Guillaume Lacombe
email: g.lacombe@cgiar.org

Alain Pierret

Impact of armed conflict on broad-scale hydrology in the Mekong Basin -Investigations in data-scarce conditions

Keywords: hydrological change, deforestation, data scarcity, causal link, Vietnam War, Mekong

The Vietnam War played a decisive role in the pre-1990s deforestation of the lower Mekong Basin, which in turn, likely influenced regional broad-scale hydrology. This note presents and discusses new analyses that strengthen this thesis. While concurrent overestimation of discharge and underestimation of rainfall, a couple of years after bombing climaxed in the early 1970s, could theoretically explain the sharp rise in water yield previously attributed to bomb-induced deforestation, new observations suggest that bombing has durably modified the landscape: by 2002, degraded forests still largely overlapped with areas heavily bombed 30 years earlier. This corroborates observed long-term hydrological changes and suggests that warfare-induced deforestation has more profound and durable hydrological effects than previously thought.

1-Introduction

It is known that, in small catchments, deforestation decreases evapotranspiration, hence increases annual basin water yield [START_REF] Brown | A review of paired catchment studies for determining changes in water yield resulting from alterations in vegetation[END_REF]. The paucity of significant results at larger scale stems from factors like land cover heterogeneity, climate variability, or counteracting changes in vegetation covers [START_REF] Bruijnzeel | Hydrological functions of tropical forests: not seeing the soil for the trees[END_REF]. Hydrological impacts of deforestation have rarely been detected in basins exceeding 10,000 km 2 , e.g. [START_REF] Mahé | The impact of land use change on soil water holding capacity and river flow modelling in the Nakambe River, Burkina-Faso[END_REF]; [START_REF] Descroix | Spatio-temporal variability of hydrological regimes around the boundaries between Sahelian and Sudanian areas of West Africa: A synthesis[END_REF]. In a 50,300 km 2 catchment of the Mekong Basin, runoff sharply rose after Vietnam War bombing climaxed in the early 1970s. Flows remained at higher than pre-war levels until the early 2000s. [START_REF] Lacombe | Conflict, migration and land-cover changes in Indochina: a hydrological assessment[END_REF] attributed these changes to bomb-induced deforestation followed by the regrowth of shallow-rooted sparse vegetation on degraded soil. While data scarcity prevented firm confirmation of this hypothesis, we present new analyses strengthening causalities.

2-Water level-discharge rating curves

Runoff in the area studied by [START_REF] Lacombe | Conflict, migration and land-cover changes in Indochina: a hydrological assessment[END_REF] was estimated by subtracting upstream discharge at Mukdahan (Mekong River) and Ubon Ratchathani (Mun River) from the Mekong discharge at Pakse (Fig. 1). At each gauging station, continuously recorded daily water levels were converted to discharge using rating curves updated annually. Since discharge records at Pakse are incomplete, water-level-specific discharges available for this station were averaged to generate the rating curves missing for the 1974-75 period, during which runoff peaked, following bombing climax. To verify whether this approach could overestimate discharge, we designed dummy rating curves producing annual runoff equivalent to that simulated by the GR2M model in 1974 and 1975, assuming pre-war catchment conditions [START_REF] Lacombe | Conflict, migration and land-cover changes in Indochina: a hydrological assessment[END_REF]). An order-2 polynomial accurately represents the water level -discharge relationship (R 2 =1.00 for all measured rating curves). While the 1974 dummy rating curve falls about 2 standard deviations below the averaged rating curve, half of the error bars of both curves overlap and mean discharge values of this hypothetical rating curve are slightly higher than the lowest discharge on record (Fig. 2). Therefore, when using the averaged rating curve, we cannot dismiss the possibility that an unrecorded change in the Mekong River cross section in 1974 could have led to an artificial runoff peak. In contrast, the 1975 dummy rating curve falls about 5.4 standard deviations below the averaged curve, without overlap of error bars between curves, and includes discharge values far lower than the lowest values on record (Fig. 2). Since the 1975 dummy rating curve yields unrealistically low discharge, the runoff peak observed in 1975 is arguably not an artefact although its exact value cannot be ascertained. However, the sharp runoff peak recorded the same year in the Mukdahan-Kong Chiam sub catchment (Fig. 2 in [START_REF] Lacombe | Conflict, migration and land-cover changes in Indochina: a hydrological assessment[END_REF], an area more than 1.5 times more intensively bombed than the overall study area, concurs to confirm the causal link between hydrological change and a war-induced ecocide of unprecedented scale [START_REF] Pralle | CHECO Southeast Asia report-Arc Light June 1967[END_REF][START_REF] Seig | Project CHECO Southeast Asia report-impact of geography on air operations in SEA[END_REF] in this 50,300 km 2 catchment.

