Anneke De Rouw
email: anneke.de_rouw@ird.fr

Nicolas Bottinelli

Sylvain Huon

Jean Luc Maeght

Guillaume Massalis

Pascal Podwojewski

Thiet Bounsamay Soulileuth

Nguyen Van

Peter Van Welzen

Keywords: Successional pathway, Grazing, Upland farming, Long-term monitoring, Afforestation, Vietnam

Forest regeneration following land abandonment is driven by historic land use affecting plant diversity and soil carbon stocks (mountainous tropical Asia)

Forest regeneration following land abandonment is driven by historic land use affecting plant diversity and soil carbon stocks (mountainous tropical Asia)

Introduction

Farmland on steep slopes is unsuitable for most forms of mechanisation, has poor access and often shallow soils. Worldwide, agriculture tends to intensify on the more fertile soils, while abandonment of farming is more common in areas with poor soils, on high altitudes, and steep slopes [START_REF] Chazdon | Tropical forest recovery: legacies of human impact and natural disturbances[END_REF]; Vu Kim Chi et al. 2013; [START_REF] Queiroz | Farmland abandonment: threat or opportunity for biodiversity conservation? A global review[END_REF]). In tropical Asia, landform is a decisive factor regarding abandonment of farmland. A clear differentiation is made between farming under hydromorphic conditions in lowlands (paddy rice) and farming under rain-fed conditions in uplands. In the rather at plains of the lowlands the modernisation of agriculture takes place with its possibility of irrigation and easy transport. Farmland in the lowlands, even on marginal soils, is never abandoned because such sites compete with infrastructure, urbanisation and industry. The uplands on the contrary, often hilly with poor access make all work extremely time-consuming. Hence, the abandon of farmland in South-east Asia concerns the uplands exclusively. Farmland is also abandoned because inappropriate use had produced excessive erosion making traditional farming no longer worthwhile [START_REF] Thai Phien | Indigenous technical knowledge in Vietnam: management of soil for agricultural development[END_REF]). Besides, the steady outmigration of young rural people to the cities, does not leave enough population to sustain the cultivation of the slopes. Most often, abandoned farmland is simply colonised by spontaneous regrowth (Arroyo-Rodriquez et al. 2017) or converted to fodder/livestock exploitation [START_REF] Valentin | Runoff and sediment losses from 27 upland catchments in Southeast Asia: Impact of rapid land use changes and conservation practices[END_REF]). In general, land abandonment is considered as an important part of land degradation process (ipbes 2018). Agriculture in South-east Asia is unique for its high number of smallholders and their practice of polyculture, often involving crop-livestock relations. These farmers subsist on paddy rice grown in lowlands and produce a surplus using the uplands thus providing the urban population with food and the industries with raw materials e.g. pulpwood, rubber, sugar.

Where the natural vegetation is forest, the land no longer cultivated will eventually revert to forest through secondary succession. Abandoned agricultural land is under no management regime; in contrast, fallows are not abandoned land. Crop-fallow systems aim to re-use the land once the development of the postharvest vegetation has su ciently restored the soil and suppressed the weeds. Yet abandoned land and fallows alike could contribute to ecological improvement through carbon storage, biodiversity conservation [START_REF] Dwyer | Carbon for conservation: Assessing the potential for win-win investment in an extensive Australian regrowth ecosystem[END_REF]) and better soil-water relationships [START_REF] Valentin | Runoff and sediment losses from 27 upland catchments in Southeast Asia: Impact of rapid land use changes and conservation practices[END_REF]). In crop-fallow systems fast fallow development is stimulated through speci c farming practices (de Rouw 1995) and at the same time these farmers recognize that a succession blocked in an early stage is likely to compromise the productivity of the land, making it useless. While there are agricultural practices that stimulate forest succession leading to the re-use of the land, so there should be practices that slow down succession, leading to abandonment (this study).

Recovery of forest structure, soil nutrient stocks and species richness is far more rapid than recovery of the original species composition [START_REF] Chazdon | Tropical forest recovery: legacies of human impact and natural disturbances[END_REF], which can take centuries to come back [START_REF] Rozendaal | Biodiversity recovery of Neotropical secondary forests[END_REF]). Hence true succession is not limited to increase in biomass and species turnover but is the improvement in plant diversity towards the original forest. This makes species composition the key tool to measure true succession (this study). The diversity of the original forest is contained in the standing biomass, the soil seed bank and the bank of vegetative regeneration i.e. stumps, rootstocks, buds or other organs (resprouter bank). Seed bank and resprouter bank are then "residuals" of the original forest in case the standing biomass suffers. Residuals assure the rapid and complete return of the original forest if enough time is allowed. When residuals are destroyed together with the above-ground biomass, then the ready and complete return of the initial species richness is compromised or may be impossible.

Agriculture, forestry and husbandry practiced on land originally covered by forest, cause not only the disappearance of the above-ground forest biomass, but affect the forest residuals, too. This relationship seems evident but is sustained by very few eld observations. In South-east Asia many hotspots of biodiversity can be found, most of them under direct threat of disappearance. In order to preserve this diversity, it is important to study how the original forest composition comes back or does not come back as a result of speci c land use practices. [START_REF] Chazdon | Tropical forest recovery: legacies of human impact and natural disturbances[END_REF]. This approach assumes that about every combination of land use will produce its proper successional stage or pathway; it suggests that the number of possible stages or pathways is endless or high. We question this argument. Our approach is to work the other way round: to survey plots with similar vegetation and then reconstruct for each plot the history of management. We suspect that an accumulation of severe, or complex or frequent disturbance produces a miserable regeneration which is represented by few successional vegetation types. In the opposite situation, following a single event, the number of successional vegetation types will be higher and more varied.

The object of this study is to describe the succession process in a tropical catchment where annual cropping stopped about 20 years ago. Our aim is to identify the speci c practices responsible for the blocked, slow or fast succession using detailed records of historical land use, complete oristic inventories, and spatial context. In contrast to most studies that use synchronous data sets, i.e. comparing plots of different ages representing a chronosequence, this study investigates diachronic data sets, i.e. the same site was observed and monitored over a long period, in our case starting before the abandon of farmland.

Methods

Study area

Dong Cao is a cultivated catchment (20°57′N, 105°29′E) of 49.7 ha, located 60 km south of Hanoi in Hoa Binh Province, Vietnam. Farmers, all smallholders, share their work time between the lowlands where they cultivate paddy rice for subsistence, and the uplands where they cultivate a variety of crops to increase their revenue in association with some husbandry, buffalo, cattle and pigs. This system, coined "Composite farming" [START_REF] Ziegler | Soil translocation by weeding on steep-slope swidden elds in northern Vietnam[END_REF]) is widespread and well-described (Nguyen The original vegetation was Lowland semi-evergreen broadleaf monsoon forest after Whitemore (1988). This formation has a majority of evergreen trees in the canopy, no incidence of re, and no occurrences of spontaneous bamboo. Considering the local climate with its persistent high air humidity measured at the site, the proportion of evergreen tree species in the original forest should have been much higher.

Land use

Sequence

Historic land use of the period before the start of MSEC in 1999 was obtained from interviews with the inhabitants of Dong Cao. The correctness of their information was con rmed by independent sources [START_REF] Wezel | Slope position effects on soil fertility and crop productivity and implications for soil conservation in upland northwest Vietnam[END_REF][START_REF] Clement | Afforestation and forestry land allocation in northern Vietnam: Analyzing the gap between policy intentions and outcomes[END_REF]. After 1999, MSEC scientists and students supplied an uninterrupted record of farmer's activities in the catchment. The timeline of major events in Dong Cao catchment is presented in Table 1, with details in Table 2.

After the export of timber, the exploitation of the uplands started with burning, piling and re-burning of what was left of the original forest, to gain space and access. For about twenty years the slopes were regularly cultivated with a variety of crops, the fertile sites and lower slopes more frequently than the steep, remote land. In 1986 the government allocated land to individual farmers, as a result cassava cropping took off, responding to the development of pig raising in the region. The demand for pig food was such that for about 15 years the catchment produced nothing else: cassava, modern varieties of maize and arrowroot, all served as pig food. The last elds (cassava) were abandoned in 2002.The opportunities for husbandry accelerated the abandon of farming because the eroded soil produced less than before.

Cassava was cultivated throughout Northern Vietnam, often on very steep slopes, causing ooding and massive erosion with sediment deposits in lowland paddy elds. Subsequently, a national policy of soil conservation measures was applied in the region. For Dong Cao this meant: subsidized tree planting and work on the contour: ditches, and line-planting of bamboo, Vetiver grass and Vernicia saplings. The initial plan to reforest the catchment by 80% was not realised. Successful afforestation was limited to weedfree sites, for instance when trees were planted immediately after the last cassava harvest. Planting trees in weedy elds several years after the last harvest, failed. Trees received no or little maintenance after planting and spacing was wide.

Mapping

To verify whether agriculture had an effect on forest succession, details of land use have to be spatial explicit. In our case remote sensing images proved of little use. Firstly, images taken before the abandonment of farming showed insu cient resolution, secondly, the high resolution images of later date suffered from artifacts. For instance, Google Earth images (2014 and later) suggested dense forest on steep slopes which was not the case, and faint, parallel lines appearing in many parts of the catchment suggested the canopies of line-planted trees, or soil conservation work on the contour (ditches, tree planting). Inspection in the eld revealed that those parallel lines had no consistent meaning and most of times it was not clear what "produced" the lines. Instead, 161 photographs with panoramic views of the catchment were used, taken by MSEC scientists working in Dong Cao (Fig. 1). Photographs with eld checks showed important features: (i) not a single tree of the original forest had been spared, (ii) the extent and intensity of cassava cropping, (iii) areas with successful and failed afforestation, (iv) details on species and spacing of the planted trees, (v) preferred areas for grazing, passing, resting of the freeroaming livestock. These spatial explicit data could be mapped (see land use map in Online Resource 2).

Land use was associated with topography and landform. In Dong Cao the left and right banks of streams and gullies are not symmetric; right banks have long and moderate slopes, left banks short and steep slopes. Almost all annual crops were grown on the right banks. Bulky products like poles of Eucalyptus and wood of Acacia, were produced at lower altitudes close to the outlet for easy evacuation, both on left and right bank.

Vegetation surveys

During surveys the catchment was crossed on foot in all major directions. A total of 33 reference samples (relevés) were made following the Braun-Blanquet method (Braun-Blanquet 1964): ve in 2002, six in 2004, two in 2010, two in 2011, ten in 2016, and eight in 2017. A sample plot was carefully selected for being representative of a larger area. Plot size was kept small (18m²) to better assure the links between species composition and habitat. Canopy trees not rooted in the plot were included in the species list. A reference sample consisted of a full species list with abundance for each species and additional information on GPS location, slope, land use, soil pro le, soil surface features, and soil fauna. All growth forms were taken into account and all age classes. Resprouter status was checked for all woody plants in the plot. The reference samples were used to produce a classi cation of vegetation types. In addition, many plotless inventories were made of common species and vegetation structure which later assisted in mapping the vegetation types in the catchment. Dried voucher specimens were made of unidenti ed species occurring in the plots and these were checked in o cial herbaria (Chiang Mai University, Thailand; Naturalis, the Netherlands). Processing of the oristic data yielded six vegetation types (Communities) and a set of diagnostic species for each Community. The mapping of the communities in Dong Cao catchment was carried out with the help of a key (below).

