lower transition dipole moments of the rhodamine derivative, and the resultant low coupling strength [35]. However, it is noteworthy that the change in the emission intensity from the weakly coupled states of TAMRA was much bigger than that for the case of the molecular beacon labelled with the FAM dye alone. Indeed, the Purcell PL intensity enhancement for emitters with a lower quantum yield is known to be stronger than that for emitters with a higher quantum yield, because the Purcell effect changes only the radiative relaxation rate [36]. donor and acceptor states. Indeed, its intensity was lower than that in the case of molecular beacons labelled with FAM alone in the donor spectral region and higher than that in the case of molecular beacons labelled with TAMRA alone in the acceptor spectral region. These effects were certainly due to the remaining highly efficient resonance energy exchange through dipole-dipole interaction upon the deposition of the molecular beacons labelled with both FAM and TAMRA dyes operating in the FRET regime inside the cavity. It is important to note that, in contrast to the case of molecular beacons labelled with the FAM dye alone, no signal from bare donor states was detected at negative detunings in the cavity photon energy range from 2.0 to 2.3 eV. The fact that the donor-acceptor dye pair outside the cavity exhibited emission from both donor and acceptor dyes with a FRET efficiency of about 80% indicated that the ratio between the donor-acceptor energy transfer rate and the donor radiative relaxation rate inside the cavity was increased. This can be explained by taking into account the suppression of the spontaneous emission of bare donor states when the cavity mode was shifted from the donor emission bandwidth. The low density of photonic states beyond the cavity mode compared to the density in the free space led to a decrease in the spontaneous emission rate and, consequently, to the effective increase in the FRET efficiency.

However, at positive detunings, we observed a PL peak determined by emission from the LPB, as in the case of the cavity containing molecular beacons labelled with the donor dye alone, which was red-shifted relative to the emission spectrum of the uncoupled dye molecules. Similarly to the previous cases, we were unable to detect any emission that could have been attributed to the UPB due to the typically fast energy relaxation from the upper polariton to the donor exciton reservoir. Thus, we omit the UPB from most of the discussion.

Nevertheless, the presence of strong coupling was clearly evidenced by anticrossing of the LPB at the cavity detunings where the cavity photon mode and donor excitons had to be degenerated. Considering the exitonic constituents as independent harmonic oscillators, we assumed that our cavity could couple together three oscillators, FAM, TAMRA, and the cavity mode. Therefore, we characterized the observed dispersion by three polariton branches.

In the case of a negatively detuned cavity, we observed an emission peak that could be attributed to the middle polariton branch (MPB). However, due to the pronounced emission from weakly coupled states and large broadening of the emission spectra, this part of the emission spectrum can hardly be used to quantitatively analyse the spectral position of the MPB.

The dependence of the polariton branches on the cavity photon energy presented in In order to determine the Hopfield coefficients, that describe the donor exciton, acceptor exciton, and photon mixing for the given coupling strengths, we further calculated the eigenfunctions of the Jaynes-Cummings Hamiltonian and represented each of them as a superposition of the initial pure photon and exciton states (see the SI for details). Figure 4 shows the Hopfield coefficients for each polariton branch and their dependences on the cavity detuning. In the analysed PL spectral region, the polariton branches displayed quite different behaviours. It can be seen from Figure 4 that the upper and the lower polaritons mainly consist of the donor exciton and photon fractions, whose ratio is reversed for both branches upon cavity tuning. On the other hand, for the MPB, the contribution from the acceptor exciton strongly dominates the other components. However, this contribution decreases with an increase in the cavity mode energy in the same way as the relative contribution of the acceptor exciton increases significantly in the LPB. Such a redistribution of fractions in the polariton branches is in accordance with the exciton-photon coupling strengths, which differ significantly for the donor and acceptor excitons, as we have mentioned above.

In order to quantitatively correlate the polariton population dependence on the detuning and the PL intensity measured experimentally, one need to introduce corrections arising from variations of both the photon fraction and the excitation intensity [25,26]. The finite elements method was used to calculate the dependence of the excitation field intensity inside the cavity on the detuning (see the SI for details). In order to make the necessary corrections, we divided the measured PL intensity of the LPB by the integral intensity of nonresonant excitation at 450 nm over the volume of the microcavity in the region of cavity detuning. Finally, the relative polariton population (Figure 5) was obtained using the following equation [25]:

(1) where is the experimentally observed intensity of PL from the LPB corrected for the excitation intensity, and is the Hopfield coefficient for the LPB defining the fraction of the cavity photon. The intensity of emission from the polariton state depends linearly on the cavity photon fraction.

As can be seen from Figure 5, the population of the LPB non-linearly increases with increasing cavity photon energy. According to the Hopfield coefficient distributions, this growth is accompanied by a linear decrease in the donor exciton fraction approximately from 0.3 to 0.6 and by a drastic increase in the acceptor exciton fraction in the LPB by almost an order of magnitude. Now, we will analyse the population and depopulation mechanisms for the LPB using an approach described in detail in Ref. [25]. The simplified consideration is based on the assumptions on fast relaxation from the UPB to the donor excitonic reservoir (which typically occurs on the femtosecond timescale [37]) and efficient FRET of most of the energy to the acceptor reservoir. Therefore, the number of states in the acceptor excitonic reservoir can be considered constant. In principle, there are three different mechanisms determining the LPB population: scattering with vibrations from both excitonic reservoirs of the donor and acceptor and direct radiative pumping. The efficiencies of the first two mechanisms strongly depend on the corresponding exciton fraction in the LPB and should be changed with the detuning. The radiative pumping mechanism is a direct absorption of the photon emitted by the weakly coupled exciton transitions, which is accounted for by the photonic fraction in the LPB.

However, it is almost negligible for cavities with a short cavity photon lifetime that contain an ensemble of molecules with a low optical density. The depopulation of the LPB occurs via radiative and non-radiative relaxations, which depend on the photon and exciton lifetimes, respectively. Thus, the mean polariton population (NLPB) in the steady state can be described by the following equation:

(

) 2
where are the proportionality constants for the terms corresponding to the LPB population through vibration scattering from the acceptor and donor reservoirs and direct radiative pumping, respectively; are the proportionality constants for the terms describing the depopulation via radiative ( ) and non-radiative ( ) relaxations. The first two terms describe the processes accompanied by the emission of molecular vibrations, which depend on the Bose-Einstein distribution , where is the bare exciton energy, is the energy of the LPB, is the temperature in kelvins, and is the Boltzmann constant. For the approximation of the population of the LPB in the steady state (Figure 5), we used the following equation, which could be easily obtained from Equation ( 2) under some reasonable assumptions described below: It is important to note that the best fit was obtained with tending to be zero. This corresponds to the low efficiency of the LPB population caused by scattering from the donor exciton reservoir, which may have been due to the relatively low rate of this process compared to the depopulation of the donor excitonic reservoir through FRET to the bare acceptor states. This mechanism was previously shown to be dominant over polariton-assisted energy transfer in mixed donor-acceptor ensembles [26]. Thus, the energy relaxation pathways in our system can be described as follows (Figure 6). The non-radiatively pumped population of the UPB rapidly decays to the donor exciton reservoir [37]. Then, due to the short distance between the donor and acceptor dye molecules in the molecular beacon, direct dipole-dipole FRET occurs with the efficiency close to unity.

The FRET efficiency for the molecular beacon placed into the microcavity was found to be increased compared to that for the molecular beacon outside the cavity (about 80%), because we did not observe any emission from the bare donor states at negative detuning, in contrast to the donor-only case. This may have been due to the decreased rate of radiative relaxation of bare states at negative detunings, leading to the increase in FRET efficiency. The most interesting is that, once the energy had been transferred to the acceptor excitonic reservoir, it started to populate the LPB, which is mostly a mixture of the donor and cavity photon fractions due to the much higher coupling strength between the donor and the cavity photon.