3-Rainfall spatial variability

Because of data scarcity, Lacombe et al. ( 2010) used a 3-station average (Mukdahan, Ubon Ratchathani and Pakse, cf. Fig. 1) to investigate temporal changes in the rainfall-runoff relationship. It can be argued that extreme rainfall in the un-gauged eastern mountainous and heavily bombed side of the study area could have contributed to the extreme runoff peak of 1975. To investigate this, we split the study area into a western part, covering about 60% of the catchment's overall surface area and including 8 rain gauges operated since 1966 (Fig. 1), and the remaining eastern part, where 3 rain gauges started being operated in 1985. Using the uninterrupted rainfall records of the western part and a range of mean annual runoff coefficients, we estimated annual rainfall depths in the eastern part yielding the 1975 runoff peak observed by [START_REF] Lacombe | Conflict, migration and land-cover changes in Indochina: a hydrological assessment[END_REF]. The same procedure was performed using a rating curve at Pakse yielding 5% lower Mekong River discharge, similar to the 1974 hypothetical rating curve presented in section 2, and inducing a 20% reduction of runoff over the study area (Fig. 3a). The Gumbel distribution model adjusted to observations from the 3 rain gauges in the eastern part between 1985 and 2003 was used to estimate the return period of the annual rainfall depths (Fig. 3b). This analysis shows that, over the eastern part, rainfall depths corresponding to reasonable return periods (i.e. < 200 years) imply dubious runoff coefficients of 70%. Further, assuming pre-war mean runoff coefficients of 45%, rainfall alone could not explain the 1975 runoff peak unless we consider that it was caused by unrecorded event(s) with a >1,000 year return period. In contrast, rainfall depths corresponding to the rating curve yielding reduced discharge at Pakse have return periods of 6 to 160 years and imply runoff coefficients of 50-60%, reconcilable with runoff observed during wet, pre-war years in the study area (e.g. years 1961 and1962;Fig. 2, Lacombe et al., 2010). [START_REF] Lacombe | Conflict, migration and land-cover changes in Indochina: a hydrological assessment[END_REF] assumed a relationship between tonnages of explosive ordnance and deforested area, based on published bomb damage assessment (BDA) (Seig,1970). Yet, without ground truth assessment, it is debatable to what extent BDA can be used to infer changes in catchment hydrology. SPOT satellite images from 2001 and 2002 (scales 1:50,000 and 1:100,000) combined with field verifications were used by the Ministry of Agriculture and Forestry of Laos [START_REF] Maf | Report on the assessment of forest cover and land use between 1992 and[END_REF] to produce a countrywide land-cover map. We merged the forest, dipterocarp and coniferous); "Degraded forest" (sparsely forested areas with very low crown density, bamboo, savannah, scrub and barren land often exhibiting a high density of bomb craters in the study area); and "Agricultural land" (rice paddy, agricultural plantations and other agricultural lands). Overlaying the distribution of locations that received > 3 tons.km -2 of explosive ordnance to this map reveals that, by 2002, the most heavily bombed areas of the studied catchment were classified as degraded forest (82%), agricultural land (12%) and forest (6%) and 70% of degraded forest corresponds to heavily bombed areas (Fig. 1). Pre-bombing land-cover maps confirm that the catchment was covered by rain forest before the war [START_REF] Seig | Project CHECO Southeast Asia report-impact of geography on air operations in SEA[END_REF], consistent with the fact that one of the main objectives of the bombing missions was to clear the forest to unmask enemies.