In 2018 and 2019, frequent returns to the eld were made to nalize the mapping of the Communities. The group of indicator (diagnostic) species assisted in the identi cation of vegetation types in the eld.

The catchment was crossed on foot with a hand-held GPS to mark off the Community areas, and checks were made in 160 points.

Soil sampling

In 2003 soil samples were collected in 109 sites following 4 transects across the catchment, covering early and late abandoned elds, young tree plantations of Acacia and Vernicia and adult Eucalyptus. Samples were collected in two layers, 0-5 cm and 5-15 cm depth. Local slope and GPS location were also recorded. Bulk densities were determined in three replicates every third site by collecting undisturbed 0-5 cm depth cores with a 100 cm 3 cylinder. These samples were oven-dried until stable weight. All other soil samples were air-dried, sieved to 2 mm to remove debris and stones and grounded to very ne powder using a hand mortar. Determination of total organic carbon was carried out at iEES-Paris by EA-IRMSe by CHN following [START_REF] Huon | Long-term soil carbon loss and accumulation in a catchment following the conversion of forest to arable land in northern Laos[END_REF]. In 2016 soil samples were collected in a 50*50 m grid covering the entire catchment. A total of 195 sites were sampled. Soils were sampled in a single layer, 0-15 cm depth and the bulk densities were determined by collecting undisturbed cores with a 100 cm 3 cylinder. At each sample site we recorded the local slope, the number of dung deposits in a radius of 10 m, and the GPS location. Soils were air-dried and sieved to 2 mm to remove debris and stones. Total organic carbon was determined following the method of Walkley and Black. In carbonated soils the Walkley and Black method underestimates soil carbon, but as these soils do not occur in Dong Cao, both analyses can be compared.

Data analysis Vegetation

To summarize the plant data we applied multivariate ordination in which similar species and samples appear close together and dissimilar entities far apart [START_REF] Gauch | Multivariate analysis in community ecology[END_REF]). The oristic data were drawn up in a two-way sample-by-species data matrix. The samples (relevés) were displayed in columns and the species in rows. Species scores were expressed as cover: 1 = < 1%, 2 = 1-5%, 3 = 5-25%, 4 = 25-50%, 5 = 50-75%, 6 = > 75%. The table arrangement was obtained from ordination (TWINSPAN) followed by re nement by hand. Species recorded both in high and low but not in medium densities, were split into pseudo-species, one corresponding to high cover (> 50%), the other to low cover (< 5%). This procedure is common in automated We used ranked reciprocal averaging (DECORNA) to identify a gradual shift of large groups of species along gradients. The programme calculates scores according to four axes, both for species and for samples, the latter being weighted mean species scores. We used only the rst axis (eigenvalue 0.71) to explore whether the shift in species abundance calculated by ordination corresponded to a pattern in other data sets.

To describe the vegetation (Kent and Coker 1992) we used Richness, being the total number of species found, and Species/area curves, being species accumulation with increasing sample area. Such curves allow to compare the species richness of communities with each other and to estimate how much of the total richness of a community was actually captured by the (limited number of) samples of that community. Using the relative abundance data for each species in a sample plot we computed Dominance/diversity curves. Species scores are then ranked in ascending order of abundance and plotted on a log 10 scale against the species cover. In order to compare communities with each other, the relative abundance was averaged over all the samples belonging to that community. Such curves show difference in evenness and dominance among communities.

The sample-by-species matrix contains no environmental or land use data, hence a vegetation map with plant communities shows purely variations in species composition. In a separate step we compared oristic data with other data. The vegetation map was digitalized and linked through a Geographical

Information System to other thematic maps of the catchment: slope angle, soil type, land use trajectory, soil conservation measures, tree plantations, and soil carbon stock.

Soil

For each sample site we calculated the carbon stocks in the top 15 cm of the soil:

(kg C m - 2 ) = TOC concentration (gC kg - 1 ) x bulk density (kg m - 3 ) x depth (m).

We calculated the porosity of the top 5 cm of the soil: P (%) = [1-(bulk density (g cm - 3 ) / 2.65 (g cm - 3 ))] x 100.

For discriminating the difference in soil carbon between plots of different land use, soil types and plant community, we applied the Student test (comparison of means of two independent samples of unequal size).

To investigate changes in soil variables over [START_REF] Feller | Physical control of soil organic matter dynamics in the tropics[END_REF] To test the effect of the environmental variables (soil carbon, local slope, soil porosity and abundance of dung deposits) on forest succession we applied the Kruskal-Wallis test using the 195 grid points. This test detects differences of location in ranked data grouped by a single classi cation. In the tests the groups were the plant communities and the test was run four times, each time with a new classi cation corresponding to the four variables: carbon, slope, porosity and dung deposits.

Results

Vegetation classi cation

The oristic analysis by table arrangement is a search for typical combinations among the common species. The clustering of species (rows) and samples (columns) resulted in six communities (see Appendix for full table and Fig. 2 for summary). A community is characterised by a distinctive species assemblage. For instance, Community 1 (Fig. 2, above left) should have species, not necessarily all, of group 1 and 2, and at the same time the species of the groups 3 through 10 should be absent. For Community 1 the species group 1 is diagnostic, in the same way the species group 3 is typical for Community 2, and so on. See Online Resource 3 for species groups with details on growth form and ecology.

The order of samples (columns left-right) follows the most important ecological gradient in the data set. In Fig. 2 this is a successional gradient with most advanced communities on the left and least advanced on the right. Likewise, the same gradient is expressed by the order of species groups (rows top-down).

Because the reference plots were sampled between 2002 and 2017 one would expect that the successional gradient equals a time-gradient with samples collected at the beginning of the abandonment (2002) on one side, and samples collected 16 years later, on the opposite side. This is not the case. The community is closed forest with three layers. The community comprises old trees of Acacia mangium and Vernicia montana, most of them planted between 1995 and 1999, both are deciduous species. In the canopy these planted trees are progressively replaced by spontaneous trees, equally deciduous. Maximum height of Acacia, Vernicia and wild trees is 24 m, 14 m and 8 m, respectively. The middle layer varies between 1 and 4 m, and the ground layer is commonly less than 0.5 m high. The middle layer is often a dense mass of shrubs, climbers and treelets. The ground layer consists of forest herbs, including ferns, and tree seedlings. The average richness per plot is 22 species.

Community 3broken forest (26 ha, 52%)

The community is a mosaic of forest and shrubs. The forest-patch and the shrub-patch do not have species in common. The forest patches are two or three layered and very variables in height, 4-20 m. The ground layer, mostly herbaceous, is about 0.5 m high. A middle layer, when present, consists of woody species and climbing ferns, 3-9 m high. The shrub-patches, 0.5-1.5 m high, are one-layered. Broken forest is dominated by species of a drier environment compared to the closed forest. The species richness in plots averages 20 species.

Community 4weedy elds and thickets

The community is a single continuous ground layer, 1-2 m high. The dense layer is a mix of tall grasses, many of them with C 4 pathway, and weeds, many of them Asteraceae. The community developed after each weeding and fully established post-harvest. Many species are annuals with Bidens pilosa dominating every plot. The community occupied the catchment after the abandonment of cropping. The species richness in plots averages 17 species.

Community 5tall herbs and shrubs(6 ha, 12%)

The community forms an herbaceous layer about 1.5 m high with isolated shrubs or shrub-like trees up to 6 m high, mostly Mallotus species. A characteristic feature is the strong dominance, over 75% of cover, by a single species. The dominant species could be Bidens or Stachytarpheta, or one of the tall perennial C 4 pathway grasses, Imperata, Panicum, or Brachiaria. The species richness in plots averages 10 species.

Community 6low grassland(5 ha, 10%)

The community consists of Paspalum conjugatum, a creeping perennial C 4 pathway grass forming loose monospeci c mats over extended areas. It grows 5 cm high when not owering. Grass alternates with the low shrub Stachytarpheta jamaicensis, growing up to 0.6 m. Mixed with the shrub, a few other C 4 pathway grasses can be found in low densities. This is a much degraded grassland and extremely species-poor vegetation of only 5 species.

Land use (1975-2019)

For each one of the 33 reference samples (red symbols in Fig. 3) we calculated how many years had been spent in a particular land use. We used the following broad classi cation: Cultivation (C), Tree planting (T), Grazing (G) and No intervention (Ab). Some categories include sub-categories: under "Cultivation" we distinguish between intermittent cultivation of annual crops with short fallow periods (C-F), and continuous cropping of cassava (Cas), and Ruzi fodder grass planting (Ru); under "Tree planting" there is successful (T) and failed tree planting (T-Ab); Grazing has three levels, heavy grazing (HeG), medium intensive grazing (MeG) and light grazing in either successful tree plantation (LiG T) or failed ones (LiG T-Ab). The abbreviations are used in Fig. 4. Some combinations did not occur in Dong Cao, for instance continuous cropping by crops other than cassava and medium grazing in tree plantations. Land uses per sample were chronically ordered and samples covered by the same plant community were clustered, thus associations between historic land use and vegetation recovery become visible (Fig. 4).

Intermittent cultivation over a period of about 20 years was the common history throughout the study area. Sites actually under Community

Soil carbon

When farmland was abandoned, the soil carbon stocks in the topsoil averaged 34.3 tC ha - 1 (sdv 5.13, n = 109, 0-15 cm depth). Stocks were unrelated (NS) to soil type, soil depth, and former land use, although there were trends. Late abandoned cassava elds were on average best provided with carbon (37.3 tC ha - 1 ) suggesting that the best soils were latest abandoned. Eucalyptus sites had lowest carbon levels (27.0 tC ha - 1 ). Early abandoned plots invaded by weeds (35.4 tC ha - 1 ) and plots planted with Acacia trees (34.3 tC ha - 1 ), had similar carbon stocks.

Soil carbon stocks were determined in 2003 (n = 109) and 2016 (n = 195). In 59 sites the locations matched, allowing the calculating of carbon change over 13 years. Figure 5 gives the carbon stocks in the matching locations, showing a positive linear relationship with elevation for both years. These trends can be explained by lower topographic positions being more frequently cultivated and had suffered more soil losses than higher positions. Carbon stocks in the top 15 cm of the soil had increased on average from 34.4 tC ha - 1 (sdv 8.31, n = 59) to 48.6 tC ha - 1 (sdv 7.75, n = 59). Over 13 years the average carbon sequestration was thus 14.2 tC ha - 1 , an increase of 1.09 tC ha - 1 year - 1 , a rate of just over 3% per year. A maximum of 2.60 tC ha - 1 year - 1 accumulation was recorded and a minimum change of 0.25 tC ha - 1 year - 1 loss. The amount of carbon accumulated was unrelated (NS) to previous land use, but again there were trends. Maximum sequestration occurred under spontaneous vegetation without tree planting, more in cases of early abandonment, average 16.9 tC ha - 1 (n = 25, rate 1.30 tC ha - 1 year - 1 ) than late abandonment, 14.6 tC ha - 1 (n = 13, rate 1.12 tC ha - 1 year - 1 ). Minimum accumulation was measured under tree plantations, more under Acacia, 12.8 tC ha - 1 (n = 10, rate 0.98 tC ha - 1 year - 1 ) than under Eucalyptus -Irvingia 11.3 tC ha - 1 (n = 11, rate 0.87 tC ha - 1 year - 1 ). Over the same period the average bulk density increased from 0.95 to 1.02 which equals a loss in soil porosity from 64-61% The change in soil compaction was slight but very signi cant (t 59 ***P < 0.001).