Thus, vibration scattering from the acceptor reservoir was shown to be the main population mechanism of the lower polariton state with the donor exciton fraction exceeding the acceptor one. It was demonstrated previously that a small absolute value of the specific exciton fraction in the polariton branch still allowed energy transfer with the corresponding excitonic reservoir [25]. In our experiments, the population of the LPB depended on the relative variation of a small fraction of the acceptor exciton in the polariton state despite the considerably higher absolute value of the Hopfield coefficient, corresponding to the donor exciton. Finally, we can state that we have engineered a strongly coupled system with donoracceptor role reversal or the "carnival effect" [27]. Indeed, we have developed a system with dominant coupling between the donor and acceptor leading to the formation of a donor-like polariton state with the lowest energy in the system. This allowed energy transfer first from the donor exciton reservoir to the acceptor exciton reservoir via standard FRET and then from the acceptor reservoir to the donor-like lower polariton state.

Conclusions

We have investigated strong coupling between the optical modes of a tuneable microcavity and the excitonic transitions of two closely located organic dye molecule labels of an oligonucleotide-based molecular beacon. The anticrossing dependence of the emission spectra of the donor-acceptor dye pair operating in the FRET regime on the detuning of the microcavity mode has been demonstrated by varying the distance between the cavity mirrors.

We have estimated the dependence of the polaritonic state population on the detuning and photon-exciton mixing, which has been calculated by fitting the experimental results with the three-level Jaynes-Cummings model and rate equations. In addition to the efficient FRET between the bare exciton states, significant alteration of the relaxation pathways by changing the photon and exciton mixing in the lower polariton state has been demonstrated.

We have confirmed that, in our system of molecular beacons labelled with organic dyes and located inside a tuneable open-access microcavity, the resonance energy transfer via direct FRET remains the dominant process despite the strong coupling of the dye excitons to the cavity mode, as has been previously demonstrated [26]. However, we have shown that the PL emission of the systems with strong dipole-dipole interaction can nevertheless be altered by strong coupling of their exciton transitions to the cavity photon. First, we have demonstrated a significant increase in the efficiency of energy transfer from the donor to the acceptor exciton reservoir, which tends to be unity inside the microcavity. Second, despite the efficient energy transfer between the donor and the acceptor exciton reservoir, we have observed emission from the LPB, which has been shown to have a considerably higher donor fraction compared to the acceptor one. Furthermore, we have proved that the LPB is populated mainly through scattering from the acceptor reservoir, despite the much larger absolute value of the donor exciton fraction than the acceptor fraction in the lower polariton. Thus, by obtaining strong coupling of the photonic mode with the exciton transition predominantly for the donor, we have demonstrated the so-called "carnival effect", where the donor and acceptor reverse their roles [29]. Consequently, energy transfer occurs first from the donor to the acceptor exciton by means of resonant dipole-dipole interaction and then from the excitonic states in the acceptor reservoir to the mainly-donor LPB. We speculate that our experimental findings, together with further investigations of the interplay between the polaritonic states and excitonic reservoirs, as well as control of the relaxation pathways, will pave the way to new In order to estimate FRET efficiency via dipole-dipole interaction we placed solution containing beacons labeled with FAM, TAMRA, and FRET pair alternately on the lower mirror of the microcavity without the upper one and excited it non-resonantly with 450 nm laser. In such way we ensured the same experimental conditions as in experiments with the media placed inside the cavity. The measured Pl spectra are shown in Figure S3. From these spectra it can be seen that FAM-only labeled hairpin shows high fluorescence (probably due to FAM high quantum yield of 97%) intensity with a peak at 525nm while TAMRA-only counterpart shows low intensity. These results correspond to higher efficiency excitation at 450 nm of FAM ( ~20% of maximum at 495 nm) compared to TAMRA (~2% of maximum at 546 nm). Both donor and acceptor labeled hairpin shows significant decrease in donor intensity and simultaneous increase in acceptor intensity, a result that indicates energy transfer from donor to acceptor. From these data FRET efficiency can be estimated using following expression [s1,s2] .

! = 1 - ! !" ! ! , ( S1
)
where E is the efficiency of FRET, FDA and FD are the donor fluorescence in the presence and absence of acceptor, respectively. These spectra lead to estimated FRET efficiency value of 80%.

S2 Experimental setup

Experimental setup is shown in Figure S4. In order to enter strong coupling regime, tunable microcavity, originally introduced in [s3] , was used. Briefly, our versatile tunable microcavity cell (VTMC) [s4] is composed of plane and convex mirror that form unstable λ/2 Fabry-Perot microcavity. One mirror is made convex in order to satisfy the plane-parallelism condition and minimize the mode volume. Plane mirror is mounted on top of a Z-piezopositioner to provide fine tuning of a microcavity length in a range up to 10 µm with a nanometer precision, while landing procedure is carried out by the high precision differential micrometer DRV3 (Thorlabs) which is indirectly connected to the convex mirror. The alignment of the plane-parallelism point and a sample is made by moving convex mirror in lateral direction by means of the XY precision positioner. A sample is deposited directly onto the plane mirror that consists of standard (18x18 mm) glass coverslips with ca 35 nm layer of aluminum metallization (Al) on their upper side. The VTMC is mounted onto inverted confocal microspectrometer consisting of an Ntegra-base (NT-MDT) with a 100X/0.80 MPLAPON lens (Olympus) on a Z-piezo-positioner, an XY scanning piezo-stage and a homemade confocal unit. The fluorescence spectra of each sample were excited by a 2.1 W 450-nm laser (L450P1600MM (Thorlabs) with LDS5-EC (Thorlabs) power supply), while for transmission spectra the MCWHF2 white LED (Thorlabs) with homemade optical condenser was used. It should be noted that in experiments laser power was far from saturation. The registration system includes an Andor Shamrock 750 monochromator equipped with an Andor DU971P-BV CCD (Andor Technology Ltd) and two 488-nm RazorEdge® ultrasteep long-pass edge filters (Semrock).

S3 Accounting for changes in pumping intensity

For correct calculation of the lower polariton branch population it was necessary to account for changes in pumping intensity during tuning the cavity length. When the exciting field is in resonance with one of the cavity eigenmodes, significant rise in field intensity inside the cavity appears. To account this effect we used numerical model [s5] implemented for calculating spectral and spatial properties of the microcavity electromagnetic modes with the finite elements method [s6] . It should be noted that higher transverse modes of the microcavity were also taken into account. It was necessary because during the excitation process light of the pumping laser was focused by the objective lens with quite high numerical aperture (NA=0.95) and subsequently pumping radiation excited the higher transverse modes (Figure S5a). In the transmission experiments illuminating light had approximately planar wavefront (Figure S5b), and so there are no appearances of higher transverse modes on the transmission spectra (Figure S6a). Using developed model, the spectral distribution of the electromagnetic energy was calculated for experimental set of cavity lengths. Then for all this set of spectra the points corresponding to the excited laser frequency were picked. These values were used as the pumping intensity at certain cavity length (Figure S6b).

Figure 1 .

 1 Figure 1. (a) The schematic of the tuneable microcavity. (b) The PL emission spectra of the solutions containing molecular beacons labelled with FAM alone (blue), TAMRA alone (red), and both FAM and TAMRA (black) dyes. All samples were located outside the microcavity. The PL spectra were excited at 450 nm. The concentrations of the dye molecules in the sample solutions were about 100 µM in all experiments. The molecular beacons are drawn not to scale.The donor-acceptor pairs of organic dyes employed in our experiments consisted of

Figure 2 .

 2 Figure 2. The PL spectra recorded at distances between the mirrors varied from 735 to 945 nm at a 15-nm step for microcavity filled with the molecular beacons labelled with TAMRA (a), FAM (b), or the FAM-TAMRA donor-acceptor pair operating in the FRET regime (c). The black solid line shows the position of the cavity mode; the vertical grey solid and dashed lines show the positions of the acceptor and donor excitons, respectively; the black dashed lines mark the calculated lower polaritonic branches. (d) The experimental (blue dots) and calculated (red dots) spectral positions of the -peak corresponding to the emission from weakly coupled states. Black arrow shows the approximate position of the emission peak corresponding to the upper polariton (b).