4-Land cover

5-Conclusions

An overestimation of discharge combined to an underestimation of rainfall, although unlikely, could theoretically explain the 1975 water yield peak attributed by [START_REF] Lacombe | Conflict, migration and land-cover changes in Indochina: a hydrological assessment[END_REF] to bomb-induced forest removal. But, the temporal co-occurrence of runoff peak in the most intensively bombed Mukdahan-Kong Chiam sub catchment together with the spatial co-occurrence of heavily bombed areas and degraded forest strengthen the hypothesis of a causal link between bombing, long-term soil and forest degradation and altered hydrology. , varying between 40% and 60% and used to compute runoff in the western side of the study area

Figure 1 Figure 2 Figure 3

 123 Figure 1 Hydro-meteorological stations, land-cover and heavily bombed area in the studied catchment

Acknowledgement

This study was funded by the CGIAR research program on water, land and ecosystems. The authors are grateful to IRD for institutional support. We thank the National Agriculture and Forestry Research Institute and the department of forestry of the Ministry of Agriculture and Forestry for the provision of the 2002 land cover map. We are grateful to the Mekong River Commission and the UXO-NRA/Mine Action Sector in Laos for the provision of data. We thank B Cappelaere, T Clayton, CT Hoanh, JL Maeght, R Moussa, A Noble and V Smakhtin for their useful suggestions and support.


Patrick Berche

A new giant bacterium reminiscent of eukaryotes

Keywords: giant bacteria, sulfide-oxidizing bacteria, Thiomargarita magnifica, Thiomargarita nabimiensis, Epulopiscium fishelsoni. 1. Introduction

A giant bacterium Thiomargarita magnifica has been described in the mangroves of Guadeloupe by a Franco-American team (Volland JM et al., 2022). Measuring 0.6 to 2 cm, the filament visible to the naked eye consists of a single cell, without a dividing septum, and a large central vacuole restricting the cytoplasm to a thin peripheral layer containing sulfur granules (sulfide-oxidizing bacteria) and membrane organelles sequestrating DNA, reminiscent of nuclei of eukaryotes. This polyploidy is also found in the other very rare giant bacteria described so far. It could be an example of transition or adaptation of eukaryotic mimicking bacteria under extreme conditions of survival.

The authors give a very detailed morphological and genomic description of this singular bacterium. By various imaging techniques, it appears that each filament consists of a single cell along most of its length, without any dividing septum, with a partial constriction of the apical pole end where buds (daughter cells) can separate from the filament. The bacterium has a large central vacuole along the filament that corresponds to 75% of the total volume of the bacterium. This vacuole contains high concentrations of nitrate, allowing prolonged survival in the absence of nitrate. The cytoplasm is thus restricted to a thin outer layer of 0.5 to 2 µm at the cell periphery. This reduction of the cytoplasm would minimize the dependence on chemical diffusion of nutrients since, unlike eukaryotes, bacteria do not have an active intracellular transport system. In the cytoplasm, numerous white vesicles of about 2 µm in diameter form refractive sulfur granules. In addition, the cytoplasm contains bubble-like membrane organelles containing nuclear material with DNA fibrils and ribosomes, like the cell nuclei of eukaryotes. These vacuole membranes contain ATP synthases producing ATP, as the mitochondria of eukaryotes. Virtually no bacteria or archaea are known to segregate their genetic material as the nucleus of eukaryotes with its nuclear membrane. The rare exceptions with membrane-enclosed nucleoids are reported in one species of Atribacteria and some species of Plancotomycetes that have a membrane compartment containing DNA(8,9) .

T. magnifica is a polyploid bacterium, like all giant bacteria previously described (10). It contains an average of 37 000 copies of the genome per mm of filaments. Its genome has been sequenced from five individual cells showing a nucleotide identity greater than 99.5%. The genome size is estimated at 11-12 Mb, compared to the average bacterial genome size of 4.2 Mb. The genome of T. magnifica contains more than 11788 genes (compared to 3935 genes on average in bacteria). The genome of this chemoautotrophic bacterium includes a large number of genes dedicated to sulfur oxidation and carbon fixation. Polyploidy is a means of decreasing selection pressure on genes, allowing intracellular gene duplications, reassortments of divergences leading to extreme intracellular genetic diversity in some giant sulfur-oxidizing bacteria (10).