Mean soil carbon stocks, soil porosity, and slope angle were very signi cantly different across communities (*** P < 0.005, n = 195, Table 3). Following the complexity gradient, carbon levels were found lowest in Closed evergreen forest (41.9 tC ha - 1 , Community 1) and highest in Low grassland (56.4 tC ha - 1 , Community 6). Soil porosity was highest in Closed evergreen forest and lowest in Low grassland.

Steepness of the terrain averaged 80% in Closed evergreen forest and declined to only 32% in Low grassland. The gradient in soil carbon followed the gradient of dung deposits which followed the complexity gradient of the communities (0.10 < P < 0.05 near-signi cant). The negative impact of livestock on forest succession which was already visible across Communities because it reduced species diversity, is now con rmed by independent environmental data: cattle and buffalos while avoiding the steepest slopes, reduce soil porosity by their trampling and enrich the soil with carbon with their dung.

Discussion

Successional pathways

In Dong Cao the succession back to forest was slow and fragmentary. For about 10 years the slopes were uniformly covered by a thick herbaceous layer with forbs and low shrubs. Our study con rms the observations of [START_REF] Chazdon | Tropical forest recovery: legacies of human impact and natural disturbances[END_REF] that following the abandonment of agricultural land on poor soil with no residual vegetation left and no local sources of seed dispersal available, forest recovery fails to initiate within 5-10 years. In 2019 closed forest covered 25% of the catchment, 52% was broken forest, and the remaining 23% carried no trees or only isolated trees. Successful recovery of the forest structure took place on 25% of the catchment surface but biodiversity recovery, the return of some of the original species of moist lowland forest, had occurred in only 2% of the catchment. The remaining forest contained species of drier environments, for instance deciduous species in the canopy instead of evergreen. Open vegetation was composed of species of broad ecological valance, adapted to a wide range of habitat conditions including still drier environments. Hence, associated with the successional gradient there is a moisture gradient, with sites that have most advanced since the abandon of farming being wettest, less advanced sites are occupied with mesic forest, and least advanced or not advanced at all, are driest [START_REF] Nikolic | Land degradation on Barren Hills: A case study in northeaster Vietnam[END_REF], this study). We observed two successional pathways on abandoned farmland. First, positive pathway, called succession, being a progressive resemblance towards the original forest species composition. But vegetation development can go "the wrong way", a negative path of decreasing similarities with the original forest, called degradation. Instead of progressive colonization by the forest species of more and more humid habitats, sites are occupied by species of drier and disturbed environments.

The following trends were observed associated with the positive pathway: increase in species number, increase in growth forms, increase in woody cover and height, gradual development to a three-layered closed forest, increase in evergreen species, increase of species of moist habitats and shady places, and the gradual replacement of heliophyle grasses by shade grasses. Ferns were precious indicators of changes in the successional process. 2017) reports annual sequestration rates between 0.2 and 0.5 tC ha - 1 year - 1 in permanently cultivated tropical uplands. A change from cropland to grassland increased the rates to 0.3-0.6 tC ha - 1 year - 1 , and tree plantations under favourable management had sequestration rates of 0.26-0.57 tC ha - 1 year - 1 (cases from Nigeria, USA, Taiwan, Indonesia). All sequestration rates were well below those measured in Dong Cao. Only one case was found, a forestry site in Sulawesi, Indonesia [START_REF] Dechert | Is soil degradation unrelated to deforestation? Examining soil parameters of land use systems in upland Central Sulawesi, Indonesia[END_REF]), with a sequestration rate of 1.12 tC ha - 1 year - 1 , similar to Dong Cao. Sulawesi and Dong Cao have in common the favourable conditions for storage: clay content > 40%, bulk densities close to 0.95 (g m - 3 ), and an initial carbon stock of 45-47 tC ha - 1 . Thus abandoned farmland undergoing spontaneous forest regeneration accumulates under favourable soil and climate conditions far more carbon in the soil than cultivated elds, even those under the best practices available.

We expect a maximum of carbon stocks under forest and a minimum under low grass for the soil carbon would follow the successional gradient of vegetation complexity and biomass accumulation. Our data, very signi cantly, indicated the opposite: 56.4 tC ha - 1 under low grassland and 41.9 tC ha - 1 under moist closed forest. Increasing carbon stocks in the topsoil matched increasing livestock activity. Livestock prefers gentle slopes where their dung accumulates. The result is a concentration of livestock in the most degraded vegetation where the dung is incorporated in the soil assisted by an abundant macrofauna. Currently, the livestock impact dominates the in uence of forest succession, i.e. the constant high dung supply in the most degraded vegetation overrules the effect of the far greater standing biomass and the greater plant diversity in the closed forest plots, where, without livestock, soil carbon stocks should be maximum.

Land use

Plots The Graphic Abstract (Fig. 6) illustrates the common pattern of human interventions in mountainous South-east Asia. It starts with the destruction of the initial forest and leads to abandonment. Forest has to be cleared for access and any form of production. Burning is the only option for smallholders to get rid of massive debris. The use of re to combat weed growth and liberate nutrients for crops stops naturally, because after a while there is no biomass left to burn. Clear-felling and removal of all tree, shrub and liana stumps is common practice to increase the planting area and to ease the evacuation of produce.

For cassava and other root crops, taking out all super cial large roots greatly facilitates the harvest of the tubers. Removal of non-woody plant material like the root systems of perennial grasses is needed for the improvement of pastures because low performing local grasses have to be replaced by more productive ones. These interventions have consequences for forest regeneration because, progressively, the residuals of the original forest are lost. Residuals are the individual organisms or their propagules that survive a disturbance event and abundance of residuals is a measure for the disturbance intensity [START_REF] Turner | Factors in uencing succession. Lessons from large, infrequent natural disturbances[END_REF].

  years we tried to match the sample locations of 2003 (n = 109) with the grid locations of 2016 (n = 195), using the geo-locations of the sample sites. Matching sample sites succeeded in 59 locations. Matching greatly improved the quality of the soil data. For comparing carbon stocks and bulk densities in 2003 with 2016 the paired t-test was used. For discriminating the difference in soil carbon between plots of different land use and between plots covered by different plant communities, we applied the Student test (comparison of means of two independent samples of unequal size).

  with identical climate/soil conditions can carry very different vegetation types, although the number of years since farming stopped was the same. Historic land use before the abandonment of farmland as logging, erosion, unsustainable agricultural practices, and res along with uncontrolled grazing are increasingly recognized as the determinant factors in secondary succession (Chazdon 2003; Nikolic et al. 2008; Arroyo-Rodriquez et al. 2017).

FiguresFigure 1

 1 Figures

  To our knowledge there are no studies that determine by eld observations the direct links between cultivation practices, forest residuals, and forest succession.Large scale and intense disturbance can produce a slow succession or no succession at all (Arroyo-Rodriquez et al. 2017). Scientists, mainly ecologists, are then looking for the dominant factors that shape succession with the aim of predicting the successional pathways[START_REF] Bellemare | Legacies of the agricultural past in the forested present: an assessment of historical land-use effects on rich mesic forests[END_REF]; Chazdon 2003; Benjamin et al. 2005; Queiroz et al. 2014; Rozendaal et al. 2019). Most often the factor land use before abandonment, is described in very general terms, i.e. logging, cultivation, grazing. The authors agree with the all importance of land use on succession yet state that it is di cult to study because, commonly, an overlay of different land uses occurs. It is then impossible to identify the particular vegetation response to a sequence of human interventions

  table arrangement and is needed to check whether the ecological role of the species in the vegetation is different in high and low abundance. Pseudo-species concerned four species: Bidens pilosa, Stachytarpheta jamaicensis, Panicum amoenum, and Paspalum conjugatum. The table arrangement yielded a classi cation of samples into vegetation types (communities) and species into species groups, including the indicator species for communities.

  The oldest samples are found somewhat in the middle, forming the Community 4, being the initial old-eld vegetation of weeds and thickets. Community 4 covered the catchment for about ten years, afterwards three communities replaced it: Community 3, broken forest, Community 5, tall shrubs and grass, and Community 6, low grass. Fifteen years after abandonment, selected areas of broken forest turned into closed forest, either Community 2, mesic forest with a canopy of deciduous species, or Community 1, moist forest with a canopy of evergreen species. In contrast, no signi cant development could be seen over the years in the Communities 5, tall shrubs and grass, and 6, low grass. Thus from a single vegetation type in 2002 ve distinct vegetation types were identi ed in 2017. All species identi ed in Dong Cao with authorities, species groups, growth form and DECORANA axis scores are in Online Resource 4. The axis scores for individual species indicate their position in the successional gradient with high scores for advanced succession and low scores for least advanced vegetation.The vegetation map (Fig.3) shows the distribution of the ve communities over the catchment in 2019: 77% of the catchment (38 ha) is engaged in forest succession, 23%(11 ha) remains open shrubby grassland. The topographic background of the map indicates more forest development at higher altitudes. On moderate slopes, i.e. right banks of streams, all ve communities can be found, but on very steep slopes, left banks of streams, only two, the Communities 3, broken forest, and 5, tall shrubs and grass. The almost at land on the southern crest is exclusively covered by Community 6, low grass. The middle layer consists of young trees, palms, wild bananas, vines and woody climbers; the ground layer of herbs, particularly ground ferns and dicots, besides seedlings of trees and shrubs. Some of the original Lowland moist forest species had returned to the site, mostly in the ground layer. Local people removed some wood for fuel, wild banana owers for food, and medicinal plants without opening the canopy. The

	community is easily identi ed by some conspicuous plants like Begonia and Wallichia siamensis, a sh-
	tail palm. The average richness per plot (18m²) is 26 species.
	Community 2 -closed mesic forest with deciduous canopy trees (12 ha, 24%)

Description of the Communities

Community details, i.e. species/area curves, dominance/diversity curves and richness are in Online Resource 5.

Community 1closed moist forest with evergreen canopy trees (1 ha, 2%)

The community is closed forest with three layers, most species are evergreen. The upper and middle layers together form a continuous canopy, leaving the ground layer in permanent deep shade. The upper layer is approximately 15 m high, the middle layer 8 m and the ground layer 0.4 m. Old trees are absent, maximum tree height is 20 m, maximum diameter of trunks 28 cm (measured at 1.6 m height).