The

  PL of molecular beacons labelled with the FAM dye alone and placed into the microcavity is shown in Figure2b. Considering the impact of the ensemble of weakly coupled molecules, one can clearly see the anticrossing behaviour of the PL emission peaks for cavity energies larger than 2.25 eV. The positions of the low-energy peaks in the emission spectra corresponding to the lower polaritonic branch (LPB) allow one to estimate the Rabi splitting energy to be about 457 meV, which is a rather high value for this type of a cavity. It is well known that the observation of the emission from the upper polariton branch (UPB) is difficult due to the fast relaxation of the UPB to the exciton reservoir[37]. In the PL experiments we have observed the emission peak that could be associated with the upper polariton for deeply negative detuning, which is marked in Figure2b. However, it is difficult to estimate the exact positions of the associated emission spectra for most other values of detuning due to the emission from bare donor molecules, the large spectral broadening, and the limited spectral range of detection in the setup used.

Figure 2c shows

  Figure2cshows the PL spectra recorded for different detunings of the microcavity

Figure 3

 3 Figure 3 confirms that strong coupling occurred in this case. Experimental data were derived

Figure 3 .

 3 Figure 3. The energies of the lower (red), middle (green), and upper (dark blue) polariton branches at different cavity detunings experimentally derived from the PL spectra (dots) and theoretically calculated (solid lines). The black dots correspond to the emission from weakly coupled dye molecules. The horizontal and inclined black dashed lines show the positions of the donor and acceptor excitons and the cavity mode energy, respectively.

Figure 3

 3 Figure 3 also shows the energy of an uncoupled cavity photon, exciton transitions for

Figure 4 .

 4 Figure 4. The Hopfield coefficients of the lower (a), middle (b), and upper (c) polaritonic branches calculated using the Jaynes-Cummings model for the microcavity filled with molecular beacons labeled with both FAM and TAMRA dyes. The grey area shows the range of the cavity mode detuning measured experimentally.

( 3 )

 3 photon lifetime (less than 10 fs). Thus, radiative decay through the photonic fraction becomes

Figure 5 .

 5 Figure 5. Experimentally observed data (black dots) and theoretically modelled results (red line) for dependences of the relative population of the lower polaritonic branch on the cavity detuning.

Figure 6 .

 6 Figure 6. Schematic energy-level diagram showing the energy relaxation pathways in the system with predominant strong coupling to the donor excitonic transition. The thickness of the horizontal lines reflects the density of the bare donor and acceptor excitonic states, as well as three new hybrid light-matter states: the upper (UPB), middle (MPB), and lower (LPB) polariton branches.

  applications of strong coupling to remote-controlled chemistry, energy harvesting, energy transfer and sensing.Experimental SectionSample preparation. All components of the oligonucleotide-based molecular beacon labelled with donor or acceptor dye alone or both dyes operating in the FRET regime were obtained from Evrogen (Evrogen Joint Stock Company, Moscow, Russia) and diluted in phosphatebuffered saline (PBS) to obtain a concentration of 100 µM. Detailed information on the chemical structures and optical properties of the dyes and all three types of molecular beacons used are presented in the SI. For experiments, 10 µL of each sample was placed on the flat bottom mirror of the microcavity and then covered with the convex upper mirror. During the experiments, the surface exposed to air was small enough to ensure a negligible rate of sample solvent evaporation and a constant sample concentration.The tuneable microcavity. The sketch of the experimental setup consisting of a tuneable microcavity and an optical excitation/collection system is shown in the SI. The setup is described in detail elsewhere[30,31]. The plane-parallelism point and the sample were aligned by moving the convex mirror in the lateral direction by means of an XY precision positioner. The curvature radius of the upper convex mirror was 77.3 mm. The sample was deposited directly onto the plane mirror, which consisted of standard (18´18 mm) glass coverslips covered with a ~35-nm layer of aluminium and a 20-nm protective layer of SiO2.Transmission and PL measurements. The tuneable microcavity as a part of the unique setup called the System for Probe-Optical 3D Correlative Microscopy (http://ckp-rf.ru/usu/486825/) was mounted on an inverted confocal microspectrometer consisting of an Ntegra-base (NT-MDT) with a 100X/0.80 MPLAPON lens (Olympus) on a Z-piezo positioner, an XY scanning piezo stage, and a homemade confocal unit. The MCWHF2 white LED (Thorlabs) with an optical condenser was used for transmission measurements. In PL measurements, we used emission of a 450-nm L450P1600MM laser (Thorlabs) with an LDS5-EC (Thorlabs) power supply, with a pump power of about 2.1 mW for excitation. The pump power in all the PL experiments was far from the saturation threshold. The recording system included an Andor Shamrock 750 monochromator equipped with an Andor DU971P-BV CCD (Andor Technology Ltd.) camera and two 488-nm RazorEdge® ultrasteep long-pass edge filters (Semrock).

Figure S2 .

 S2 Figure S2. Absorption and photoluminescence spectra of solutions of the TAMRA dye (a); FAM dye (b); FAM and TAMRA dyes (c).

Figure S3 .

 S3 Figure S3. PL emission spectra of the solutions containing molecular beacons labelled with FAM (blue), TAMRA (red), FAM and TAMRA (black) outside of the cavity.

Figure S4 .

 S4 Figure S4. Experimental setup

Figure S5 .

 S5 Figure S5. The principal configuration of a) the excitation and b) the transmission experiments.

Figure S6 .

 S6 Figure S6. Panel (a) shows calculated spectrum of electromagnetic energy inside the microcavity with (red) and without mode selection (blue). The grey area represents corresponding experimental transmission spectrum. Panel (b) shows the pumping intensity dependence on the cavity mode frequency.

Acknowledgements

This study was supported by the Ministry of Education and Science of the Russian Federation (grant no. 14.Y26.31.0011). I.N. acknowledges the support from the Ministry of Higher Education, Research and Innovation of the French Republic and the University of Reims Champagne-Ardenne. Y.R. acknowledges the support from the Basque Government (grant no. IT1164-19). We thank Vladimir Ushakov for the help with technical preparation of the manuscript.

Supporting Information

The Supporting Information is available from the Wiley Online Library or from the author. It includes: detailed descriptions of the properties of the molecular beacon samples, tuneable microcavity setup, and PL/Transmission collection system; calculations of the pumping intensity dependence on the cavity detuning; and description of the Jaynes-Cummings model.

S1 Materials

6-Carboxyfluorescein (FAM) was selected as donor molecule, Carboxytetramethylrhodamine (TAMRA) as acceptor molecule. The most common way to fix the required for efficient FRET distance between chromophores is to use oligonucleotide-based molecular beacons as in this case distance between donor and acceptor is of the order of the diameter of the DNA double helix, which is 2 nm. In this study, donor and acceptor were conjugated with self-complementary oligonucleotide, 5'-TGG AGC GTG GGG ACG GCA AGC AGC GAA CTC AGT ACA ACA TGC CGT CCC CAC GCT CCA-3'. Donor-and acceptor-only labeled hairpins were also obtained and studied as controls. (Figure S1). Oligonucleotide sequence of 57 units with 18 base pairs was selected so that to ensure hairpin stability and small distance between donor and acceptor. Molecular weights of donoronly, acceptor-only and both donor and acceptor labeled hairpins are MFAM=17477.47, MTAMRA=17568.68 and MFAM_TAMRA=17945, respectively. All components were obtained from Evrogen (Evrogen Joint Stock Company, Moscow, Russia) and diluted in phosphate buffered saline (PBS) to obtain concentration of 100 µM. For experiments, we used 10 µL of each component. Photoluminescence and absorption spectra of the compounds were first studied outside of the cavity (Figure S2). Here we used different excitation wavelength depending on the absorption spectra of each compound.