Giant bacteria

Why means this gigantism ? Giant bacteria belong to a few highly specialized bacterial groups. They live under extreme conditions of pH, temperature or pressure, and often thrive in carbon and nutrient rich environments. For most bacteria, it is advantageous to be very small, because their metabolism depends on the diffusion of molecules across the cytoplasmic membrane, a process that is very fast and efficient only over short distances of a few micrometers or less. Longer distances greatly slow down the diffusion of molecules. Eukaryotic protozoa, which are 10 to 20 µm long and can sometimes reach 3 to 4 cm (11), have overcome these constraints by developing protein transporters and a system of organelles to organize subcellular functions. This could explain the strategy of giant bacteria which tends to increase their surface area relative to their volume, by restricting their cytoplasm. In the case of T. magnifica, we also observe compartmentalization, especially of DNA, as in eukaryotes. Many questions remain unanswered, notably about the metabolism and functions of the organelles of these giant bacteria. As the authors suggest, this could be solved by culturing this bacterium with a growth time of two weeks, similar to that of other species of Thiomargarita sp. (4).

Research on the biology of giant bacteria is another unexpected step towards understanding the evolution of biological complexity of living cells, leading to the emergence of eukaryotic cells. Do the innovations of life come from the adversity of hostile environments?

Une nouvelle bactérie géante réminiscente des eucaryotes Résumé

Une bactérie géante Thiomargarita magnifica a été décrite dans les mangroves de la Guadeloupe par une équipe franco-américaine (Volland JM et al., 2022). Mesurant 0,6 à 2 cm, le filament visible à l'oeil nu est constitué d'une seule cellule sans septum de division, et d'une vaste vacuole centrale restreignant le cytoplasme à une mince couche périphérique contenant des granules de soufre (bactérie oxydant les sulfures) et des organelles membranaires séquestrant l'ADN, ressemblant aux noyaux des eucaryotes. Cette polyploïdie est également retrouvée chez les autres très rares bactéries géantes décrites jusqu'ici. Il pourrait s'agir d'un exemple d'adaptation des bactéries mimant les eucaryotes dans des conditions extrêmes de survie.

Mots-clés : bactéries géantes, bactéries oxidant les sulfures, Thiomargarita magnifica, Thiomargarita nabimiensis, Epulopiscium fishelsoni.

Introduction

Depuis leur découverte par Antoni van Leeuwenhoek en 1674, on a longtemps cru que les bactéries ne mesuraient que quelques µm de longueur. Cela reste vrai dans la très grande majorité des cas. Mais, on a observé dans les dernières décennies des bactéries géantes, souvent confondues au départ avec des organismes procaryotes. Ces bactéries peuvent atteindre une taille de quelques dizaines à quelques centaines de µm. Elles ont été décrites dans au moins quatre phyla (1,2). Certaines géantes sont même visibles à l'oeil nu. En 1993, Esther Angert et al. de l'Indiana University a mis au jour une bactérie géante (Epulopiscium fishelsoni) dans le tractus intestinal d'un poisson-chirurgien de la mer Rouge (Acanthurus nigrofuscus). D'abord identifié comme un protiste eucaryote, il s'agissait en réalité d'une bactérie sulfureuse à Gram positif mesurant 200-700 µm de longueur (3). En 1997, Heide Schultz et al. du Max Planck Institute for Marine Microbiology, a découvert à Walvis Bay en Namibie dans les sédiments marins très inhabituels, sans oxygène, très riche de sulfures et de matières organiques formées de diatomées morts. Cette bactérie mesure 100 à 300 µm de diamètre en moyenne, mais peut atteindre 750 µm. A l'oeil nu, ces bactéries cocoïdes ont l'aspect de perles blanches. Il s'agit d'une bactérie sulfureuse nommée Thiomargarita nabimiensis, capable d'oxyder l'hydrogène sulfuré (H2S) en soufre élémentaire et d'utiliser le nitrate comme accepteur d'électrons. On peut visualiser des vacuoles contenant le soufre et les nitrates et permettant aux bactéries de survivre à de longues périodes de restrictions en nitrate et en hydrogène sulfuré (4). Cette bactérie comme d'autres de ce groupe, possède aussi des introns auto-épissés, typiquement retrouvés uniquement chez les eucaryotes (5).