  1, closed evergreen forest, were subsequently abandoned; they have no history of tree planting, grazing or cassava. Community 2, closed deciduous forest, is found on sites where Acacia and Vernicia trees were planted; here too, no history of continuous cultivation of cassava or grazing. Sites with broken forest, Community 3, experienced extra disturbance after the period of intermittent cultivation, e.g. continuous cassava and light grazing by cattle and buffalos. It is noteworthy that very different actions lead to the same result: broken forest with Community 3. This also accounts for Community 4. Sites covered by tall herbs and shrubs, Community 5, have experienced still more disturbance after the period of intermittent cultivation, i.e. prolonged period of cassava, fodder production, grazing, all produced the same treeless vegetation. The Community 6 is directly linked to

livestock. Heavy grazing, probably starting in 1993 (interview data) is the continuous practice. This species-poor grassland had remained unchanged since our rst visit to the site in 2000.

  The negative pathway was associated with: decrease in growth forms, decrease in height, decrease to extinction of woody cover, appearance of C 4 pathway grasses gaining dominance and replacing the C 3 pathway heliophyle grasses, decrease to extinction of all trees, shrubs, lianas of humid and moist forest and establishment of trees, shrubs, lianas of drier and disturbed environments. The impact of cattle and buffaloes was absent to slight in closed forest, slight to medium in broken forest and medium to strong in open vegetation. The spread of the degraded vegetation matches the pattern of preferential grazing along gentler slopes, while the attest sites were the longest exploited and the deepest degraded.LivestockIn Dong Cao, forest development was negatively associated with livestock: the less impact of cattle and buffalo the more rapid the positive succession to moist evergreen forest took place. But past a certain threshold of passing, resting, grazing, trampling, a negative development sets in to seriously degraded grassland. The practice of free-roaming livestock was found to (i) slow down succession(Burgers et al. 2005; Fukushima et al. 2008; Wangpakattanawong et al. 2010, this study), (ii) decrease the forestbiodiversity (Burgers et al. 2005; Nikolic et al. 2008, this study), and to (iii) deviate the process from forest succession to degradation[START_REF] Nikolic | Land degradation on Barren Hills: A case study in northeaster Vietnam[END_REF], this study).The negative path was due to buffalos in[START_REF] Nikolic | Land degradation on Barren Hills: A case study in northeaster Vietnam[END_REF], northern Vietnam and buffalos and cattle in Dong Cao. In[START_REF] Nikolic | Land degradation on Barren Hills: A case study in northeaster Vietnam[END_REF] the degradation was worse than in Dong Cao because it led to stable woodland, a kind of dry savannah, very different from the climax "Lower montane rainforest with bamboo". The human impact on vegetation was greater, too: (i) the hill slopes were deforested in the 1960s, about 15 years earlier than in Dong Cao; (ii) livestock populations were higher; (iii) the farmers over-cultivated the land even more than the farmers of Dong Cao did; and (iv) there was no incidence of tree planting. These practices led to a more pronounced degradation and xerophytization of the environment compared to Dong Cao, as can be judged from both species lists. Thereby the climate is cooler with a more pronounced dry season, making it more di cult for lower montane species to establish. In Dong Cao the same process of degradation is less advanced, due to the continuous high humidity, higher average temperatures, lower livestock densities, some planting of trees and, most importantly the cessation of annual cropping for almost 20 years. Farmland in Nikolic et al. (2008) study site was not abandoned but de facto has become unsuitable for annual crops. Farmers state that all sites require a long period of rest during which the spontaneous vegetation develops before they can be cultivated again: an estimated 30 years for herbaceous covers resembling our open bush and low grass land (our Community 4), an estimated 20 years for bushy vegetation with sparse wood (our Community 5), and about 10 years for woody shrub land (similar to Community 3).Did tree planting accelerate the forest succession? Tree plantations catalyzed the process of forest succession only where a ground cover developed, thus preventing erosion and increasing soil fertility. Under Eucalyptus the soil remains bare and when abandoned and the stumps removed, a stable, species-poor community installs of tall herbs, shrubs and an isolated tree, resisting the colonization of trees. The other tree species naturally develop ground cover. When planted in medium densities and a regular pattern, i.e. Acacia mangium, Vernicia montana and Irvingia malayana, grassy patches are avoided and a closed canopy develops. Little by little native trees join the canopy and replace the planted trees. However, when planted in a wide grid or in irregular patterns, i.e. Styrax tonkinensis, Canarium tramdenum, and Chukrasia tabularis, open spaces are invaded by herbaceous covers. These patches attract livestock, feeding on the herbs and appreciating the shade from the trees. In Dong Cao the observation is that tree planting, even in cases of poor establishment, prevented the massive invasion of C 4 grasses, the development of livestock and hence the negative successional pathway.Plant diversitySpecies of disturbed environments, weeds, forbs, pioneer trees, are widely dispersed, hence the same diagnostic species identi ed in this study, indicate the same environment elsewhere, for instance the communities of early successional stages in Dong Cao, matched those found elsewhere in Vietnam (see species lists in[START_REF] Ziegler | Hydrological consequences of landscape fragmentation in mountainous northern Vietnam: evidence of accelerated overland ow generation[END_REF][START_REF] Nikolic | Land degradation on Barren Hills: A case study in northeaster Vietnam[END_REF]). In contrast, the communities of advanced successional stages, like Community 1 in this study, and the old-growth forest types described by Ziegler et al. (2004) and[START_REF] Nikolic | Land degradation on Barren Hills: A case study in northeaster Vietnam[END_REF], are all different from each other. Zieglers' site was slightly drier than Dong Cao and Nikolics' site was located at an higher altitude (700 m). As forest succession advances exclusive species belonging to the local climax tend to establish. In the three sites the soil/climate conditions were different, so the species composition of their respective advanced secondary forest was different, too. This illustrates the very high plant diversity in South-east Asia.Plant diversity does not always increase when farmland is abandoned, for Asia[START_REF] Queiroz | Farmland abandonment: threat or opportunity for biodiversity conservation? A global review[END_REF] found in 60% of case studies a drop in species richness following abandonment. This could be a question of sampling technique. Few studies include plants of all ages and growth forms (this study), most account only for dominant species, or ignore species with low cover, low height, or life forms other than trees. This greatly limits the recorded richness. In Dong Cao, species richness after abandonment of farming always increased. Community richness in elds and recently abandoned elds was estimated about 40 species. When this vegetation remains open with herbs, shrubs and some isolated trees, the total richness increased to about 60 species. In case a forest with a discontinuous canopy develops, the community richness increased to about 70 species. Though species richness increased, the conservation value of all these very common species is very low. In Dong Cao, only closed forest produced some conservation recovery in the occurrence of the species groups 1 and 2 (Community 1).In good structured clay soils carbon sequestration is high[START_REF] Bottinelli | Anecic earthworms generate more topsoil than they contribute to erosion -Evidence at catchment scale in northern Vietnam[END_REF]) and more carbon accumulates in uncultivated soils than in cultivated land(Feller and Beare 1998, this study). After abandonment soil carbon accumulation in Dong Cao was high, an average rate of 1.09 tC ha - 1 year - 1 in the top 15 cm soil measured over a thirteen-year interval.[START_REF] Minasny | Soil carbon 4 per mille[END_REF] reports case studies of carbon sequestration in soils after adoption of the best management practices available. Ignoring peat soils,Minasny et al. (

	Soil carbon sequestration

Acknowledgements

We wish to thank the National Institute for Soil and Fertilizers in Hanoi, Vietnam for their constant support since the rst surveys in Dong Cao. The works of the Master of Science students Julien Renaud and Chuleewan Boonchamni were particularly appreciated. For their dedicated help with plant identi cation we thank the late James Maxwell, Chiang Mai Herbarium, Thailand, Gaby Schmelzer, Wageningen University, and M.M.J. van Balgooy, B.E.E. Duifjes, and P.H. Hovenkamp, from Naturalis Biodiversity Center, Leiden, The Netherlands. The research was part of the M-Tropics programme, coordinated by the Institut de Recherche pour le Développement (IRD, France).

Funding

The authors declare that no funding was received for conducting this study except by the French taxpayers. Online Resource_1 (pdf 784 ko), Online Resource_2 (pdf 785 ko), Online Resource_3 (pdf 707), Online Resource_4 (pdf 722), Online Resource_5 (pdf 568)

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Con ict of interest

The authors have no relevant nancial or non-nancial interests to disclose.

Supplementary Information

Below are the links to the electronic supplementary material:

Tables Table 1 to

The Graphic abstract summarizes the sequence of human interventions when tropical forest is cleared for cultivation. Only the key practices are represented, those directly responsible for destruction of forest residuals and therefore the major contributors to slowing down the succession process and reducing plant diversity. With each downward step the cumulative disturbance is such that regeneration becomes more and more di cult and plant communities increasingly species-poor. It also shows the resilience of the environment to human damage and its capacity to recover from disturbances. Only long-cycle shifting cultivation with a single burning allows the preservation of most of the primary forest species.

Supplementary Files

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J Salort

F Gauthier

B Chabaud

O Bourgeois

J.-L Garden

R Du Puits

A Thess

P.-E Roche

Convection at very high Rayleigh number: signature of transition from a micro-thermometer inside the flow

Introduction

In 2001, a change in the statistics of temperature fluctuations in Rayleigh-Bénard convection was reported at very high Rayleigh number (Ra ∼ 10 12 ) [START_REF] Chavanne | Turbulent Rayleigh-Bénard[END_REF]. This change was concomitant with an enhancement of the heat transfer which had been interpreted [START_REF] Chavanne | Observation of the ultimate regime in Rayleigh-Bénard convection[END_REF] as the triggering of Kraichnan Convection Regime [START_REF] Kraichnan | Turbulent thermal convection at arbitrary Prandtl numbers[END_REF]. But a systematic study of finite probe size effect showed that the 200 µm probe used in the 2001 study was about three times too large to be free from finite size correction [START_REF] Gauthier | Temperature fluctuations in the Ultimate Regime of Convection[END_REF], calling for a confirmation of these results. We report new measurements of temperature fluctuations performed with a probe ten times smaller than the one used in 2001.

In this proceeding, we discuss experimental aspects of this experiment and complementary measurements made in the "Barrel of Ilmenau". The first experiment was conducted with cryogenic helium and the second with air. Once combined, those two experiments lead to evidence of a signature of a transition in the local temperature fluctuations, supporting the conclusion of the 2001 study.

Micron-size thermometer

The temperature probe is a 17-µm glass fiber, on which a 1 µm layer of NbN [START_REF] Bourgeois | Liquid nitrogen to room-temperature thermometry using niobium nitride thin films[END_REF] was deposited and annealed. The fiber and its stainless steel frame was glued on a wooden tripod. The fiber is placed two millimeters above the bottom plate of a 43-cm-high 10-cm diameter convection cell (see figure 1). It is connected to two pairs of copper wires for the 4-wire-measurement. We were careful not to over-heat the fiber with the measuring current to avoid hot-wire artefact. As can be seen on figure 2, the fiber starts to self-heat meaningfully for input current of order of 1 µA. All subsequent measurements were done with an input current of 0.5 µA. The temperature response of the probe is calibrated versus Ge thermometers in homogeneous temperature conditions. We find a temperature sensitivity ∂ ln R/∂ ln T ≃ 0.7 at 5 K (see inset in figure 2). The performance is limited by a resistance noise with spectral density 1.5 × 10 -6 /f , where f is the frequency in Hertz, corresponding to a rms noise of order 1 mK over the bandwidth of interest. This noise prevented operation above Ra = 5 × 10 13 in order to maintain Boussinesq conditions.