S4 Calculation of the coupling strengths and Hopfield coefficients

Spectral properties of the experimental system based on donor-acceptor pair placed inside the microcavity were researched using the Jaynes-Cummings Hamiltonian that describes interaction between a cavity mode and dipole moments of emitters. For our hybrid system this Hamiltonian is as follows:

) where ℏωc, ℏωD and ℏωA -energies of microcavity electromagnetic mode, donor and acceptor excitons, respectively; a(a + )-photon annihilation (creation) operator, ! !"#$% (# = %, ') =

-coupling strength, where dD and dA -dipole moments of energy transition of donor and acceptor, ε0 -vacuum permittivity, V -volume of electromagnetic mode;

), |! ' ⟩ and |3 ' ⟩ -ground and excited state wavefunctions of emitters. It should be noted here that expression (S1) doesn't have the term describing direct interaction between dipole moments of chromophores. This is due to this process is much slower than energy exchange between emitters and cavity mode for coupling strengths taking place in the current research [s7,s8] . The Hamiltonian also can be written in matrix representation:

We diagonalize this matrix using QuTiP [s9] what let us obtain eigenstates and eigenvalues of this Hamiltonian. Values of energies of microcavity electromagnetic mode, donor and acceptor excitons were extracted from the experimental data. For finding the coupling strengths of our hybrid system we used differential evolution method [s10] , stochastic method for obtaining extrema of function of several variables. In order to obtain correct experimental coupling strengths, we minimized the absolute value of difference between Hamiltonian eigenvalues and energies corresponding to experimental spectra maxima. As a result of this procedure we obtain the magnitudes of ! *"#$% and ! +"#$% that are equal to 435 meV and 41 meV.

In order to find the Hopfield coefficients, we obtained the eigenfunctions of Hamiltonian with coupling strengths corresponding to our experimental data. Every eigenfunction was represented as superposition of functions of pure photon and exciton states. The coefficients of the decomposition define Hopfield fractions of the polaritonic states.


Aurélie Salavert
email: salavert@mnhn.fr

Emilie Gouriveau
email: emilie.gouriveau@univ-fcomte.fr

Erwan Messager
email: erwan.messager@univ-smb.fr

Vincent Lebreton
email: lebreton@mnhn.fr

Dmytro Kiosak
email: dkiosak@ukr.net.

Salavert Aurélie

Gouriveau Emilie

Messager Erwan

Lebreton Vincent

Kiosak Dmytro

Multi-proxy Archaeobotanical Analysis from Mesolithic and Early Neolithic Sites in South-west Ukraine

Keywords: archaeobotany, Neolithic, Late Mesolithic, Linearbandkeramik, forest-steppe, Ukraine 1-Introduction

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

investigations into the eastern extension of this widespread LBK culture are still limited [START_REF] Saile | Zur Bandkeramik zwischen Pruth und Südlichem Bug[END_REF]). In the same way, concepts about the disappearance of local Late Mesolithic cultures and their role in the emergence of Neolithic communities are based on the surface discoveries of Mesolithic artefacts, and suffer from an insufficient number of radiocarbon dates (Biagi and Starinini 2016).

This study presents the results of multi-proxy archaeobotanical analyses (charcoal, seeds/fruits, phytoliths) carried out on the LBK site of Kamyane-Zavallia in south-west Ukraine (Fig. 1). In order to further explore the characterization of the environmental framework of the Neolithic transition in this area, archaeobotanical analyses was undertaken on the Late Mesolithic stratigraphic unit (SU) at Melnychna-Krucha; a multilayered site occupied from 7250 to 5600 cal BC and located 18 km southeast of Kamyane-Zavallia (Fig. 1). Despite the modest dataset, these preliminary archaeobotanical results provide new data for the environmental setting and subsistence of the Late Mesolithic groups and early farmers, which are poorly investigated in this region [START_REF] Monah | The spread of cultivated plant in the region between the Carpathians and Dniester, 6 th -4 th millennium cal BC[END_REF]. So far, no study based on archaeological charcoal has been published for the Late Mesolithic (Kukrek) and Early Neolithic (Bug-Dniester, LBK) in south-west Ukraine. The nearest ones were conducted at Maidanetske, a Trypillian mega-site inhabited during the first half of the 4 th millennium (Kirleis andDreibrodt 2016, Dal Corso et al. 2019). Multi-proxy archaeobotanical analyses were also conducted at Buran-Kaya IV rock-shelter (Crimea) on Neolithic layers dated from the first half of the 6 th millennium BC [START_REF] Salavert | First results of archaeobotanical analysis from Neolithic layers of Buran Kaya IV (Crimea, Ukraine)[END_REF]. Moreover, the cultivated plants, weeds and phytoliths of Kamyane-Zavallia provide additional data to the recent archaeobotanical study of Ratniv-2 (Motuzaité-Matuzevičiūtė and Telizhenko 2017) and contribute to the preliminary definition of the LBK crop package and plant processing in the eastern LBK culture.

Figure 1: Location of Melnychna-Krucha (Late Mesolithic/Bug-Dniester) and Kamyane-Zavallia (LBK) as well as others sites mentioned in the text (black circles). Extension of the Bug-Dniester (BD) and Linear pottery/Linearbandkeramik (LBK) cultures.

2-Environmental setting

Today, the climate of south-western Ukraine is humid continental, characterized by large seasonal temperature contrasts [START_REF] Peel | Updated world map of the Köppen-Geiger climate classification, Hydrology and Earth System Sciences Discussions[END_REF]. At Zavallia, near the LBK site, the average temperature increases from -4.5°C in January to 20.2°C in July. The average annual precipitation is about 550 mm.

Melnychna-Krucha and Kamyane-Zavallia are located close to the current interface of the foreststeppe and steppe provinces of Eastern Europe (Fig. 2). The steppe zone is characterized by the rarety or limited extension of tree cover and the predominance of grasses (Poaceae). In south-western Ukraine, the forest-steppe is a mosaic of open grasslands and Carpinus-Quercus (hornbeam-oak) forests (Fig. 2). Woodland patches can potentially occur anywhere, though with a decrease in their extension from north to south [START_REF] Chibilyov | Steppe and forest-steppe[END_REF]. Melnychna-Krucha is surrounded by pastoral meadow steppes dominated by Poaceae and with patches of deciduous broad-leaved forests. The site sits on riverine sediments close to the Southern Bug River. Kamyane-Zavallia is located on the loessic first terrace of the Southern Bug River, covered by podzolized chernozem soils. Today, a large agricultural plain spreads directly around this site, and woodlands are barely visible.

In the western Ukraine area, the few existing pollen sequences that covers the Early Atlantic period (6900-4700 cal BC) are not well dated (Pashkevich 1982[START_REF] Kremenetski | Holocene vegetation and climate history of southwestern Ukraine[END_REF][START_REF] Vinogradova | The Holocene Palaeovegetation of South-western Ukraine: A Review[END_REF]. These limited pollen studies indicate that around 5000 cal BC, the forest-steppe vegetation was characterized by thermophilous mixed-oak woodlands, mainly composed of Corylus (hazelnut), Ulmus (elm), Tilia (lime tree), Quercus and some Carpinus [START_REF] Albert | What was the ecological impact of a Trypillia megasite occupation? Multi-proxy palaeo-environmental investigations at Nebelivka, Ukraine[END_REF].

Figure 2: Vegetation units in the Southern Bug (modified from [START_REF] Bohn | Karte der natürlichen Vegetation Europas[END_REF].

Archaeological settings

3-1 Melnychna-Krucha

The deepest stratigraphic units (SU3 and SU2) of the 2012 test trench revealed lithic artefacts belonging to the pre-pottery occupation of the Kukrek tradition [START_REF] Kiosak | Mesolithic "Heritage" and Neolithic in Southwest Ukraine[END_REF]. The two radiocarbon dates, obtained on charcoal fragments sampled from SU3, fall into the second half of the 7 th millenium [START_REF] Kiosak | Revisiting the chronology of two Neolithic sites in Eastern Europe: new radiocarbon dates from Melnychna Krucha and Kamyane-Zavallia (Southern Buh region, Ukraine)[END_REF]Tabl. 1, Fig. 3). The Kukrek culture corresponds to one of the Late Mesolithic complexes of south-western Ukraine [START_REF] Biagi | The Mesolithic of the northwestern Pontic region. New AMS dates for the origin and spread of the blade and trapeze industries in southeastern Europe[END_REF].