Découverte de Thiomargarita magnifica

Jean-Marie Volland et al. viennent de publier dans Science la découverte d'une nouvelle bactérie géante qu'ils appellent Thiomargarita magnifica (6). Il s'agit d'un travail collaboratif entre des équipes françaises de l'université des Antilles, du Muséum d'Histoire naturelle, de Sorbonne Université, et des équipes américaines de l'université de Californie, Berkeley. Cet organisme singulier avait été trouvé en 2009 par le Dr Olivier Gros, un des co-auteurs, biologiste de l'université des Antilles. Dans les forêts de mangrove de la Guadeloupe, il avait observé des filaments blancs de 0,9 cm à 2 cm, attachés aux feuilles immergées d'une plante aquatique (Rhizophora mangle) (7). En réalité il s'agissait d'une bactérie sulfureuse de la famille des γ-proteobacteria, comme T. namibiensis.

Les auteurs font une description morphologique et génomique très détaillée de cette singulière bactérie. Par diverses techniques d'imagerie, il apparaît que chaque filament est constitué d'une seule cellule sur presque toute sa longueur, sans septum de division, avec une constriction partielle de l'extrémité du pôle apical où des bourgeons (cellules-filles) se séparent du filament. La bactérie présente une vaste vacuole centrale tout au long du filament qui correspond à 75% du volume total de la bactérie. Cette vacuole contient de fortes concentrations de nitrates, ce qui permet une survie prolongée en l'absence de nitrates. Le cytoplasme est ainsi restreint à une mince couche externe de 0,5 à 2 µm en périphérie de la cellule. Cette réduction du cytoplasme minimiserait la dépendance à la diffusion chimique des nutriments puisque, à la différence des eucaryotes, les bactéries n'ont pas de système de transport intracellulaire actif. Dans le cytoplasme, on distingue au microscope électronique de nombreuses vésicules claires d'environ 2 µm de diamètre qui forment des granules réfringents de soufre. De plus, le cytoplasme contient des organelles membranaires en forme de bulles contenant du matériel nucléaire avec des fibrilles d'ADN et des ribosomes, comme les noyaux cellulaires des eucaryotes. Ces membranes contiennent des ATP synthases qui produisent de l'ATP, à l'instar des mitochondries des eucaryotes. Pratiquement aucune bactérie ni archée n'est connue pour ségréger son matériel génétique, à la façon du noyau des eucaryotes qui possède une membrane nucléaire. Les rares exceptions sont des nucléoïdes entourés de membranes dans une espèce de Atribacteria et certaines espèces de Plancotomycetes qui présentent un compartiment membranaire contenant de l' ADN (8,9).

Comme toutes les bactéries géantes déjà décrites (10), T. magnifica est une bactérie polyploïde. Elle contient en moyenne 37 000 copies du génome par mm de filaments. Son génome a été séquencé à partir de cinq cellules individuelles montrant une identité nucléotidique supérieure à 99,5%. La taille du génome est estimée à 11-12 Mb, comparativement à la moyenne des génomes bactériens de 4,2 Mb. Le génome de T. magnifica contient plus de 11788 gènes (contre 3935 gènes en moyenne chez les bactéries). Le génome cette bactérie chimioautotrophe comprend un grand nombre de gènes dévolus à l'oxydation du soufre et à la fixation du carbone. La polyploïdie est un moyen de décroître la pression de sélection sur les gènes, permettant des duplications de gènes intracellulaires, des réassortiments, des divergences menant à une diversité génétique intracellulaire extrême dans certaines bactéries géantes oxydant le soufre (10).