��� ���

I [µA] R/R 0 (T ) 4
The fluctuation data was acquired using the electronic setup shown on figure 2 : the input current is provided by the generator of a NF Lock-In Amplifier LI-5640 (V = 500 mV, f = 7825 Hz). The voltage drop across the fiber is pre-amplified (JFET, Gain 10 4 , typical noise input voltage 700 pV/ √ Hz). We substract the mean-value of the temperature signal using a Singer inductor bridge and an adjustable RC filter to compensate for the phase in order to use all the dynamics of the Lock-in. The demodulated signal is filtered by a General Purpose Kemo 1208/20/41LP low-pass filter (8 pole Elliptic filter with a flat passband, frequency cutoff 200 Hz) to avoid aliasing and then acquired by a NI Analog-Digital Converter NI6289 (18 bits).

Results and interpretation

Following Chavanne et al. [START_REF] Chavanne | Turbulent Rayleigh-Bénard[END_REF], we consider the exponent ξ 2 of the 2 nd order structure function of the temperature fluctuations T (t) :

ξ 2 = d log (T (t + τ ) -T (t)) 2 t d log τ (1)
where the brackets represent time averaging. The dependence of ξ 2 versus the time increment τ holds the same information as a temperature spectrum. Fig. 3-a shows ξ 2 versus the dimensionless increment τ /τ 0 where τ 0 = (h/2Nu) 2 /κ is a caracteristic time scale of the flow. A change of shape occurs above Ra ≈ 10 13 , which is also the threshold Ra for which a heat transfer transition is measured in this cell, as shown on fig. 3-b. The observed jumps of N u(Ra) are characteristic of the multiple possible configurations of the mean large scale circulation in elongated cells. They are known to hardly affect the temperature spectra, and therefore ξ 2 , although they can be responsible for a few tens of % offset of the x-axis of fig. 3-a through the N u dependence of τ 0 .

For Ra ≥ 10 12 , h/2Nu ≤ 400 µm < 2 mm: the probe is clearly outside the thermal boundary layer. We also studied the systematic dependance of ξ 2 (τ ) versus the probe-plate distance below the transition (Ra = 3.2 × 10 11 ) in the "Barrel of Ilmenau" in the range 0.1 < z/(h/2Nu) < 20.3. study reported in [START_REF] Gauthier | Temperature fluctuations in the Ultimate Regime of Convection[END_REF] showed that the variation of this effective plate-probe distance cannot explain the transition observed on the temperature statistics.

The observed change in shape of ξ 2 is consistent with Chavanne et al.'s qualitative observations, confirming a posteriori that their observation was not an artefact although it was partly altered by a finite size effect [START_REF] Chavanne | Turbulent Rayleigh-Bénard[END_REF].

Conclusion

The regime observed (and named "Ultimate Regime") in [START_REF] Chavanne | Turbulent Rayleigh-Bénard[END_REF] is thus characterised by 3 specific signatures : an integral one (heat transfer enhancement) and localized ones on the thermal boundary layer [START_REF] Gauthier | Evidence of a boundary layer instability at very high Rayleigh number[END_REF] and inside the flow as shown in this paper (see also [START_REF] Gauthier | Temperature fluctuations in the Ultimate Regime of Convection[END_REF]). These signatures are compatible with Kraichnan's prediction [START_REF] Kraichnan | Turbulent thermal convection at arbitrary Prandtl numbers[END_REF] but more work is certainly needed to understand the basic mechanism of the flow instability at very high Ra.

Fig. 1 .

 1 Fig. 1. (a) Temperature probe on its tripod above the bottom plate of the 43-cmhigh cryogenic Rayleigh-Bénard cell. Inset: detail on the web-like fiber support. (b) Drawing and electronic microscope picture of the sensitive element

Fig. 2 .

 2 Fig. 2. (a)Self-heating of the micro-thermometer for various experimental conditions. ρ = 1.6 kg/m 3 , T ≈ 6.5 K ( ); ρ = 6.5 kg/m 3 , T ≈ 6.0 K ( ); ρ = 19 kg/m 3 , T ≈ 6.0 K ( ); ρ = 40 kg/m 3 , T ≈ 6.0 K ( ). Inset : Resistance of the fiber versus temperature. (b) Electronic diagram of the acquisition system.

5 • 10 - 2 6 • 10 - 2 RaNu/Ra 1/ 3 ( a )

 5223a 9 10 10 10 11 10 12 10 13 10 14 10 15 Second-order increments. (b) Compensated heat-transfert data.

Fig. 3 .

 3 Fig. 3. Data measured in the 43-cm Rayleigh-Bénard cell.

Acknowledgements : We thank B. Hébral, F. Chillà and more especially B. Castaing for numerous discussions. The assistance of T. Fournier, P. Diribarne and P. Lachkar in instrumentation is gratefully acknowledged. A Procope collaboration was made possible to Deutscher Akademischer Austauschdienst (D/0707571) and Ministère des Affaires Etrangères (17858YD)


E Lévêque

C F Barenghi

P Diribarne

P.-E Roche

Turbulent cascade of a quantum fluid at finite temperature

Introduction : Motivation and Model

Quantum fluids (liquid 3 He and 4 He, atomic Bose-Einstein condensates) consist of two co-penetrating fluids: the superfluid, associated to the quantum ground state, and the normal fluid, associated to thermal excitations. In this work the turbulent inertial cascade undergone by a quantum fluid at small but finite temperature above absolute zero is simulated by DNS of two coupled Navier-Stokes equations [START_REF]Quantized vortices in Helium II[END_REF]. Following the two-fluid model of Landau and Tisza, one equation accounts for the viscous normal fluid, while the other equation models superfluid dynamics on scales larger than the inter-vortex spacing.

The originality of our approach consists in introducing an artificial superfluid viscosity -as a turbulence closure -to model non-viscous processes taking place on scales smaller than the inter-vortex spacing, such as phonon emission. For simplicity, the fluids' coupling -often called "mutual friction"is a simplified version of the coupling of the HVBK model [START_REF]Quantized vortices in Helium II[END_REF]. With obvious notation, the governing equations of the two fluids are :

Dvs Dt = -1 ρs ∇p s + ν s ∇ 2 v s -ρn ρ B 2 |ω s |(v n -v s ) Dvn Dt = -1 ρn ∇p n + ν n ∇ 2 v n + ρs ρ B 2 |ω s |(v n -v s )
where the subscripts "n" and "s" stand for "normal fluid" and "superfluid", ω s is the superfluid vorticity, B = 2 and the velocity fields are solenoidal. This model allows us to take advantage of optimized parallelized codes in exploring the interaction between the two fluids. This paper is more focused on numerical aspects, the physical interpretation will be discussed elsewhere.

Numerical simulations refer to the integration of the previous dynamical equations in a cubic domain with periodic boundary conditions in the three directions. The simulations rely on a parallel (distributed-memory) pseudo-spectral solver [START_REF] Leveque | [END_REF]. Three grid sizes are considered, 128 3 , 256 3 and 512 3 respectively. The velocity Fourier modes are advanced in time by a second-order Adams-Bashforth scheme.

The ratio between the density of the normal fluid and the density of the superfluid is ρ n /ρ s = 0.1, 1 or 10 respectively for "low", "intermediate" and "high" temperatures. The ratio between the kinematic viscosities is fixed at ν n /ν s = 4, which is a compromise chosen to separate the dissipative cut-off wavenumbers of the two fluids and maximize the extension of the inertial range of the superfluid.

In order to ensure (statistical) stationarity, a constant-energy flux is supplied to the Fourier modes in the shell of wave-vectors 1.5 < |k| < 2.5. This external energy flux applies to the normal fluid in the high-temperature and intermediate-temperature cases, and to the superfluid in the low-temperature case. This forcing scheme permits a rapid relaxation to a steady state, which is attained when the total dissipation fluctuates around the imposed energy input flux.

The transient from the initial state is monitored by the space-averaged enstrophies < ω 2 n >, < ω 2 s > and < (ω sω n ) 2 >. The first three subplots of Fig. 1 show this transient.

Results

The fourth subplot of Fig. 1 shows that correlation between ω 2 s and ω 2 n is larger than 97% in steady state, indicating a strong locking of the two fluids. Fig. 2 illustrates this locking in physical space : the vorticity structuresslightly more intense in the superfluid -are indeed overlapping. A similar conclusion was reached recently in a numerical simulation performed at high temperature without coupling from the superfluid to the normal fluid [3].

Fig. 3 shows velocity power density spectra under various conditions; for both fluids there is clear evidence of an inertial range compatible with a -5/3 Kolmogorov-like scaling. This figure also shows the power spectra of the slip velocity v sv n . We find that this quantity peaks at small scales, where ν n efficiently damps the kinematic energy of the normal fluid. The spectra in the inset are obtained at different temperatures but with fixed ν s , ν n and total injected energy. They show that the extend of the inertial range varies with temperature, showing that the Reynolds number -defined using the separation of large and small scales -is a temperature dependent parameter in our two-fluid system.

Conclusion

The locking between the superfluid and the normal fluid which we observe, as well as the Kolmogorov scaling, are consistent with the present theoretical understanding of quantum turbulence [4]. This work extends the numerical evidence over a wide temperature range, using a self-consistent coupling model between the two fluids. Two new features which we observe are the peaking of the slip velocity at small scales, and the temperature dependence of the Reynolds numbers for fixed energy injection and kinematic viscosities.

Fig. 1 .

 1 Fig. 1. Subplots 1,2 and 3 illustrate for different temperature the convergence to the steady state by monitoring the total enstrophy of the superfluid < ω 2 s > (top curve), normal fluid < ω 2 n > (intermediate curve) and the slip enstrophy < (ωsωn) 2 > (lower curve). These simulations were performed with grid resolution 512 3 . Subplot 4 shows the correlation between the superfluid and normal fluid enstrophy fields during the transient to the steady state. These simulations were performed with grid resolution 256 3 and 512 3 .

Fig. 2 .!!Fig. 3 .

 23 Fig. 2. Local enstrophy of the superfluid (left) and normal fluid (right) at high temperature (ρn/ρs = 10). Grid resolution is 256 3 . This visualisation is generated with "Vapor" freeware. (www.vapor.ucar.edu)

Acknowledgements : We thank L. Chevillard and J. Salort for useful discussions and support. CPU resources were provided by local computing facilities (PSMN at Ens-Lyon) and french supercomputing centers (CINES and CCRT-CEA). Supports from EPSRC (EP/D040892) and ANR TSF are acknowledged.


François Catzeflis

Christiane Denys
email: denys@mnhn.fr.