3-2 Kamyane-Zavallia

The excavation focused on an elongated pit, in which numerous ceramic and lithic artefacts were mixed with domestic and wild animal bones (determination of O.P. Sekerska), pieces of burnt clay, charred macroremains, grinding stones and bone tools (Kiosak et al. 2014a(Kiosak et al. , 2014b;;[START_REF] Kiosak | Mesolithic "Heritage" and Neolithic in Southwest Ukraine[END_REF][START_REF] Kiosak | Kamyane-Zavallia, the Easternmost Linear Pottery Culture Settlement Ever Excavated[END_REF]. No postholes have been detected so far (Fig. 4). The fine pottery mostly bears Notenkopf-style decoration that is a regional variation of LBK pottery. The Romanian Dudeşti-style ornamentation of some of the potsherds could illustrate long distance contact with the eastern LBK settlements [START_REF] Saile | Zur Bandkeramik zwischen Pruth und Südlichem Bug[END_REF]. The two radiocarbon dates from animal bone and charcoal fragments mostly encompass the last quarter of the 6 th millenium (Tabl.1) and correspond well with the duration of the Notenkopf-style phase in the eastern area of LBK distribution [START_REF] Kiosak | Revisiting the chronology of two Neolithic sites in Eastern Europe: new radiocarbon dates from Melnychna Krucha and Kamyane-Zavallia (Southern Buh region, Ukraine)[END_REF].

3-Material and method

3-1 Macroremains

3-1-1 Sampling

At Melnychna-Krucha, the botanical material sampled from the SU3 corresponded to a layer of greyish loess-like loam with finds scattered throughout its depth and with no clear walking surface. In this context, macroremains are mixed with wild animal bones and Late Mesolithic lithic material [START_REF] Kiosak | Revisiting the chronology of two Neolithic sites in Eastern Europe: new radiocarbon dates from Melnychna Krucha and Kamyane-Zavallia (Southern Buh region, Ukraine)[END_REF]. The charcoal, originating from local vegetation, probably corresponds to several burning activities (i.e. to firewood) or even natural fires [START_REF] Chabal | Forêts et sociétés en Languedoc (Néolithique final, Antiquité tardive) : l'anthracologie, méthode et paléoécologie[END_REF]. The sampling strategy consisted of taking small-size soil samples (average of ca. 1, 5 litres per sample) in the 2012 testtrench section reopened during the 2014 field mission. We focused on areas with charcoal inclusions without apparent taphonomic disturbances (i.e. plant roots, burrows). Other samples were handpicked from previous campaigns where the charcoal material had been detected with the naked eye. No large charred seeds or fruits were noticed during excavations. In total, 21 samples, including the four smallsize samples, were studied (Tabl. 2).

Figure 4: Plan of the pit (object 1) and stratigraphy at Kamyane-Zavallia, location of samples for radiocarbon dating and projected positions of the phytoliths samples (in Kiosak and Salavert 2018: fig. 5). The macroremains and dating material were sampled from horizon D. The stratigraphic section corresponds to the western wall of square 7 (Sq7). At Kamyane-Zavallia, samples came from the elongated pit. Macroremains were dispersed in the sediment and mixed with LBK domestic artefacts that cannot be strictly associated with the primary functions of the structure. The macroremains could result from several heterogeneous activities, without any functional or temporal links between them. Regarding charcoal, several plant groups, at different states of maturity, were represented within one sample, according to vegetation dynamics and the gathering practices of past societies for firewood. A total of 23 samples provided charred macroremains (Tabl. 2), ten of which were handpicked during the 2013 excavation. The others corresponded to large (ca. ten litres each) or small volume samples collected during the excavation, or sampled from the north wall section of the pit in 2014.

Lab code

3-1-2 Extractions, identifications and countings

In 2014, the large and small volume soil samples were water sieved (250 µm) then floated directly onsite (Fig. 5). Charcoal fragments were fractioned according to the three wood anatomical planes and identified under an optical reflection microscope at x100 to x500 magnification, with the help of a comparison atlas [START_REF] Schweingruber | Anatomie europäischer Hölzer/Anatomy of European Woods[END_REF]) and the charcoal reference collection from the Muséum national d'Histoire naturelle in Paris. At both sites, most of the charcoal fragments measured around or less than 1 mm in their transversal section.

The charcoal fragments from each sample were counted for each taxon, with the dominant taxa being assumed to be the most harvested [START_REF] Chabal | Forêts et sociétés en Languedoc (Néolithique final, Antiquité tardive) : l'anthracologie, méthode et paléoécologie[END_REF]. Usually, the most common species is the lead species of a community, such as Quercus sp. in the deciduous oak forest or Alnus sp. (alder) in the mature riparian forest [START_REF] Chabal | La représentativité paléo-écologique des charbons de bois archéologiques issus du bois de feu. In Vernet J.-L. (éds.) Les charbons de bois, les anciens écosystèmes et le rôle de l'Homme[END_REF]. At Melnychna-Krucha, most of samples were handpicked, but this method does not document a reliable overview of past forest diversity. At Kamyane-Zavallia, the grouping of all the samples from pit 1 ensured the maximum of palaeoecological diversity. In addition, the number of taxa occurrences were calculated (i.e. each taxon determined in a given sample is counted as a single individual whatever the number of fragments), to assess, for each taxa, the minimum number of logs burnt at both sites. This method valorizes the taxa that are regularly present in small quantities on sites [START_REF] Delhon | Les assemblages de charbons « concentrés » : une piste pour améliorer leur représentativité[END_REF]. Regarding charred seeds and fruits, remains were scarce and badly preserved. The number of remains corresponds mainly to fragmented remains. Few cereal seeds were sub-complete. Most of the time, bad preservation of the material did not allow identification to the species level, especially for cereals.

Figure 5: The sieving process applied to small and large volume samples collected at Melnychna-Krucha and Kamyane-Zavallia.

3-2 Phytoliths

At both sites, the sediment was sampled along the test-pit profiles for phytolith analysis. The samples were not systematically collected at regular intervals because of the complex stratigraphy of the pit filling. In Melnychna-Krucha, 20 samples were collected from the archaeological layers of the test-pit section (Fig. 3). In Kamyane-Zavallia, 9 samples were collected in the detritic pit. One additional sample collected in the deposit under the pit was used as a control sample for this site (Fig. 4).

Phytoliths were extracted from sediment samples using HCl and H2O2 baths, sieving, clay removal and densimetric separation [START_REF] Lentfer | A comparison of three methods for the extraction of phytoliths from sediments[END_REF]. After cleaning, the residue was suspended in immersion oil and mounted on glass slides.

The slides were observed under a Leica DM 1000 ™ Microscope at x650 magnification. Each phytolith was classified according to its morphology, following several systems [START_REF] Twiss | Morphology classification of grass phytoliths[END_REF][START_REF] Mulholland | Grass opal phytolith production: a basis for archaeological interpretation in the northern plains. Archaeobotany through phytolith analysis symposium[END_REF][START_REF] Fredlund | Modern phytolith assemblages from the North American Great Plains[END_REF] Table 3: Phytolith morphotypes (following ICPN 2.0, ICPT et al., 2019), with their synonyms in the ICPN 1.0., their taxonomic attributions and the corresponding literature (In brackets, the morphotype name used in the cited literature).

4-Results

4-1 Melnychna-Krucha (Late Mesolithic, 6460-6100 cal BC)

4-1-1 Charcoal

Among the three taxa identified, Fraxinus dominated with 62 % of the total amount of fragments, followed by Quercus (34 %). Ulmus remains were minimal (4 %). The occurrences showed the same rank for the tree taxa (Fig. 6). No seeds/fruits were identified.