Les bactéries géantes

Que signifie ce gigantisme ? Les bactéries géantes appartiennent à quelques groupes bactériens hautement spécialisés. Elles vivent dans des conditions extrêmes de pH, de température ou de pression, et prospèrent souvent dans des environnements riches de carbone et de nutriments. Pour la plupart des bactéries, il est avantageux d'être très petites, car leur métabolisme dépend de la diffusion des molécules à travers la membrane cytoplasmique, un processus très rapide et efficace seulement sur de courtes distances de quelques micromètres ou moins. De plus grandes distances ralentissent fortement la diffusion des molécules. Les protozoaires eucaryotes qui mesurent 10 à 20 µm et peuvent parfois atteindre 3 à 4 cm (11), se sont affranchies de ces contraintes en élaborant des transporteurs protéiques et un système d'organelles pour organiser les fonctions subcellulaires. Cela pourrait expliquer la stratégie des bactéries géantes qui tend à augmenter leur surface par rapport à leur volume, tout en en restreignant leur cytoplasme. Dans le cas de T. magnifica, on observe en plus une compartimentalisation de l'ADN, comme chez les eucaryotes. De nombreuses questions restent en suspens, notamment sur le métabolisme et les fonctions des organelles de ces bactéries géantes. Comme le suggèrent les auteurs, elles pourraient être résolues par la mise en culture de cette bactérie dont le temps de croissance serait deux semaines, similaire à celui d'autres espèces de Thiomargarita (4).

Les recherches sur la biologie des bactéries géantes permettent un pas de plus vers la compréhension de l'évolution de la complexité biologique des cellules vivantes, aboutissant à l'emergence des cellules eucaryotes. Les innovations de la vie viennent-elles de l'adversité des milieux hostiles ? Conflits d'intérêts : L'auteur n'a aucun conflit d'intérêts à déclarer.

  Conflicts of interest : The author has no conflict ofinterest to declare. 1. Pr émérite Patrick Berche Membre de l'Académie de médecine, Université Paris-Cité, Email : patrick.berche@u-paris.fr References 1. H.N. Schulz, Jørgensen B.B., Big Bacteria, Ann. Rev. Microbiol., 55 (2001), p.105-137. 2. D. Ionescu, M. Bizic, Giant Bacteria, In: eLS. John Wiley & Sons, Ltd: Chichester, 2019. 3. E.R. Angert, K.D. Clements, N.R. Pace, The largest bacterium, Nature, 362 (1993), p.239-241. 4. H.N. Schulz , T. Brinkhoff., T.G. Ferdelman, M.H. Mariné, A. Teske, B.B. Jorgensen, Dense populations of a giant sulfur bacterium in Namibian shelf sediments. Science, 284 (1999), p. 493-495. 5. V. Salman, R. Amann, D.A. Shubb , H.N. Schulz-Vogta, Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria, PNAS, 109 (2012) p. 4203-4208. 6. J.M. Volland et al., A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles, Science 376 (2022), p. 1453-1458. 7. F. Muller, T. Brissac, N.Le Bris, H.Felbeck, O.Gros, First description of giant Archaea (Thaumarchaeota) associated with putative bacterial ectosymbionts in a sulfidic marine habitat. Environ. Microbiol. 12 (2010), p. 2371-2383. 8. T. Katayama, et al, Isolation of a member of the candidate phylum 'Atribacteria' reveals a unique cell membrane structure. Nature Communications, 11 (2020) Article N°6381. 9. C. Greening , T. Lithgow , Formation and function of bacterial organelles, Nat Rev Microbiol. 18 (2020), p. 677-689. 10. J.E. Mendell, K.D. Clements, J.H. Choat, E.R. Angert, Extreme polyploidy in a large bacterium. PNAS, 105 (2008), p. 6730-6734. 11. J. Dumais, K. Serikawa, D.F. Mandoli, Acetabularia: A unicellular model for understanding subcellular localization and morphogenesis during development. J Plant Growth Regul , 19 (2000), p.253-264.