Laurent Granjon
email: laurent.granjon@ird.fr

Obituary

Christiane Denys* and Laurent Granjon* François Catzeflis https://doi.org/10.1515/mammalia-2022-0091

François Catzeflis left us in Montpellier on November 25, 2021 after a long battle against cancer. He was married and had three children and one grandchild. François was an Associate Editor of Mammalia since 1999, and in that position particularly responsible for manuscripts on South American mammals.

Born in Belgium on August 3, 1953 he was the first of four children of an agricultural engineer. François spent his first 10 years in Africa, where his father worked. After having moved to Switzerland, he obtained his scientific high school diploma in Sion in 1974.

He was introduced to ornithology during a holiday at the Zwin Reserve, Belgium. After high school, he joined an ornithological group and began to be concerned with the protection of nature and the environment.

François obtained his PhD on the systematics of European Soricidae under the supervision of Professor Peter Vogel from the University of Lausanne in 1984. A postdoctoral stage at Yale University and the Peabody Museum, New Haven, Connecticut, USA allowed him to learn the molecular technique of DNA/DNA hybridisation under the direction of Professor Charles G. Sibley. He came back to France in 1987 to join François Bonhomme's research team working on the evolutionary biology of mice at the Institut des Sciences de l'Evolution of Montpellier (ISEM), directed by Professor Louis Thaler. There he was encouraged by Professor Jean-Jacques Jaeger to create a research group on the molecular phylogeny of rodents, and was finally recruited by CNRS in 1988. By that time, he polished the techniques and methods of DNA/DNA hybridisation and published many key papers on African rodent phylogeny based on this technique. He was also at the outset, or involved in, many other issues dealing with mammalian, and especially rodent, evolution based on a variety of molecular markers. However, the laboratory work did not fully satisfy François, and when in 1994 the opportunity came up to participate in fieldwork in French Guiana he seized it and fell in love with the tropical forest and its magnificent biodiversity. Since that time, he managed to go into the field for at least one month per year until 2017, in French Guiana but also in Surinam and Venezuela. On these occasions, he devoted all his time to fieldwork and declared himself "unavailable" to Mammalia. In this context, he collected and studied many rodents, marsupials (especially opposums) and bats from the Amazonian Forest and published many scientific articles as well as a book entitled Guide des marsupiaux et rongeurs de Guyane in collaboration with the Institut Pasteur de Guyane (2014). He also conducted a survey on the "Manicou" (Didelphis marsupialis) in Martinique, with regular visits to the island between 2005 and 2015.

In parallel with all his activities, François Catzeflis developed one of the first banks of mammal tissues in ethanol. He was always keen to keep the link between the tissues and the voucher specimens, and deposited officially in the MNHN Zoologie Mammifères & Oiseaux collection in Paris many specimens of mammals from French Guiana and other parts of South America.

As early as in 2000, he became an environmental activist of Greenpeace and as such was always motivated to participate in forest and ocean protection and defence as well as anti-GMO campaigns. In French Guiana he stood up against illegal gold washing, and locally in his village near Montpellier he participated in citizen actions for the environment and organised ornithological excursions.

François Catzeflis has been a pioneer in molecular systematic studies in Europe and was a great naturalist. He contributed to the improvement of knowledge of the evolutionary biology of rodents and mammals in general as well as biodiversity of South American forests. His contributions in these fields will stay permanently as significant ones. As an editor of Mammalia, he was a very dedicated, constant and friendly collaborator.

François was a passionate researcher and nature lover. The mind-opening discussions we have had with him on topics as varied as molecular versus morphology conflicts in rodent phylogeny, the use of genetic information in integrative taxonomy, the ecology of South American forest small mammals, collection management, African deforestation or on how to get involved to guarantee a better future for the younger generation were always instructive and exciting. We will remember François for a long time.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Conflict of interest statement: The authors declare no conflicts of interest regarding this article.


Fernando Vilela

Mélanie Chenon

Christophe Velours

Jessica Andreani

Paola Llinas
email: paola.llinas@i2bc.paris-saclay.fr

Julie Ménétrey
email: julie.menetrey@i2bc.paris-saclay.fr

Insights into the activation of Kinesin1 from the molecular characterisation of JIP3/4 binding to Kif5b

Keywords: JIP3, JIP4, RH1 domain, Kif5, Kinesin1

Whereas our understanding of kinesin auto-inhibition mechanisms is improving faster, important insights into kinesin activation mechanisms such as those controlled by cargo-motor adaptors are still missing. JIP3 and JIP4 are versatile motor-cargo adaptors for kinesin1 and dynein-dynactin motors enabling bi-directional transport on microtubules. JIP3 activates kinesin1 heavy chains, independently of kinesin1 light chains. In this report, we characterize the molecular details of the binding of the kinesin1 heavy chain, Kif5b to the motor-cargo adaptors, JIP3 and JIP4, using biophysical approaches. The definition of the exact binding site of Kif5b, as well as the specificity of interaction between JIP3 and JIP4 provide new insights into kinesin1 activation.

INTRODUCTION

Kinesin1 is a molecular motor that transport cargos, such as vesicles, organelles, mRNA and protein assemblies along microtubules (MTs) throughout the cell. When not transporting cargo, kinesin1 is maintained in an autoinhibited state, which prevents futile ATP consumption and congestion on MTs.

Activation of kinesin1 is controlled by the binding of both MT-associated proteins and motor-cargo adaptors [START_REF] Cross | Motor-cargo adaptors at the organelle-cytoskeleton interface[END_REF][START_REF] Hooikaas | MAP7 family proteins regulate kinesin-1 recruitment and activation[END_REF]. Recruitment of a single [START_REF] Watt | JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation[END_REF] or a coordination of two motor-cargo adaptors [START_REF] Blasius | Two binding partners cooperate to activate the molecular motor Kinesin-1[END_REF] can trigger kinesin1 activation.

Although we know a lot about kinesin1 inhibition, we still lack important insights concerning the regulating mechanisms such as the structural basis of this motor activation by cargo-motor adaptors.

JIP3 and JIP4 (JNK-Interacting Protein 3 and 4) are close paralogs identified as MT-based motor-cargo adaptors [START_REF] Bowman | Kinesin-Dependent Axonal Transport Is Mediated by the Sunday Driver (SYD) Protein[END_REF][START_REF] Cavalli | Sunday Driver links axonal transport to damage signaling[END_REF]. JIP3 is mainly found in neurons and has emerged as a regulator of axonal transport [START_REF] Edwards | An organelle gatekeeper function for Caenorhabditis elegans UNC-16 (JIP3) at the axon initial segment[END_REF][START_REF] Rafiq | JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton[END_REF][START_REF] Sun | c-Jun NH2-terminal Kinase (JNK)-interacting Protein-3 (JIP3) Regulates Neuronal Axon Elongation in a Kinesin-and JNK-dependent Manner[END_REF][START_REF] Watt | JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation[END_REF], while JIP4 is ubiquitously expressed and involved in organelle positioning [START_REF] Boecker | Increased LRRK2 kinase activity alters neuronal autophagy by disrupting the axonal transport of autophagosomes[END_REF][START_REF] Willett | TFEB regulates lysosomal positioning by modulating TMEM55B expression and JIP4 recruitment to lysosomes[END_REF], endosomal membrane reshaping [START_REF] Marchesin | ARF6-JIP3/4 regulate endosomal tubules for MT1-MMP exocytosis in cancer invasion[END_REF] and recycling [START_REF] Montagnac | Decoupling of Activation and Effector Binding Underlies ARF6 Priming of Fast Endocytic Recycling[END_REF]. Such functions in intracellular transport are based on the ability of JIP3 and JIP4 (JIP3/4) to coordinate kinesin1 [START_REF] Bowman | Kinesin-Dependent Axonal Transport Is Mediated by the Sunday Driver (SYD) Protein[END_REF][START_REF] Nguyen | JLP Associates with Kinesin Light Chain 1 through a Novel Leucine Zipper-like Domain[END_REF][START_REF] Sun | Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility[END_REF] and dynactin/dynein motors [START_REF] Cavalli | Sunday Driver links axonal transport to damage signaling[END_REF][START_REF] Montagnac | ARF6 Interacts with JIP4 to Control a Motor Switch Mechanism Regulating Endosome Traffic in Cytokinesis[END_REF] motors enabling bi-directional transport on MTs. Furthermore, JIP3 activates kinesin1, enhancing its motility along MTs [START_REF] Sun | Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility[END_REF].

Kinesin1 is an heterotetramer composed of a homodimer of heavy chains (KHC, with the three paralogs Kif5a-c in human) associated to two light chains (KLC; four paralogs, KLC1-4). KHC consists of the N-terminal motor domain (MD) followed by a long stalk formed by several coiled-coils and the C-terminal Tail encompassing a coiled-coil region and an unstructured region named, the distal tail. Kinesin1 interacts with JIP3/4 through a dual mode of binding: the Tail region of KHC binds to the Nterminal region of JIP3/4 [START_REF] Sun | Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility[END_REF], while KLC binds to the middle part of JIP3/4 [START_REF] Nguyen | JLP Associates with Kinesin Light Chain 1 through a Novel Leucine Zipper-like Domain[END_REF]. Direct interaction of JIP3 with KHC, independently of KLC, is functional and enhances the motility of kinesin1 along MTs [START_REF] Sun | Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility[END_REF][START_REF] Watt | JIP3 Activates Kinesin-1 Motility to Promote Axon Elongation[END_REF]. The motility of KHC is regulated enabling the interaction of a short motif within the distal tail, called the Isoleucine-Alanine-Lysine (IAK) motif, with the MD. This interaction prevents structural rearrangements required for MT-binding and the ATPase cycle [START_REF] Cai | Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells[END_REF][START_REF] Dietrich | The kinesin-1 motor protein is regulated by a direct interaction of its head and tail[END_REF][START_REF] Hackney | Kinesin's IAK tail domain inhibits initial microtubulestimulated ADP release[END_REF][START_REF] Kaan | The Structure of the Kinesin-1 Motor-Tail Complex Reveals the Mechanism of Autoinhibition[END_REF][START_REF] Seiler | Cargo binding and regulatory sites in the tail of fungal conventional kinesin[END_REF]. KLC is also involved in the autoinhibition of kinesin1 [START_REF] Vale | Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain[END_REF][START_REF] Verhey | Light Chain-dependent Regulation of Kinesin's Interaction with Microtubules[END_REF], a recent study reported that KLC inhibits kinesin1-MT interaction independently from the proposed intramolecular interaction within KHC [START_REF] Chiba | Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity[END_REF]. Whereas the binding of JIP3 to KLC2 has been characterized structurally [START_REF] Cockburn | Insights into Kinesin-1 Activation from the Crystal Structure of KLC2 Bound to JIP3[END_REF], such molecular details are not available for the KHC:JIP3/4 interaction. These data are required to understand how this interaction enables kinesin1 activation Here, we present the molecular characterization of JIP3 and JIP4 binding to Kif5b using purified proteins. Our biophysical experiments define the minimal binding site of Kif5b and JIP3/4 for the interaction and provide critical information on the specificity of Kif5b for JIP3 and JIP4. Altogether, our data provide new insights into KHC activation by JIP3.