Fig. 6: Results of the charcoal analyses of Melnychna-Krucha (analysis: Salavert A.) and Kamyane-Zavallia (analysis: Gouriveau E., Salavert A.).

4-1-2 Phytoliths

The sediment samples collected were very poor in phytoliths. Among the 20 samples, only four (Fig. 7) yielded more than 100 phytoliths (excluding silica spicules). Samples present very similar assemblages. Grasses (Poaceae) were the dominant taxon. Among this group, Pooideae, represented by sinuate trapeziform and some rondel classes, was the best recorded subfamily. Bilobate was recorded in three samples but in very low quantities. Globular was recorded in every sample. Dendritic elongate phytoliths and silica skeletons, the two morphotypes that are generally abundant at archaeological sites where cereals were processed [START_REF] Berlin | Ptolemaic agriculture,"Syrian wheat", and Triticum aestivum[END_REF][START_REF] Portillo | Husbandry practices and livestock dung at the Numidian site of Althiburos (el Médéina, Kef Governorate, northern Tunisia): the phytolith and spherulite evidence[END_REF], were completely absent from the Melnychna-Krucha assemblages. The assemblages were marked by the abundance of silica spicules (Fig. 7), an aquatic environment marker. 4-2 Kamyane-Zavallia (LBK, 5290-4960 cal BC)

4-2-1 Charcoal and seeds/fruits remains

Seven woody taxa were identified. Fraxinus (77 %) and Quercus (20 %) were the most abundant trees.

Carpinus (1.5 %) was also identified, as were four minor taxa whose identification is uncertain due to the small size of the fragments: cf. Ulmus, Prunus (e.g. cherry, blackthorn), cf. Sambucus (elder) and cf. Cornus (dogwood). The occurrence valorizes Quercus, which was almost as frequent as Fraxinus (Fig. 6).

For seeds/fruits analysis, cereals with Triticum turgidum cf. subsp.

4-2-2 Phytoliths

Phytoliths were well recorded, except in the sample located under the pit (Fig. 7). This control sample, from a natural deposit (non-anthropogenous), did not yield any phytoliths. We can therefore assume that the assemblages identified in the archaeological layers were not inherited from older assemblages.

As for Melnychna-Krucha, Pooideae (represented by sinuate trapeziform and some rondel classes) was the best recorded Poaceae subfamily. Globular morphotypes were rarely identified.

Among the Poaceae phytoliths, the morphotype elongate dendritic, coming from inflorescences (glumes, lemma, palea) of Poaceae [START_REF] Ball | An approach to identifying inflorescence phytoliths from selected species of wheat and barley[END_REF], was recorded in every pit sample. In the upper samples only a small amount was recorded, but in the bottom of the pit it was abundant (from 14 to 27 %). This class of phytolith is scarcely recorded in natural soil assemblages; however, it is often wellrepresented in archaeological sites where cereals have been processed [START_REF] Berlin | Ptolemaic agriculture,"Syrian wheat", and Triticum aestivum[END_REF][START_REF] Portillo | Husbandry practices and livestock dung at the Numidian site of Althiburos (el Médéina, Kef Governorate, northern Tunisia): the phytolith and spherulite evidence[END_REF]. Thus, the presence of numerous dendritic phytoliths in the bottom of the pit reveals cereal processing at Kamyane-Zavallia. The class silica skeletons, representing fragmented epidermal cells, was identified by only one fragment in the deepest pit sample.

First inter-site comparison

Comparisons between the archaeobotanical assemblages from both sites, especially charcoal assemblages, were difficult to undertake due to the differences in the nature of the samples collected and the number of macroremains identified (Tabl. 2). There was no obvious difference in the leading taxa in the charcoal assemblages from the two sites (Fig. 6): Fraxinus and Quercus dominated and corresponded to more then 90 % of the charcoal assemblage. At this stage of the research, the highest diversity noticed at Kamyane-Zavallia may be due to the higher number of identified charcoal. Not surprisingly, this LBK site bears several indicators of productive economy; in contrast to the lack of botanical macro-and microremains in the SU3 of which do not indicate productive subsistence (Tabl.5). Table 5: Comparison of the absence (0) and presence (x) of production economy indicators at Melnychna-Krucha (MK) and Kamyane-Zavallia (KZ).

MK (Late

5-Interpretation

5-1 Contribution and limit of charcoal and phytoliths analysis to reconstruct past vegetation

At Melnychna-Krucha and Kamyane-Zavallia, charcoal analyses provided a list of taxa that informs about the adjacent territories exploited by human groups, mainly for the collection of firewood. Some of the charcoal could also correspond to wood collected initially for other purposes (construction, crafts) and then later used as fuel. Although the charcoal analyses did not provide direct evidence on the exploited biomasswhether the woodland cover was associated with open (steppe), semi-closed (steppe-forest) or closed (forest) environments around the sitesphytolith assemblages are essentially produced by herbaceous taxa (such as Poaceae) in temperate ecosystems. Unfortunately, on an archaeological site, it is usually difficult to separate the phytoliths coming from crops from those coming from wild plants. At the Maidanetske site (Trypillia culture), the Stipa-type phytoliths [START_REF] Blinnikov | Phytoliths in plants and soils of the interior Pacific Northwest, USA[END_REF][START_REF] Fredlund | Modern phytolith assemblages from the North American Great Plains[END_REF] permitted the identification of wild grasses coming from a steppe environnement (Dal [START_REF] Corso | Where are the cereals? Contribution of phytolith analysis to the study of subsistence economy at the Trypillia site Maidanetske (ca. 3900-3650 BCE), central Ukraine[END_REF], but this morphotype was not recognized in the Melnychna-Krucha and Kamyane-Zavallia assemblages.

5-2 Firewood resources during the Late Mesolithic (ca. 6500-6100 cal BC) and Early Neolithic (ca.

5200-5000 cal BC) in Southern Bug

The main taxa used for firewood were quite similar at Melnychna-Krucha and Kamyane-Zavallia, although occupied 1000 years apart: Fraxinus and Quercus dominated both charcoal assemblages.

These taxa, as well as Ulmus, could have grown together in the alluvial deciduous forest, probably on the riverbank of the Southern Bug River, in close vicinity of both sites. The presence of heliophilic shrubs (Prunus, cf. Sambucus, cf. Cornus) growing in forest edges may indicate a locally open environment during the LBK, but further analyses are crucial to discuss a possible human impact on the woodland at this period.

The Late Mesolithic and LBK occupations fall into the Early Atlantic palynozone (6900-4700 cal BC). Pollen analyses from the Dovjok swamp, located in the current forest-steppe on the flood plain of the Murafa River (Fig. 1), shows that the vegetation was dominated by arboreal taxa, with Quercus, Corylus, Ulmus and Tilia in the first half (8000-6000 BP) of the Atlantic period [START_REF] Kremenetski | Holocene vegetation and climate history of southwestern Ukraine[END_REF].

Tilia, and more particularly Corylus, are important taxa in palynological records [START_REF] Albert | What was the ecological impact of a Trypillia megasite occupation? Multi-proxy palaeo-environmental investigations at Nebelivka, Ukraine[END_REF]), but they were not identified among the charcoal fragments in our study. Regarding Tilia, its rarity in charcoal records compared to its representation in palynological diagrams is common at Neolithic sites, particularly in temperate Europe [START_REF] Bakels | The botanical shadow of two early Neolithic settlements in Belgium: carbonized seeds and disturbances in a pollen record[END_REF][START_REF] Van Der Sloot | Le Mésolithique et le Néolithique du site Saint-Lambert à Liège dans leur contexte chronologique, géologique et environnemental. Synthèse des données et acquis récents[END_REF][START_REF] Salavert | Environnement végétal et collecte du bois de feu au Néolithique dans le sud du bassin de l'Escaut (5 200-2 200 BC)[END_REF].

Several hypothesessuch as the poor flame resistance of Tilia wood, or its use for handcrafts or medicinesmay explain what led to its abandonment for firewood and subsequent absence in charcoal assemblages [START_REF] Salavert | Environnement végétal et collecte du bois de feu au Néolithique dans le sud du bassin de l'Escaut (5 200-2 200 BC)[END_REF]. Today, Corylus is one of the most dominant trees in the undergrowth of deciduous mixed forests in the Eastern European plain [START_REF] Serebryanny | Mixed and deciduous forests[END_REF]) therefore, the absence of Corylus in archaeological charcoal records could suggest its scarcity surrounding Melnycha-Krucha and Kamyane-Zavallia. Alternatively, it may not have been gathered for firewood by Late Mesolithic and first farmer populations. However, it is premature to generalize the absence of Corylus or the use of Tilia at both sites pending further charcoal analysis.

Carpinus was present but probably still not very abundant around Kamyane-Zavallia at the end of the 6 th millennium. Mature Carpinus trees develop in shaded or semi-shaded conditions, and could have formed part of the forest undergrowth with the other minor taxa identified at Kamyane-Zavallia (cf.

Cornus, Prunus and cf. Sambucus). Carpinus is recorded since the Early Holocene in the Black Sea region [START_REF] Filipova-Marinova | First high-resolution marinopalynological stratigraphy of Late Quaternary sediments from the central part of the Bulgarian Black Sea area[END_REF][START_REF] Feurdean | Origin of the forest steppe and exceptional grassland diversity in Transylvania (central-eastern Europe)[END_REF][START_REF] Shumilovskikh | Vegetation and environmental dynamics in the southern Black Sea region since 18 kyr BP derived from the marine core 22-GC3[END_REF][START_REF] Messager | Late Quaternary record of the vegetation and catchment-related changes from Lake Paravani (Javakheti, South Caucasus)[END_REF]. In eastern Ukraine, a few pollen grains of Carpinus were identified at the Kamyana Mohyla site before 4600 cal BC [START_REF] Gobet | Vegetation shifts at the monu mental Ukrainian si te of Ka myana Mohyla during the Neolithisa tion period[END_REF]). In the regional pollen record of Dovjok, Carpinus is frequently recorded with low values [START_REF] Kremenetski | Holocene vegetation and climate history of southwestern Ukraine[END_REF]. This early occurence in the Black Sea region could be related to the short distance of refuge areas for Carpinus. The seedlings of Carpinus are light demanding and the tree could have benefitted from natural (or human made) open space like forest edges to spread in the Carpathian-Alpine region [START_REF] Magyari | The Holocene expansion of beech (Fagus sylvatica L.) and hornbeam (Carpinus betulus L.) in the eastern Carpathian basin[END_REF]. Based on archaeological charcoal, it is impossible to infer on the tree cover during Mesolithic and Neolithic times.

5-3 Late Mesolithic subsistences and LBK plant economy

No evidence of the use of edible plants was recorded at Melnychna-Krucha, which may be due to the small size of the excavation, the sampling strategy and the type of site. Phytolith assemblages did not reveal the presence of cereal and rather reflect wild grasses living on the riverside than steppic grasses.

Numerous fish remains and wild animal bones have been identified, on the site suggesting the exploitation of the local fresh-water environment and a diet oriented toward aquatic resources and game [START_REF] Kiosak | Revisiting the chronology of two Neolithic sites in Eastern Europe: new radiocarbon dates from Melnychna Krucha and Kamyane-Zavallia (Southern Buh region, Ukraine)[END_REF]. Thus, the Late Mesolithic layers of Melnychna-Krucha record a broad-spectrum of local resource acquisition, terrestrial and aquatic, near the Bug River.

At Kamyane-Zavallia, Triticum cf. dicoccum, T. cf. monococcum and weeds are typical of the LBK crop package [START_REF] Kreuz | Archaeobotanical perspectives on the beginning of agriculture north of the Alps[END_REF][START_REF] Bakels | The Western European loess belt: agrarian history, 5300 BC-AD 1000[END_REF]Salavert 2011, Motuzaite Matuzevičiūtė and[START_REF] Matuzevičiūtė | The First Farmers of Ukraine: an Archaeobotanical Investigation and AMS Dating of Wheat Grains from the Ratniv-2 Site[END_REF]. Regarding cf. Hordeum, this cereal is sometimes considered as a weed in Central and Western temperate Europe because of its rarity on LBK sites in this area [START_REF] Kreuz | Closed forest or open woodland as natural vegetation in the surroundings of Linearbandkeramik settlements?[END_REF][START_REF] Salavert | Plant economy of the first farmers of centralBelgium (Linearbandkeramik, 5200-5000B.C.)[END_REF]). Ratniv-2, occupied slightly earlier then Kamyane-Zavallia, provided one seed of Hordeum vulgare (Motuzaite [START_REF] Matuzevičiūtė | The First Farmers of Ukraine: an Archaeobotanical Investigation and AMS Dating of Wheat Grains from the Ratniv-2 Site[END_REF]. The further development of archaeobotanical analyses in eastern LBK sites will assess whether the presence of Hordeum could be considered as a crop. This would be an originality, compared to the rest of the LBK territory.

Furthermore, the significant contribution of elongate dendritic phytoliths in the pit infill is a good signal of cereal processing [START_REF] Berlin | Ptolemaic agriculture,"Syrian wheat", and Triticum aestivum[END_REF][START_REF] Portillo | Husbandry practices and livestock dung at the Numidian site of Althiburos (el Médéina, Kef Governorate, northern Tunisia): the phytolith and spherulite evidence[END_REF]. Among the potential cereal processes, dehusking or winnowing are more likely because the remains of threshing processes (silica skeleton) are almost absent. These activities could have taken place in the village, maybe at a household level. No charred chaff remains were identified in the elongate pit at Kamyane-Zavallia, whereas several chaff fragments (glumes of hulled wheat) were present at Ratniv-2 (Motuzaite Matuzevičiūtė and Telizhenko 2017). On this latter site, archaeobotanical samples came from a fireplace in a pit house that may correspond to single-activity refuse [START_REF] Kreuz | Searching for "single-activity refuse" in Linearbandkeramik settlements. An archaeobotanical approach[END_REF], enabling better preservation of the fragile chaff elements than in the detritic context at Kamyane-Zavallia. Among the weed macroremains identified at Kamyane-Zavallia, Chenopodium album type and Fallopia convolvus can both grow in cereal plots and field edges. Chenopodium album is common in summerand winter-sown fields in temperate areas and can also be used for human consumption [START_REF] Golea | Discuții asupra prezenței speciei Chenopodium album în așezările preistorice din România[END_REF][START_REF] Mueller-Bieniek | ChenopodiumSeeds in Open-Air Archaeological Sites -How to Not Throw the Baby Out with the Bathwater[END_REF]).

Conclusion

The composition of tree vegetation, or wood acquisition strategies, was quite similar at Melnychna- Acknowledgements: fieldwork (2014) was supported by the project « Interactions et dynamiques des sociétés et des environnements à la transition Mésolithique-Néolithique dans les steppes du sud-ouest de l'Ukraine » (coord. A. Salavert, program ATM DS-E MNHN). We would like to thank the anonymous reviewers for their meaningful advice. Figure 4: Plan of the pit (object 1) and stratigraphy at Kamyane-Zavallia, location of samples for radiocarbon dating and projected positions of the phytoliths samples (in Kiosak and Salavert 2018: fig. 5). The macroremains and dating material were sampled from horizon D. The stratigraphic section corresponds to the western wall of square 7 (Sq7).

List of figures/tables

Figure 5: The sieving process applied to small and large volume samples collected at Melnychna-Krucha and Kamyane-Zavallia. Table 1: Radiocarbon dates from Melnychna-Krucha SU3 (MK, Late Mesolithic) and Kamyane-Zavallia (KZ, LBK).

Table 2: Archaeobotanical dataset of Melnychna-Krucha (MK) and Kamyane-Zavallia (KZ).

Table 3: Phytolith morphotypes (following ICPN 2.0, ICPT et al., 2019), with their synonyms in the ICPN 1.0., their taxonomic attributions and the corresponding literature (In brackets, the morphotype name used in the cited literature).