RESULTS AND DISCUSSION

Biophysical characterization of Kif5b-Tail and JIP3/4-Nter fragments

In order to perform biophysical studies on JIP3/4 binding to Kif5b and define the minimal binding sites of both proteins, we designed various fragments of the Tail region of Kif5b (Kif5b-Tail) and of the N-terminal part of JIP3 and JIP4 (JIP3/4-Nter), and validated their structural integrity using Circular Dichroism (CD), Analytical UltraCentrifugation (AUC) and/or Multi-Angle Light Scattering (MALS),

The Tail region of Kif5b consists of the last part of the cc2 coiled-coil (namely cc2b) encompassing the KLC-binding site, followed by the cc3 coiled-coil and the 50-aa unstructured distal tail (dt). We conceived three fragments of the Tail region of human Kif5b (Fig. 1A and Table S1): (i) the cc2b coiledcoil followed by the cc3 coiled-coil and the distal tail (cc2b-cc3-dt fragment), (ii) a truncated form lacking the distal tail (cc2b-cc3 fragment) and (iii) the cc3 coiled-coil alone (cc3 fragment). CD experiments showed that the cc2b-cc3 and cc3 fragments are mainly alpha-helical, while for the cc2b-cc3-dt fragment, the turn/random content increases significantly, consistent with the presence of the unstructured distal tail (see details in Supplementary information). Furthermore, AUC experiments indicate that the three fragments are mainly dimers in solution (data not shown). The N-terminal part of JIP3/4 encompasses an RH1 (RILP-Homology 1) domain which assembles as a dimer of two antiparallel helices forming a four-helix bundle. The RH1 domain is flanked by a short unstructured region in its N-terminus (N) and followed by a long Leucine Zipper coiled-coil region (LZI) (Fig. 1C). For both JIP3 and JIP4, we used a previously characterized fragment that encompasses the RH1 domain followed by the entire LZI coiled-coil (RH1-LZI fragment) [START_REF] Vilela | Structural characterization of the RH1-LZI tandem of JIP3/4 highlights RH1 domains as a cytoskeletal motor-binding motif[END_REF], as well as three new fragments (Figure 1C and Table S2). The first one contains the N-terminus region (N-RH1-LZI fragment) and the two others were truncated after the third heptad repeat of LZI coiled-coil (RH1-lz3 and N-RH1-lz3 fragments). MALS experiments demonstrated that the presence of the N-terminus, as well as the truncation of the last eleven heptads of the LZI have no impact on the dimeric form of JIP3 and JIP4

(see details in Supplementary information). Previously, we showed that the RH1 domain is not stable in the absence of the LZI coiled-coil [START_REF] Vilela | Structural characterization of the RH1-LZI tandem of JIP3/4 highlights RH1 domains as a cytoskeletal motor-binding motif[END_REF]. Our new results indicate that the first three heptad repeats of LZI are sufficient to stabilise the dimeric form of JIP3 and JIP4. Collectively, these results showed that all Kif5b-Tail fragments and JIP3/4-Nter fragments used here, exhibit the expected structural integrity required for binding assays.

The cc3 coiled-coil of Kif5b is the binding site for JIP3

To characterize the mode of binding of JIP3 to Kif5b, we used the different fragments of both proteins to analyse their interaction in solution by Microscale Thermophoresis (MST). First, we confirmed that both proteins interact together using the longest Kif5b-Tail and JIP3-Nter fragments. The Kif5b-cc2b-cc3-dt fragment binds to the JIP3-N-RH1-LZI fragment with a Kd of 18.2 ± 2.9 nM (Fig. 2A, Table 1). When the N-terminus of JIP3 is removed (RH1-LZI fragment), the Kd is 3.7-fold higher than that of the N-RH1-LZI fragment (Fig. 2A and Table 1) indicating a role, although minor, for this unstructured region in the interaction. However, whether the N-terminus is directly in contact with Kif5b or is involved in the stabilisation of the RH1 domain of JIP3/4 remains to be determined. When the LZI is truncated after the third heptad repeat (N-RH1-lz3 fragment), the Kd is the same as that of the N-RH1-LZI fragment (Fig. 2A and Table 1) revealing no role of the full LZI in the interaction. Therefore, our data identify the RH1-lz3 region of JIP3 as the main binding site for Kif5b. This is consistent with previous data defining a minimal Kif5-binding site to residues 50 to 80, which overlaps to the RH1 domain [START_REF] Sun | Sunday Driver/JIP3 binds kinesin heavy chain directly and enhances its motility[END_REF].

On the other hand, to define the minimal region of Kif5b required for JIP3 binding, the interaction between the N-RH1-lz3 fragment of JIP3 and truncated Kif5b-Tail fragments were analysed and compared. When the distal tail is removed, Kif5b shows a 4.5-fold decrease in binding affinity (cc2b-cc3-dt vs cc2b-cc3; Fig. 2B andTable 1). However, no significant interaction was detected with the distal tail alone at the tested concentrations (in absence of the cc2b-cc3 part; Fig. 2B andTable 1). These data indicate that the distal tail might play a minor role in JIP3 binding, possibly in stabilising the cc3 coiledcoil or the JIP3-Kif5b assembly. When the cc2b part is removed, Kif5b exhibits similar binding affinity for JIP3 (cc2b-cc3 vs cc3; Fig. 2b andTable 1), showing that the cc2b region is not involved in JIP3 binding. Altogether, this interaction analysis defines the cc3 region of Kif5b as the main binding site for JIP3. Thus, these results indicate that the RH1-lz3 domain of JIP3/4 makes direct interaction with the cc3 region of Kif5b and that two unstructured regions from JIP3 (N-terminus) and Kif5b (distal tail) have minor impacts on this interaction, possibly in stabilizing the proteins alone or their complex.

Finally, the physicochemical forces responsible for the interaction were assessed by analysing the binding between the JIP3-N-RH1-lz3 and the Kif5b-cc2b-cc3-dt fragments at three different concentrations of NaCl. No significant difference is observed for the binding affinity between 200 mM and 500mM NaCl, but a 10-fold difference is observed when saline concentration is reduced to 75 mM NaCl (Fig. 2C and2D and Table 1). These results show that the interaction between JIP3-Nter and cc3 has a major hydrophobic component, as it is maintained at 500 mM NaCl. The increase in the affinity at lower ionic strength indicates the formation of electrostatic interactions also, consistent with the electrostatic properties of both JIP3-N-RH1-lz3 (pI 4.8) and the Kif5b-cc2b-cc3-dt (pI 10.2) fragments.

To summarize, this study showed that JIP3 bind to the cc3 coiled-coil of Kif5b. The cc3 region of Kif5 is a well-known binding site for cargos or adaptors, especially those using coiled-coil regions for the interaction, like the SNARE proteins SNAP25 and SNAP23, the ribosome receptor p180, the Glutamate-receptor-interacting protein GRIP1, the Milton/TRAK adaptor and the tropomyosin [START_REF] Diefenbach | The Heavy Chain of Conventional Kinesin Interacts with the SNARE Proteins SNAP25 and SNAP23[END_REF][START_REF] Diefenbach | The ribosome receptor, p180, interacts with kinesin heavy chain, KIF5B[END_REF][START_REF] Dimitrova-Paternoga | Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex[END_REF][START_REF] Glater | Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent[END_REF][START_REF] Setou | Glutamate-receptor-interacting protein GRIP1 directly steers kinesin to dendrites[END_REF].

Kif5b exhibits higher affinity for JIP3 than JIP4

Because JIP3 and JIP4 have some sequence differences in the Kif5-binding site (Fig. 3A), we wondered if Kif5b may exhibit binding specificity between the two paralogs. MST binding assays showed that Kif5b-cc2b-cc3-dt fragment binds to JIP4-N-RH1-lz3 fragment with a Kd of 760 ± 160 nM, which indicates a 40-fold lower affinity compared to the equivalent JIP3 fragment (Fig. 3B and Table S2). This result points to a higher specificity of Kif5b for JIP3 compared to JIP4. However, it should be noted that the Kd of the JIP4:KHC interaction is similar to what was measured for motor light chains (KLC [START_REF] Cockburn | Insights into Kinesin-1 Activation from the Crystal Structure of KLC2 Bound to JIP3[END_REF] or DLIC [START_REF] Celestino | JIP3 interacts with dynein and kinesin-1 to regulate bidirectional organelle transport[END_REF]) bound to JIP3/4. Consistently, functional studies showed that JIP4 is recruited by kinesin1 heavy chain and is involved in kinesin-based transport process [START_REF] Sato | JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration[END_REF][START_REF] Suzuki | Functional role of c-Jun NH2-terminal kinase-associated leucine zipper protein (JLP) in lysosome localization and autophagy[END_REF]. Thus, we suggest that such a binding affinity difference between JIP3 and JIP4 could account in variations of kif5b regulation mechanisms, such as motor activation.

To analyse further the structural basis of this specificity, we investigated the impact of sequence differences between JIP3 and JIP4 on the binding affinity for Kif5b using truncated fragments and sitedirected mutants. Sequence differences are observed in the N-terminus, in the RH1 domain especially into the loop that connect the a1 and a2 helices (a1-a2 loop), the a2 helix, as well as in the hinge region between the RH1 domain and the LZI and at the beginning of the LZI (Fig. 3A.). First, the N-terminus region diverges with several small insertions in JIP3 compared to JIP4 (Fig. 3A). The JIP4-RH1-lz3 fragment which lacks the N-terminus binds to the Kif5b-cc2b-cc3-dt fragment with a Kd of 705 ± 192 nM which is equivalent to that measured for the JIP4-N-RH1-lz3 fragment (Fig. 3B and Table 1), indicating that the N-terminus of JIP4 has no impact on Kif5b binding. This result diverges from that of JIP3 for which the truncation of the N-terminus leads to a 3.7-fold binding difference (Fig. 2A and Table 1). Thus, the sequence differences between JIP3 and JIP4 at the N-terminus contribute, albeit in a minor way, to the binding specificity of Kif5b for JIP3/4. Then, site-directed mutagenesis was performed on the JIP3-N-RH1-lz3 fragment in order to change for JIP4 sequence (Fig. 3A): (1) His45-Cys46 were mutated to Gly-Arg (H45G-C46R), (2) Asn61 to Ala (N61A), (3) Leu-Ser-Glu-Asn (70-73) to Phe-Ala-Gln-Asp (L70F-S71A-E72Q-N73D) and (4) Glu77 to Gln (E77Q). MST experiments were performed to determine the binding affinity of these four JIP3-N-RH1-lz3 mutants for Kif5b-cc2b-cc3-dt (Fig. 3C and Table 1). Data showed that there is no difference in affinity between the wild-type JIP3-N-RH1-lz3 fragment and the N61A mutant. A recent report showed that the V60Q mutation of JIP3 does not prevent the interaction with the mouse Kif5c [START_REF] Celestino | JIP3 interacts with dynein and kinesin-1 to regulate bidirectional organelle transport[END_REF]. This confirms that the region 60-61 of JIP3 is not involved in Kif5 binding. Then, only a 2-to 3-fold difference in affinity is observed for the E77Q and L70F-S71A-E72Q-N73D mutants, and a 9-fold difference for the H45G-C46R mutant. However, whether these residues are in direct contact with Kif5b or are involved in the stabilisation of JIP3/4 structure remains to be determined. Thus, our data show that sequence differences in the a1-a2 loop are the main contributions for the higher affinity of Kif5b for JIP3, while those in the N-terminus and the a2 helix contribute only marginally.