Table 4: Charred seeds identified in the elongated pit (object 1) at Kamyane-Zavallia, analysis: Salavert A.

Table 5: Comparison of the absence (0) and presence (x) of production economy indicators at Melnychna-Krucha (MK) and Kamyane-Zavallia (KZ).

Figure 3 :

 3 Figure 3: Stratigraphy of the north wall of square 3 from Melnychna-Krucha with projected position of the two samples for radiocarbon dating in the SU3 and phytolith samples positions (modified from Kiosak and Salavert 2018: fig. 2).

Figure 7 :

 7 Figure 7: Microremain assemblages (phytoliths+spicules+diatoms) from Melnychna-Krucha and Kamyane-Zavallia (analysis: Messager E).

Figure 8 :

 8 Figure 8: Kamyane-Zavallia, ventral face (up) and profile (down) of T. cf. dicoccum (A), Triticum cf. monococcum. (B) and cf. Hordeum sp. (C).

Figure 1 :

 1 Figure 1: Location of Melnychna-Krucha (Late Mesolithic/Bug-Dniester) and Kamyane-Zavallia (LBK) as well as others sites mentioned in the text (black circles). Extension of the Bug-Dniester (BD) and Linear pottery/Linearbandkeramik (LBK) cultures.

Figure 2 :

 2 Figure2: Vegetation units in the Southern Bug (modified from[START_REF] Bohn | Karte der natürlichen Vegetation Europas[END_REF].

Figure 3 :

 3 Figure 3: Stratigraphy of the north wall of square 3 from Melnychna-Krucha with projected position of the two samples for radiocarbon dating in the SU3 and phytolith samples positions (modified from Kiosak and Salavert 2018: fig. 2).

Fig. 6 :

 6 Fig.6: Results of the charcoal analyses of Melnychna-Krucha (analysis: Salavert A.) and Kamyane-Zavallia (analysis: Gouriveau E., Salavert A.).

Figure 7 :

 7 Figure 7: Microremain assemblages (phytoliths+spicules+diatoms) from Melnychna-Krucha and Kamyane-Zavallia (analysis: Messager E).

Figure 8 :

 8 Figure 8: Kamyane-Zavallia, ventral face (up) and profile (down) of T. cf. dicoccum (A), Triticum cf. monococcum. (B) and cf. Hordeum sp. (C).

Depth (cm) Sample Support Conventional Age 14 C age BP Age cal BC 2σ

	Poz-67121	130	KZ1	Animal bone (undetermined)	6200 ± 40	5295-5045
	Table 1: Radiocarbon dates from Melnychna-Krucha SU3 (MK, Late Mesolithic) and Kamyane-
	Zavallia (KZ, LBK).
				MK	KZ
				(Late Mesolithic)	(LBK)
		Handpicked		17	10
		Sieved (volume)		4	13
		Volume of sediment	5	104
		Total		21	23
		Charcoal fragments	79	540
		Number of wood taxa	3	7
		Charred seeds/fruits	0	35
	Poz-67497	170	MK3	Charcoal (Fraxinus)		7380 ± 40	6380-6100
	Poz-67496	200	MK2	Charcoal (Angiosperm)	7520 ± 5050	6461-6253
	Poz-67554	82	KZ2	Charcoal (Fraxinus)		6130 ± 40	5211-4962

Table 2 :

 2 Archaeobotanical dataset of Melnychna-Krucha (MK) and Kamyane-Zavallia (KZ).

  and the International Code for Phytolith Nomenclature(ICPN Working Group et al. 2019). The observed phytoliths were classified into 10 different categories (Tabl.3). The morphotype silica skeletons (linked phytoliths) corresponding to fragments of silicified Poaceae epidermis were also identified[START_REF] Rosen | Preliminary identification of silica skeletons from Near Eastern archaeological sites: an anatomical approach[END_REF]). These represent the remains of fragmented pieces of culms, leaves and inflorescences of Poaceae plants. Sponge spicules and diatoms were also observed. Each morphotype's relative abundance was calculated as a percentage of the sum of classified phytoliths. This sum, used for percentage calculations, was based on the total number of phytoliths counted per sample (more than 200 for each sample). Silica skeletons, diatoms and sponge spicules were excluded from the total phytolith sum. The representation of each of these groups was expressed as a percentage of the sum of phytoliths plus the group sum [i.e. % of diatoms = N diatoms/ (Ʃ phytoliths + N diatoms) x 100].

	Morphotypes (ICPN 2.O)	ICPN 1.0	Main taxonomic attribution	Bibliography
	Elongate entire	Elongate	Poaceae	Twiss et al. 1969 (elongate); Alexandre et al. 1997 (elongated)
	Elongate dendritic	Elongate dendritic	Poaceae	Berlin et al. 2003
	Acute bulbosus	Acicular hair cell 1	Poaceae	Twiss et al. 1969 (point-shaped); H.-Y. Lu et al. 2006 (long point)
	Bulliform flabellate & Blocky	Cuneiform bulliform cell & Parallelepipedal bulliform	Poaceae	Twiss et al. 1969 (fan-shaped); Strömberg 2002 (keystone-shaped bulliform)
	Rondel & Trapezoid	Rondel & Trapeziform short cell	Poaceae	Fredlund et Tieszen 1994 (keeled, conical, pyramidal); Mulholland 1989 (rondel)
	Saddle	Saddle	Poaceae, Chloridoideae	Mulholland 1989 (saddle); Fredlund et Tieszen 1994 (saddle)
	Crenate	Sinuate trapeziform	Poaceae, Pooideae	Brémond et al. 2004 (crenate); Barboni et al. 2007; Mulholland 1989 (sinuate)
	Bilobate	Bilobate	Poaceae, Panicoideae/Arundinoideae	Brown 1984 (bilobate); Mulholland 1989 (dumbbell)
	Spheroid psilate & ornate	Globular	cf. Dicotyledonous	Alexandre et al. 1997 (circular rugose); Runge 1999 (spherical)
	Acute bulbosus 2	Acicular-hair cell 2	No taxonomic value	Piperno 2006 (hair cell); Pearsall 2000 (hair cell)

Table 4 :

 4

	Number of samples	16
	Volume of sediments (l.)	116
	Number of macroremains	35
	Hordeum/Triticum sp.	8
	Triticum cf. dicoccum	6
	Cf. Hordeum sp.	3
	Triticum cf. monococcum	1
	Chenopodium album type	14
	Fallopia convolvulus	2
	Caryophyllaceae type	1

dicoccon (T. cf. dicoccum, emmer), Hordeum sp./Triticum sp. (barley/wheat), Triticum cf. monococcum (einkorn) and cf. Hordeum sp., represent 50 % of the dataset (Tabl. 4). Whole seeds of Chenopodium album type (fat-hen), fragments of Fallopia convolvulus (wild buckwheat) and of Caryophyllaceae type (Fig. 8) represent the weeds/wild taxa. Charred seeds identified in the elongated pit (object 1) at Kamyane-Zavallia, analysis: Salavert A.

  Krucha and Kamyane-Zavallia. Late Mesolithic groups and LBK farmers mainly exploited Fraxinus and Quercus for firewood. The Late Mesolithic populations seemed to settle in a forest-steppe environment close to forested areasto hunt game and collect firewoodand areas rich in aquatic resources. At the LBK site, the development of edge shrubs as a result of anthropogenic impacts from the activities of contemporaneous or previous human groups (i.e. Bug-Dniester) will have to be verified by further charcoal analyses in the future. The discovery of a grinding stone in the pit of Kamyane-Zavallia, together with cultivated plants and cereal processing evidence, reinforces the strong agrarian economy of the LBK inhabitants in the eastern margins of the LBK cultural extension.These first multi-proxy analyses of a Late Mesolithic occupation and LBK site located in Southern Bug River gives the potential of botanical macroremains and phytoliths studies to characterize the local environment and use of plant resources. Location and cultural dynamics make the south-west Ukraine a key area to better understand the development of human societies of the 7 th and 6 th millennium in Eastern Europe.