Does JIP3 binding to Kif5b induce structural rearrangements?

In order to detail the structural basis of JIP3 recruitment by Kif5b, we tried to predict a model of the Kif5b:JIP3 complex using AlphaFold2 (Evans et al., 2022;Jumper et al., 2021;Mirdita et al., 2022).

However, despite varying the boundaries of the Kif5b and JIP3 sequences, the stoichiometry of the complex, the multiple sequence alignments used for prediction and the AlphaFold parameters (monomer or multimer version), we did not converge on a reliable model. The difficulties to predict a structural model for Kif5b:JIP3 complex could be explained by the coiled-coil regions involved in the interaction.

Coiled-coils are versatile structural motifs [START_REF] Truebestein | Coiled-coils: The long and short of it[END_REF]) that can change their multimeric arrangement upon partner binding, as shown for instance by the recent structural study of the actin binding protein tropomyosin 1 (aTm1) bound to the cc3 region of Khc [START_REF] Dimitrova-Paternoga | Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex[END_REF]. The Khc-binding region of aTm1 consists of a dimeric coiled-coil that dissociates in presence of Khc-cc3 and a single helix associates with the Khc-cc3 dimeric coiled-coil to form a tripartite coiled-coil complex [START_REF] Dimitrova-Paternoga | Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex[END_REF]. Another type of structural rearrangement could be a change in the register of the coiled-coils, as was observed in the dynein stalk domain and the dynein activator BicD2 [START_REF] Carter | Structure and Functional Role of Dynein9s Microtubule-Binding Domain[END_REF][START_REF] Liu | Bicaudal-D uses a parallel, homodimeric coiled coil with heterotypic registry to coordinate recruitment of cargos to dynein[END_REF]. Further work will be necessary to decipher the structural basis of the binding mode of Kif5b to JIP3/4.

Insights into the activation of KHC by JIP3

The characterization of JIP3 binding to Kif5b presented in this study provides new insights into KHC activation by JIP3, independently of KLC binding. Our data have shown that JIP3 is recruited by the cc3 part of KHC, with no direct interaction with the distal tail. Therefore, the auto-inhibitory IAK motif of Kif5b, embedded in the distal tail, does not contribute to the interaction with JIP3. Thus, our results exclude a model in which JIP3 would release the MD:Tail interaction by competing with the MD for the IAK motif binding, and rather favour an allosteric model. One possibility would be a proximity effect between the JIP3:KHC-cc3 and the MD:IAK assemblies triggering steric hindrance. Another, non-exclusive, allosteric model for KHC activation might be due to structural rearrangements in the cc3 part of KHC upon JIP3 binding that will affect the distal tail region, leading to IAK motif dissociation from the MD. Further work will be required to characterize the allosteric mechanism of KHC activation

MATERIALS AND METHODS

Microscale Thermophoresis -JIP3-Nter fragments were labelled using the Protein Labelling Kit Blue-NHS (NanoTemper Technologies). The labelling reaction was performed according to the manufacturer's instructions in the supplied labelling buffer applying a concentration of 20 μM protein (molar dye:protein ratio ≈ 3:1) at room temperature for 30 min. The labelled protein fragments were stored in MST buffer (20 mM Hepes pH 7, 150 mM NaCl). For the binding assays, the labelled JIP3-Nter fragments were adjusted to 20 nM with an assay buffer containing 50 mM Tris pH 8.0, 200 mM NaCl, 5 mM MgCl2, 0.05 % Tween 20. Kif5b fragments were buffer exchanged to the assay buffer and a series of 16 1:1 dilution was prepared. For the measurement, each Kif5b fragment dilution was mixed with one volume of labelled JIP3-Nter fragments. The samples were loaded into Monolith NT.115 Premium Capillaries MO-K025 (NanoTemper Technologies). MST was measured using a Monolith NT.115 instrument (NanoTemper Technologies) at 25°C. Instrument parameters were adjusted to 40 % LED power and low MST power. Data of three independent replicates were analysed using the signal from an MST-on time at 1.5-2.5 s (MO.Affinity Analysis software version 2.3, NanoTemper Technologies). At 75 mM NaCl, there is a fluorescence signal variation due to the interaction so the data must be analyzed using this signal and not the thermophoresis. JIP3-N-RH1-lz3 is negatively charged and KIF5b-cc2-cc3-dt is positively charged, the diminution of NaCl concentration may allow additional interactions in a region near to the fluorophore, that should be masked at higher ionic strength. Error bars indicate the standard deviation of three replicates. (Evans et al., 2022;Jumper et al., 2021;Mirdita et al., 2022). Sequence differences between JIP3 and JIP4 are shown in "red" and correspond to the mutations studied in this work (B and C). The region of JIP3-Nter corresponding to residues 50-80 is indicated by a bold black square and the hinge region between the RH1 domain and the LZI region by a dash-line black square. (B) Superposition of the dose-response curves from the titration of labelled JIP3-N-RH1-lz3 (black), JIP4-N-RH1-lz3 (brown dots) and JIP4-RH1-lz3 (brown) with Kif5b-cc2-cc3-dt. The concentration of the labelled JIP3 and JIP4 fragments was kept constant at 20 nM while the concentration of KIF5b-cc2-cc3-dt was varied between 1.2.10 -4 to 16 uM. (C) Superposition of the dose-response curves from the titration of labelled JIP3-N-RH1-lz3 and the JIP3 to JIP4 mutants with Kif5b-cc2-cc3-dt.

Table 1. MST interaction experiments between various

  mM Tris pH 8, 10 mM MgCl2, 0.05% tween 20 and NaCl (concentration indicated in the table). * Same measurement. #Kd measured from initial fluorescence dose-response curves.

Figure 1 .

 1 Figure 1. Structural integrity of Kif5b-Tail and JIP3/4-Nter fragments. (A) Schema of the full length Kif5b. MD: Motor Domain; NC: Neck Coil; cc: coiled-coil; dt: distal tail. Sequence limits of human Kif5b are indicated at the beginning of the known domains. The dash lined-box (bottom) shows the fragments of the C-terminal part of Kif5b used in this study. (B) Model of Kif5b-cc2b-cc3-dt using the AlphaFold2-based pipeline ColabFold with the AlphaFold-Multimer version (Evans et al., 2022; Jumper et al., 2021; Mirdita et al., 2022). (C) Schema of the full length JIP3/4 proteins. The JIP3/4 partners are indicated in red. The dash lined-box (bottom) shows the fragments of the N-terminal part of JIP3/4 used in this study.

Figure 2 .

 2 Figure 2. MST characterization of the between Kif5b-Tail and JIP3-Nter. (A) Superposition of the dose-response curves from the titration of JIP3-N-RH1-LZ1 (grey line), JIP3-RH1-LZ1 (grey dashes) and JIP3-N-RH1-lz3 (black) with KIF5b-cc2-cc3-dt. The concentration of the labelled JIP3-Nter fragments was kept constant at 20 nM, while the concentration of the KIF5b-cc2-cc3-dt was varied between to 1.2.10 -4 to 4 uM. (B) Superposition of the dose-response curves from the titration of labelled JIP3-N-RH1-lz3 with Kif5b-Tail fragments: cc2b-cc3-dt (black), cc2b-cc3 (cyan), cc3 (light blue) and Trx-dt (dark blue). The concentration of the labelled JIP3-N-RH1-lz3 was kept constant at 20 nM while the concentration of the tail fragments was varied from 5.2.10 -4 to 17 uM (kif5b-cc2b-cc3), 3.2.10 -4 to 10.6 uM (Kif5b-cc3) and 1.2.10 -4 to 4 uM (Kif5b-cc2b-cc3-dt). (C) Superposition of the dose-response curves from the titration of JIP3-N-RH1-lz3 with KIF5b-cc2-cc3-dt at different ionic strength: 200 mM NaCl (black) and 500 mM NaCl (light orange). (D) Initial fluorescence doseresponse curve from the titration of JIP3-N-RH1-lz3 with KIF5b-cc2-cc3-dt at 75 mM NaCl (yellow).At 75 mM NaCl, there is a fluorescence signal variation due to the interaction so the data must be analyzed using this signal and not the thermophoresis. JIP3-N-RH1-lz3 is negatively charged and KIF5b-cc2-cc3-dt is positively charged, the diminution of NaCl concentration may allow additional interactions in a region near to the fluorophore, that should be masked at higher ionic strength. Error bars indicate the standard deviation of three replicates.

Figure 3 .

 3 Figure 3. Differences in the interaction the N-terminal region of JIP3 and JIP4 with the tail of Kif5b analyzed by microscale thermophoresis (MST). (A) Sequence comparison of the N-terminal region of JIP3 and JIP4 with a schematic representation of the folding of this region. (Inset) Model of JIP3-N-RH1-lz3 fragment using the AlphaFold2-based pipeline ColabFold with the AlphaFold-Multimer version(Evans et al., 2022; Jumper et al., 2021; Mirdita et al., 2022). Sequence differences between JIP3 and JIP4 are shown in "red" and correspond to the mutations studied in this work (B and C). The region of JIP3-Nter corresponding to residues 50-80 is indicated by a bold black square and the hinge region between the RH1 domain and the LZI region by a dash-line black square. (B) Superposition of the dose-response curves from the titration of labelled JIP3-N-RH1-lz3 (black), JIP4-N-RH1-lz3 (brown dots) and JIP4-RH1-lz3 (brown) with Kif5b-cc2-cc3-dt. The concentration of the labelled JIP3 and JIP4 fragments was kept constant at 20 nM while the concentration of KIF5b-cc2-cc3-dt was varied between 1.2.10 -4 to 16 uM. (C) Superposition of the dose-response curves from the titration of labelled JIP3-N-RH1-lz3 and the JIP3 to JIP4 mutants with Kif5b-cc2-cc3-dt.

Acknowledgments

We thank Drs. Philippe Chavrier, Kristen Verhey and Reza Khayat for the gift of the JIP3, JIP4 and Kif5b plasmids, respectively. This work has benefited from the expertise of Magali Aumont-Nicaise and the Macromolecular interaction measurements Platform of I2BC supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05. JA is supported by the Agence Nationale de la Recherche through grant ESPRINet (ANR-18-CE45-0005-01). We are grateful to Drs. Davy Martin and Human Rezaei (INRA, VIM-UR0892, Jouy-en-Josas) for access to their CD equipment. Advice given by Pierre Soule (NanoTemper Technologies GmbH) have been a great help for MST experiments. We thank Dr. Benoît Gigant for useful discussions, as well as for careful reading and comments on the manuscript.

Competing financial interests:

The authors declare no competing interests.

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